CN101297042A - 四碳醇的发酵生产 - Google Patents
四碳醇的发酵生产 Download PDFInfo
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Abstract
提供了发酵生产四碳醇的方法。具体地,通过表达异丁醇生物合成途径的重组细菌的发酵生长来生产丁醇,优选异丁醇。
Description
相关申请的交叉参考
依据U.S.C.§119,本申请要求2005年10月26日申请的U.S.临时申请系列No.60/730290的优先权。
发明领域
本发明涉及工业微生物学和醇类生产的领域。更具体地,通过重组微生物的工业发酵来产生异丁醇。
发明背景
丁醇是重要的工业化学物质,用作燃料添加剂,塑料工业中的原料化学物质以及食品和香料工业中的食品级萃取剂。每年通过石油化学方法产生100至120亿磅的丁醇,并且对这种日用化学物质的需求很可能增加。
异丁醇化学合成的方法是已知的,如氧化合成,一氧化碳的催化氢化(Ullmann’s Encyclopedia of Industrial Chemistry,第6版,2003,Wiley-VCHVerlag GmbH and Co.,Weinheim,Germany,Vol.5,pp.716-719)以及甲醇和n-丙醇的Guerbet缩合(Carlini等,J.Mol.Catal.A:Chem.220:215-220(2004))。这些方法使用源自石油化学,并且通常是昂贵的,环境不友好的原料。从植物产生的原料生产异丁醇将最小化温室气体释放并将表示本领域中的进展。
异丁醇是作为酵母发酵的副产物通过生物产生的。它是作为通过该组真菌不完全代谢氨基酸的结果形成的“杂醇油”的组分。从L-缬氨酸的分解代谢特异性地产生异丁醇。作为氮源收集L-缬氨酸的胺基后,通过所谓的Ehrlich途径将所得到的α-酮酸脱羧基并还原成异丁醇(Dickinson等,J.Biol.Chem.273(40):25752-25756(1998))。在饮料发酵过程中获得的杂醇油和/或其组分的产量通常是低的。例如,报道了啤酒发酵中产生的异丁醇浓度低于百万分之16(Garcia等,ProcessBiochemistry 29:303-309(1994))。将外源L-缬氨酸添加至发酵中提高了异丁醇的产量,如Dickinson等所述的,上文,其中报道了通过在发酵中提供20g/L浓度的L-缬氨酸获得了3g/L异丁醇的产量。然而,将缬氨酸用作原料对于工业规模的异丁醇生产成本过高。直接从糖生物合成异丁醇在经济上是可行的,并将表示本领域中的进展。没有设计重组微生物来生产异丁醇的报道。
因此,存在对环境负责的,成本有效的作为单一产物的异丁醇生产方法的需求。本发明通过提供表达异丁醇生物合成途径的重组微生物生产宿主解决了这种需求。
发明概述
本发明提供了具有工程化异丁醇生物合成途径的重组微生物。该工程化的微生物用于异丁醇的商业性生产。因此,在一个实施方案中,本发明提供了重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至异丁醛(途径步骤d),和
v)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
在另一个实施方案中,本发明提供了重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f)
v)异丁酰-CoA至异丁醛(途径步骤g),和
vi)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
在另一实施方案中,本发明提供了重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至缬氨酸(途径步骤h)
v)缬氨酸至异丁胺(途径步骤i),
vi)异丁胺至异丁醛(途径步骤j),和
vii)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
在另一实施方案中,本发明提供了生产异丁醇的方法,其包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至异丁醛(途径步骤d),和
v)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
在另一实施方案中,本发明提供了生产异丁醇的方法,其包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f)
v)异丁酰-CoA至异丁醛(途径步骤g),和
vi)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
在另一实施方案中,本发明提供了生产异丁醇的方法,其包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a)
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b)
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c)
iv)α-酮基异戊酸至缬氨酸(途径步骤h)
v)缬氨酸至异丁胺(途径步骤i),
vi)异丁胺至异丁醛(途径步骤j),和
vii)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
在可替换的实施方案中,本发明提供了通过本发明的方法产生的含有异丁醇的发酵培养基。
附图和序列表简述
从以下形成本申请一部分的详述,附图和附带的序列描述可以更全面地了解本发明。
图1显示了四个不同的异丁醇生物合成途径。标记为“a”,“b”,“c”,“d”,“e”,“f”,“g”,“h”,“i”,“j”和“k”的步骤表示以下所述的底物至产物的转化。
以下的序列符合37C.F.R.1.821-1.825(“含有核苷酸序列和/或氨基酸序列公开内容的专利申请的要求-序列规定”)并符合世界知识产权组织(WIPO)标准ST.25(1998)以及EPO和PCT的序列表要求(细则5.2和49.5(a-bis),和行政指令的208条和附录C)。用于核苷酸和氨基酸序列数据的符号和格式遵照37C.F.R.§1.822中列出的规定。
表1
基因和蛋白质SEQ ID编号的概括
描述 | SEQ ID NO:核酸 | SEQ ID NO:肽 |
肺炎克雷伯氏菌(Klebsiella pneumoniae)budB(乙酰乳酸合酶) | 1 | 2 |
枯草芽孢杆菌(Bacillus subtillis)alsS(乙酰乳酸合酶) | 78 | 178 |
乳酸乳球菌(Lactococcus lactis)als(乙酰乳酸合酶) | 179 | 180 |
大肠杆菌(E.coli)ilvC(乙酰醇酸还原异构酶) | 3 | 4 |
酿酒酵母(S.cerevisiae)ILV5(乙酰醇酸还原异构酶) | 80 | 181 |
海藻甲烷球菌(M.maripaludis)ilvC(酮醇酸还原异构酶) | 182 | 183 |
枯草芽孢杆菌ilvC(乙酰醇酸还原异构酶) | 184 | 185 |
大肠杆菌ilvD(乙酰醇酸脱水酶) | 5 | 6 |
酿酒酵母ILV3(二醇酸脱水酶) | 83 | 186 |
海藻甲烷球菌ilvD(二醇酸脱水酶) | 187 | 188 |
枯草芽孢杆菌ilvD(二醇酸脱水酶) | 189 | 190 |
乳酸乳球菌kivD(支链α-酮酸脱羧酶),密码子最佳化 | 7 | 8 |
乳酸乳球菌kivD(支链α-酮酸脱羧酶) | 191 | 8 |
乳酸乳球菌kdcA(支链α-酮酸脱羧酶) | 192 | 193 |
鼠伤寒沙门氏菌(Salmonella typhimurium)(吲哚丙酮酸脱羧酶) | 194 | 195 |
丙醇丁酮梭菌(Clostridium acetobutylicum)pdc(丙酮酸脱羧酶) | 196 | 197 |
大肠杆菌yqhD(支链醇脱氢酶) | 9 | 10 |
酿酒酵母YPR1(2-甲基丁醛还原酶) | 198 | 199 |
酿酒酵母ADH6(NADPH-依赖性肉桂醇脱氢酶) | 200 | 201 |
丙醇丁酮梭菌bdhA(NADH-依赖性丁醇脱氢酶A) | 202 | 203 |
丙醇丁酮梭菌bdhB(丁醇脱氢酶) | 158 | 204 |
枯草芽孢杆菌bkdAA(支链酮酸脱氢酶E1亚基) | 205 | 206 |
枯草芽孢杆菌bkdAB(支链α-酮酸脱氢酶E1亚基) | 207 | 208 |
枯草芽孢杆菌bkdB(支链α-酮酸脱氢酶E2亚基) | 209 | 210 |
枯草芽孢杆菌lpdV(支链α-酮酸脱氢酶E3亚基) | 211 | 212 |
恶臭假单胞菌(P.putida)bkdA1(酮酸脱氢酶E1-α亚基) | 213 | 214 |
恶臭假单胞菌bkdA2(酮酸脱氢酶E1-β亚基) | 215 | 216 |
恶臭假单胞菌bkdB(转酰酶) | 217 | 218 |
恶臭假单胞菌lpdV(硫辛酰胺脱氢酶) | 219 | 220 |
拜氏梭菌(C.beijerinckii)ald(辅酶A酰化醛脱氢酶) | 221 | 222 |
丙醇丁酮梭菌adhe1(醛脱氢酶) | 223 | 224 |
丙醇丁酮梭菌adhe(醇-醛脱氢酶) | 225 | 226 |
恶臭假单胞菌nahO(乙醛脱氢酶) | 227 | 228 |
嗜热栖热菌(T.thermophilus)(乙醛脱氢酶) | 229 | 230 |
大肠杆菌avtA(缬氨酸-丙酮酸转氨酶) | 231 | 232 |
地衣芽孢杆菌(B.licheniformis)avtA(缬氨酸-丙酮酸转氨酶) | 233 | 234 |
大肠杆菌ilvE(支链氨基酸转氨酶) | 235 | 236 |
酿酒酵母BAT2(支链氨基酸转氨酶) | 237 | 238 |
嗜热碱甲烷杆菌(M.thermoautotrophicum)(支链氨基酸转氨酶) | 239 | 240 |
天蓝色链霉菌(S.coelicolor)(缬氨酸脱氢酶) | 241 | 242 |
枯草芽孢杆菌bcd(亮氨酸脱氢酶) | 243 | 244 |
生绿链霉菌(S.viridifaciens)(缬氨酸脱氢酶) | 245 | 246 |
反硝化产碱菌(A.denitrificans)aptA(ω-氨基酸:丙酮酸转氨酶) | 247 | 248 |
真养雷氏菌(R.eutropha)(丙氨酸-丙酮酸转氨酶) | 249 | 250 |
S.oneidensis(β丙氨酸-丙酮酸转氨酶) | 251 | 252 |
恶臭假单胞菌(β丙氨酸-丙酮酸转氨酶) | 253 | 254 |
肉桂地链霉菌(S.cinnamonensis)icm(异丁酰-CoA变位酶) | 255 | 256 |
肉桂地链霉菌icmB(异丁酰-CoA变位酶) | 257 | 258 |
天蓝色链霉菌SCO5415(异丁酰-CoA变位酶) | 259 | 260 |
天蓝色链霉菌SCO4800(异丁酰-CoA变位酶) | 261 | 262 |
除虫链霉菌(S.avermitilis)icmA(异丁酰-CoA变位酶) | 263 | 264 |
除虫链霉菌icmB(异丁酰-CoA变位酶) | 265 | 266 |
SEQ ID NO:11-38,40-69,72-75,85-138,144,145,147-157,159-176是本文所述实施例中使用的寡核苷酸克隆,筛选或测序引物的核苷酸序列。
SEQ ID NO:39是实施例16中所述的cscBKA基因簇的核苷酸序列。
SEQ ID NO:70是实施例13中所述的葡萄糖异构酶启动子1.6GI的核苷酸序列。
SEQ ID NO:71是实施例13中所述的1.5GI启动子的核苷酸序列。
SEQ ID NO:76是实施例17中所述的GPD启动子的核苷酸序列。
SEQ ID NO:77是实施例17中所述的CYC1终止子的核苷酸序列。
SEQ ID NO:79是实施例17中所述的FBA启动子的核苷酸序列。
SEQ ID NO:81是实施例17中所述的ADH 1启动子的核苷酸序列。
SEQ ID NO:82是实施例17中所述的ADH1终止子的核苷酸序列。
SEQ ID NO:84是实施例17中所述的GMP启动子的核苷酸序列。
SEQ ID NO:139是蔗糖水解酶(CscA)的氨基酸序列。
SEQ ID NO:140是D-果糖激酶(CscK)的氨基酸序列。
SEQ ID NO:141是蔗糖透性酶(CscB)的氨基酸序列。
SEQ ID NO:142是实施例20中所述的质粒pPF988DssPspac的核苷酸序列。
SEQ ID NO:143是实施例20中所述的质粒pPF988DssPgroE的核苷酸序列。
SEQ ID NO:146是实施例20中所述的pPF988Dss载体片段的核苷酸序列。
SEQ ID NO:177是实施例21中所述的质粒pPF988整合载体的核苷酸序列。
SEQ ID NO:267是实施例21中所述的质粒pC194的核苷酸序列。
发明详述
本发明涉及使用重组微生物生产异丁醇的方法。本发明满足各种商业和工业需求。丁醇是具有多种应用的重要工业日用化学品,其中其作为燃料或燃料添加剂的潜能是特别重要的。尽管只是四碳醇,丁醇具有与汽油相似的能含量并可以与任何矿物燃料混合。喜欢将丁醇作为燃料或燃料添加剂,因为它在标准内燃机中燃烧时只产生CO2并且几乎没有SOX或NOX。此外,丁醇比迄今为止最优选的燃料添加剂乙醇的腐蚀性小。
除了作为生物燃料或燃料添加剂的用途,丁醇具有影响新兴燃料电池工业中氢分配问题的潜能。目前燃料电池受到与氢运输和分配相关的安全问题的困扰。对于氢含量可以将丁醇容易地重组,并可以通过现有的加油站分配,以燃料电池或车辆需要的纯度。
最后,本发明从源自植物的碳源生产异丁醇,避免了与丁醇生产的标准石化方法相关的负面环境影响。
以下的定义和缩写用于权利要求和说明书的解释。
如在此所用的术语“发明”和“本发明”是非限制性的术语,不是用来指特定发明的任何单个实施方案,而是包括如说明书和权利要求中所述的所有可能的实施方案。
术语“异丁醇生物合成途径”指的是产生异丁醇的酶途径。
在此可交替使用的术语“乙酰乳酸合酶”和“乙酰乳酸合成酶”指的是催化丙酮酸转化成乙酰乳酸和CO2的酶。优选的乙酰乳酸合酶已知为EC编号2.2.1.69(Enzyme Nomenclature(酶命名)1992,AcademicPress,San Diego)。这些酶可从各种来源获得,包括但不限于,枯草芽孢杆菌(GenBank No:CAB 15618(SEQ ID NO:178),Z99122(SEQID NO:78),各自为NCBI(国立生物技术信息中心)氨基酸序列,NCBI核苷酸序列),肺炎克雷伯氏菌(GenBank No:AAA25079(SEQID NO:2),M73842(SEQ ID NO:1)),和乳酸乳球菌(GenBank No:AAA25161(SEQ ID NO:180),L16975(SEQ ID NO:179))。
在此可交替使用的术语“乙酰醇酸异构还原酶”和“乙酰醇酸还原异构酶”指的是使用NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸酯)作为电子供体催化乙酰乳酸转化成2,3-二羟基异戊酸的酶。优选的乙酰醇酸异构还原酶已知为EC编号1.1.1.86,并且可从大量的微生物阵列中获得序列,这些微生物包括但不限于,大肠杆菌(GenBank No:NP_418222(SEQ ID NO:4),NC_000913(SEQ ID NO:3)),酿酒酵母(GenBank No:NP_013459(SEQ ID NO:181),NC_001144(SEQID NO:80)),海藻甲烷球菌(GenBank No:CAF30210(SEQ ID NO:183),BX957220(SEQ ID NO:182)),和枯草芽孢杆菌(GenBank No:CAB 14798(SEQ ID NO:158),Z99118(SEQ ID NO:184))。
术语“乙酰醇酸脱水酶”指的是催化2,3-二羟基异戊酸转化成α-酮基异戊酸的酶。优选的乙酰醇酸脱水酶已知为EC编号4.2.1.9。这些酶可从大量的微生物阵列中获得序列,这些微生物包括但不限于,大肠杆菌(GenBank No:YP_026248(SEQ ID NO:6),NC_000913(SEQID NO:5)),酿酒酵母(GenBank No:NP_012550(SEQ ID NO:186),NC_001142(SEQ ID NO:83)),海藻甲烷球菌(GenBank No:CAF29874(SEQ ID NO:188),BX957219(SEQ ID NO:187)),和枯草芽孢杆菌(GenBank No:CAB14105(SEQ ID NO:190),Z99115(SEQ ID NO:189))。
术语“支链α-酮酸脱羧酶”指的是催化α-酮基异戊酸转化成异丁醛和CO2的酶。优选的支链α-酮酸脱羧酶已知为EC编号4.1.1.72并可从各种来源获得,包括但不限于,乳酸乳球菌(GenBank No:AAS49166(SEQ ID NO:193),AY548760(SEQ ID NO:192);CAG34226(SEQID NO:8),AJ746364(SEQ ID NO:191),鼠伤寒沙门氏菌(GenBankNo:NP_461346(SEQ ID NO:195),NC_003197(SEQ ID NO:194)),和丙醇丁酮梭菌(GenBank No:NP_149189(SEQ ID NO:197),NC_001988(SEQ ID NO:196))。
术语“支链醇脱氢酶”指的是催化异丁醛转化成异丁醇的酶。优选的支链醇脱氢酶已知为EC编号1.1.1.265,但也可以归类于其他醇脱氢酶(特别是,EC 1.1.1.1或1.1.1.2)。这些酶使用NADH(还原型烟酰胺腺嘌呤二核苷酸)和/或NADPH作为电子供体,并从各种来源可获得,包括但不限于,酿酒酵母(GenBank No:NP_010656(SEQ ID NO:199),NC_001136(SEQ ID NO:198);NP_014051(SEQ ID NO:201),NC_001145(SEQ ID NO:200)),大肠杆菌(GenBank No:NP_417484(SEQ ID NO:10),NC_000913(SEQ ID NO:9)),和丙醇丁酮梭菌(GenBank No:NP_349892(SEQ ID NO:203),NC_003030(SEQ IDNO:202);NP_349891(SEQ ID NO:204),NC_003030(SEQ ID NO:158))。
术语“支链酮酸脱氢酶”指的是催化α-酮基异戊酸转化成异丁酰-CoA(异丁酰-辅酶A)的酶,使用NAD+(烟酰胺腺嘌呤二核苷酸)作为电子受体。优选的支链酮酸脱氢酶已知为EC编号1.2.4.4。这些支链酮酸脱氢酶由四个亚基构成,并且所有亚基的序列可从大量微生物阵列中获得,这些维生物包括但不限于,枯草芽孢杆菌(GenBank No:CAB14336(SEQ ID NO:206),Z99116(SEQ ID NO:205);CAB14335(SEQ ID NO:208),Z99116(SEQ ID NO:207);CAB14334(SEQ IDNO:210),Z99116(SEQ ID NO:209);和CAB14337(SEQ ID NO:212),Z99116(SEQ ID NO:211))和恶臭假单胞菌(GenBank No:AAA65614(SEQ ID NO:214),M57613(SEQ ID NO:213);AAA65615(SEQ ID NO:216)。M57613(SEQ ID NO:215);AAA65617(SEQID NO:218),M57613(SEQ ID NO:217);和AAA65618(SEQ ID NO:220),M57613(SEQ ID NO:219))。
术语“酰化醛脱氢酶”指的是催化异丁酰-CoA转化成异丁醛的酶,使用NADH或NADPH作为电子供体。优选的酰化醛脱氢酶已知为EC编号1.2.1.10和1.2.1.57。这些酶可从多个来源获得,包括但不限于,拜氏梭菌(GenBank No:AAD31841(SEQ ID NO:222),AF157306(SEQ ID NO:221)),丙醇丁酮梭菌(GenBank No:NP_149325(SEQID NO:224),NC_001988(SEQ ID NO:223);NP_149199(SEQ IDNO:226),NC_001988(SEQ ID NO:225)),恶臭假单胞菌(GenBankNo:AAA89106(SEQ ID NO:228),U13232(SEQ ID NO:227)),和嗜热栖热菌(GenBank No:YP_145486(SEQ ID NO:230),NC_006461(SEQ ID NO:229))。
术语“转氨酶”指的是催化α-酮基异戊酸转化成L-缬氨酸的酶,使用丙氨酸或谷氨酸作为胺供体。优选的转氨酶已知为EC编号2.6.1.42和2.6.1.66。这些酶可从多种来源获得。丙氨酸依赖性酶的来源实例包括,但不限于,大肠杆菌(GenBank No:YP_026231(SEQ IDNO:232),NC_000913(SEQ ID NO:231))和地衣芽孢杆菌(GenBankNo:YP_093743(SEQ ID NO:234),NC_006322(SEQ ID NO:233))。谷氨酸依赖性酶的来源实例包括,但不限于,大肠杆菌(GenBank No:YP_026247(SEQ ID NO:236),NC_000913(SEQ ID NO:235)),酿酒酵母(GenBank No:NP_012682(SEQ ID NO:238),NC_001142(SEQ ID NO:237))和嗜热碱甲烷杆菌(GenBank No:NP_276546(SEQ ID NO:240),NC_000916(SEQ ID NO:239))。
术语“缬氨酸脱氢酶”指的是催化α-酮基异戊酸转化成L-缬氨酸的酶,使用NAD(P)H作为电子供体和氨作为胺供体。优选的缬氨酸脱氢酶已知为EC编号1.4.1.8和1.4.1.9,并可从各种来源获得,包括但不限于,天蓝色链霉菌(GenBank No:NP_628270(SEQ ID NO:242),NC_003888(SEQ ID NO:241))和枯草芽孢杆菌(GenBank No:CAB14339(SEQ ID NO:244),Z99116(SEQ ID NO:243))。
术语“缬氨酸脱羧酶”指的是催化L-缬氨酸转化成异丁胺和CO2的酶。优选的缬氨酸脱羧酶已知为EC编号4.1.1.14。这些酶可在链霉菌中找到,如例如,生绿链霉菌(GenBank No:AAN10242(SEQ ID NO:246),AY116644(SEQ ID NO:245))。
术语“ω转氨酶”指的是催化异丁胺转化成异丁醛的酶,使用合适的氨基酸作为胺供体。优选的ω转氨酶已知为EC编号2.6.1.18并可从各种来源获得,包括但不限于,反硝化产碱菌(AAP92672(SEQ ID NO:248),AY330220(SEQ ID NO:247)),真养雷氏菌(GenBank No:YP_294474)(SEQ ID NO:250),NC_007247(SEQ ID NO:249)),Shewanella oneidensis(GenBank No:NP_719046(SEQ ID NO:252),NC_004347(SEQ ID NO:251)),和恶臭假单胞菌(GenBank No:AAN66223(SEQ ID NO:254),AE016776(SEQ ID NO:253))。
术语“异丁酰-CoA突变酶”指的是催化丁酰-CoA转化成异丁酰-CoA的酶。这种酶使用辅酶B12作为辅因子。优选的异丁酰-CoA突变酶已知为EC编号5.4.99.13。在各种链霉菌中找到这种酶,包括但不限于,肉桂地链霉菌(GenBank No:AAC08713(SEQ ID NO:256),U67612(SEQ ID NO:255);CAB59633(SEQ ID NO:258),AJ246005(SEQID NO:257)),天蓝色链霉菌(GenBank No:CAB70645(SEQ ID NO:260),AL939123(SEQ ID NO:269);CAB92663(SEQ ID NO:262);AL939121(SEQ ID NO:261));和除虫链霉菌(GenBank No:NP_824008(SEQ ID NO:264),NC_003155(SEQ ID NO:263);NP_824637(SEQID NO:266),NC_003155(SEQ ID NO:265))。
术语“兼性厌氧菌”指的是在好氧和厌氧环境中都可以生长的微生物。
术语“碳底物”或“可发酵碳底物”指的是能够被本发明的宿主微生物代谢的碳源,并且是选自单糖,寡糖,多糖和单碳底物或其混合物的碳源。
术语“基因”指的是能够表达为特定蛋白质的核酸片段,任选包括编码序列之前(5’非-编码序列)和之后(3’非-编码序列)的调控序列。“天然基因”指的是与其自身调控序列在自然状态发现的基因。“嵌合基因”指的是不是天然基因的任何基因,包括不是在自然状态一起发现的调控序列和编码序列。因此,嵌合基因可以包括来自不同来源的调控序列和编码序列,或调控序列和编码序列源自相同的来源,但以不同于自然状态发现的方式排列。“内源基因”指的是处于其在生物体基因组中的天然位置的天然基因。“外源基因”或“异源基因”指的是通常不是在宿主生物体中发现的而是通过基因转移引入宿主生物体中的基因。外源基因可以包括插入非天然生物体中的天然基因,或嵌合基因。“转基因”是已经通过转化程序引入基因组中的基因。
如在此所用的,术语“编码序列”指的是编码特定氨基酸序列的DNA序列。“合适的调控序列”指的是位于编码序列上游(5’非-编码序列),之内或下游(3’非-编码序列)的核苷酸序列,并且其影响相连编码序列的转录,RNA加工或稳定性,或翻译。调控序列可以包括启动子,翻译前导序列,内含子,多腺苷酸化识别序列,RNA加工位点,效应子结合位点和茎-环结构。
术语“启动子”指的是能够控制编码序列或功能性RNA表达的DNA序列。通常,编码序列位于启动子序列的3’端。启动子可以完全源自天然基因,或由源自自然状态发现的不同启动子的不同序列构成,或甚至包括合成的DNA片段。本领域技术人员应当理解不同的启动子可以指导不同组织或细胞类型中的,或在不同的发育阶段的,或应答不同的环境或生物条件的基因表达。使基因在大部分时间在大部分细胞类型中得到表达的启动子通常称为“组成型启动子”。进一步认识到因为在大部分情况中,调控序列的确切分界线还没有得到完全限定,因此不同长度的DNA片段可能具有相同的启动子活性。
术语“可操纵地连接”指的是单个核酸片段上的核酸序列连接,使得一个的功能受到另一个的影响。例如,在其能够影响编码序列的表达时,启动子可操纵地连接编码序列(即,编码序列处于启动子的转录控制下)。可以将编码序列以正义或反义方向可操纵地连接调控序列。
如在此所用的术语“表达”指的是源自本发明核酸片段的正义(mRNA)或反义RNA的转录和稳定累积。表达还可以指mRNA翻译成多肽。
如在此所用的术语“转化”指的是核酸片段转移至宿主生物体中,导致遗传上稳定的遗传。将含有转化的核酸片段的宿主生物体称为“转基因”或“重组”或“转化的”生物体。
术语“质粒”,“载体”和“盒”指的是额外的染色体序列,其通常携带不是细胞重要代谢的一部分的基因,且通常是环状双链DNA片段的形式。这样的序列可以是源自任何来源的单链或双链DNA或RNA的自主复制序列,基因组整合序列,噬菌体或核苷酸序列,线性或环状的,其中各种核苷酸序列已经连接或重组成独特的构建体,其能够将用于选定的基因产物的启动子片段和DNA序列以及合适的3’未翻译序列引入细胞中。“转化盒”指的是含有外源基因并具有除了促进特定宿主细胞转化的外源基因以外的序列的特定载体。“表达盒”指的是含有外源基因并具有除了允许该基因在外源宿主中提高的表达的外源基因以外的序列的特定载体。
如在此所用的术语“密码子简并性”指的是遗传密码中允许核苷酸序列改变而没有影响所编码多肽的氨基酸序列的性质。本领域技术人员公知核苷酸密码子使用中由特定宿主细胞呈现的“密码子偏好”确定了给定的氨基酸。因此,合成用于在宿主细胞中提高表达的基因时,理想的是设计基因使得密码子使用的频率接近宿主细胞的优选密码子使用的频率。
术语“密码子最佳化”,当涉及用于各种宿主转化的基因和核酸分子的编码片段时,指的是基因或核酸分子编码片段中的密码子改变,这种改变反应出宿主生物体的典型密码子使用而没有改变由DNA编码的多肽。
在此使用的标准重组DNA和分子克隆技术是本领域公知的,并描述于Sambrook,J.,Fritsch,E.F.和Maniatis,T.,Molecular Cloning:ALaboratory Manual(分子克隆:实验室手册),第二版,Cold SpringHarbor Laboratory Press,Cold Spring Harbor,NY(1989)(此后称为“Maniatis”);和Silhavy,T.J.,Bennan,M.L.和Enquist,L.M.,Experiments with Gene Fusions(使用基因融合的表达),Cold SpringHarbor Laboratory Press.Cold Spring Harbor,NY(1984);和Ausubel,F.M.等,Current Protocols in Molecular Biology(分子生物学中的通用实验方案),Greene Publishing Assoc.and Wiley-Interscience出版(1987)。
异丁醇生物合成途径
利用碳水化合物的微生物使用Embden-Meyerhof-Parnas(EMP)途径,Entner-Doudoroff途径和戊糖磷酸酯循环作为主要的代谢途径来提供用于生长和维持的能量和细胞前体。这些途径具有共同的中间产物甘油醛-3-磷酸,并且最终直接或结合EMP途径形成丙酮酸。随后,丙酮酸通过各种方式转化成乙酰-辅酶A(乙酰-CoA)。乙酰-CoA用作关键的中间产物,例如,在产生脂肪酸,氨基酸和次级代谢产物中。糖转化成丙酮酸的组合反应产生了能量(例如,腺苷-5’-三磷酸,ATP)和还原等价物(例如,还原型烟酰胺腺嘌呤二核苷酸,NADH,和还原型烟酰胺腺嘌呤二核苷酸磷酸酯,NADPH)。NADH和NADPH必须再循环成其氧化形式(各自为NAD+和NAPD+)。在无机电子受体(例如,O2,NO3 -和SO4 2-)的存在下,还原等价物可以用来扩大能量池;或者,可以形成还原碳副产物。
本发明能够通过提供四个完整的反应途径使用重组微生物从碳水化合物来源产生异丁醇,如图1中所示的。其中三个途径包括丙酮酸通过一系列的酶步骤转化成异丁醇。优选的异丁醇步骤(图1,步骤a至e),包括以下的底物至产物的转化:
a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的,
b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催化的,
c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催化的,
d)α-酮基异戊酸至异丁醛,例如由支链酮酸脱羧酶催化的,
e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。
该途径结合了已知在公认的缬氨酸生物合成(丙酮酸至α-酮基异戊酸)和缬氨酸分解代谢(α-酮基异戊酸至异丁醇)途径中涉及的酶。因为许多缬氨酸生物合成酶还在异亮氨酸生物合成途径中催化类似的反应,因此底物特异性在选择基因来源中是一个主要的考虑因素。为此,对于乙酰乳酸合酶的主要目标基因是来自芽孢杆菌(alaS)和克雷伯氏菌(budB)的那些。已知这些特定的乙酰乳酸合酶参与这些生物体中的丁二醇发酵,并显示出对丙酮酸的亲和性高于酮丁酸(Gollop等,J.Bacteriol.172(6):3444-3449(1990);Holtzclaw等,J.Bacteriol.121(3):917-922(1975))。第二个和第三个途径步骤各自是由乙酰醇酸还原异构酶和脱水酶催化的。已经从各种来源表征了这些酶,如例如,大肠杆菌(Chunduru等,Biochemistry 28(2):486-493(1989);Flint等,J.Biol.Chem.268(29):14732-14742(1993))。已知优选的异丁醇途径的最后两个步骤在酵母中发生,酵母可以使用缬氨酸作为氮源,并且在过程中分泌异丁醇。α-酮基异戊酸可以通过各种酮酸脱羧酶转化成异丁醛,如例如丙酮酸脱羧酶。为了防止丙酮酸远离异丁醇产生的方向错误,对丙酮酸的亲和性降低的脱羧酶是理想的。迄今为止,本领域中已知存在两种这样的酶(Smit等,Appl.Environ.Microbiol.71(1):303-311(2005);de la Plaza等,FEMS Microbiol.Lett.238(2):367-374(2004))。两种酶都来自乳酸乳球菌的菌株并对酮异戊酸的偏好超过丙酮酸50-200倍。最后,在酵母中已经鉴定了多种醛还原酶,许多具有重叠的底物特异性。已知偏好支链底物胜于乙醛的那些包括但不限于,醇脱氢酶VI(ADH6)和Ypr1p(Larroy等,Biochem.J.361(Pt1):163-172(2002);Ford等,Yeast 19(12):1087-1096(2002)),两者都使用了NADPH作为电子供体。最近已经在大肠杆菌中鉴定了用支链底物激活的NADPH-依赖性还原酶,YqhD(Sulzenbacher等,J.Mol.Biol.342(2):489-502(2004))。
丙酮酸转化成异丁醇的另一个途径包括以下的底物至产物的转化(图1,步骤a,b,c,f,g,e):
a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的,
b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催化的,
c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催化的,
f)α-酮基异戊酸至异丁酰-CoA,例如由支链酮酸脱氢酶催化的,
g)异丁酰-CoA至异丁醛,例如由酰化醛脱氢酶催化的,
e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。
该途径中的头三个步骤(a,b,c)和上述的那些相同。通过支链酮酸脱氢酶的作用将α-酮基异戊酸转化成异丁酰-CoA。虽然酵母只使用缬氨酸作为氮源,但许多其他生物体(真核生物和原核生物)可以也可以使用缬氨酸作为碳源。这些生物体具有支链酮酸脱氢酶(Sokatch等,J.Bacteriol.148(2):647-652(1981)),其产生异丁酰-CoA。异丁酰-CoA通过酰化醛脱氢酶可以转化成异丁醛。已经描述了明串珠菌(Leuconostoc)和丙酸杆菌(Propionibacterium)中用支链底物激活的脱氢酶,但还没有克隆(Kazahaya等,J.Gen.Appl.Microbiol.18:43-55(1972);Hosol等,J.Ferment.Technol.57:418-427(1979))。然而,还可能的是已知作用于直链酰基-CoA(即,丁酰-CoA)的酰化醛脱氢酶也作用于异丁酰-CoA。然后异丁醛通过支链醇脱氢酶转化成异丁醇,如以上对于第一个途径中所述的。
丙酮酸转化成异丁醇的另一个途径包括以下的底物至产物的转化(图1,步骤a,b,c,h,i,j,e):
a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的,
b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催化的,
c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催化的,
h)α-酮基异戊酸至缬氨酸,例如由缬氨酸脱氢酶或转氨酶催化的,
i)缬氨酸至异丁胺,例如由缬氨酸脱羧酶催化的,
j)异丁胺至异丁醛,例如由ω转氨酶催化的,和
e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。
该途径中的头三个步骤(a,b,c)和上述的那些相同。该途径需要加入缬氨酸脱氢酶或合适的转氨酶。缬氨酸(和或亮氨酸)脱氢酶催化还原性胺化并使用氨;氨的Km值在毫摩尔范围中(Priestly等,Biochem J.261(3):853-861(1989);Vancura等,J.Gen.Microbiol.134(12):3213-3219(1988),Zink等,Arch.Biochem.Biophys.99:72-77(1962);Sekimoto等,J.Biochem(日本)116(1):176-182(1994))。转氨酶通常使用谷氨酸或丙氨酸作为氨基供体,并已经从各种生物体得到了表征(Lee-Peng等,J.Bacteriol.139(2):399-345(1979);Berg等,J.Bacteriol.155(3):1009-1014(1983))。丙氨酸-特异性酶是理想的,因为除去胺基时,该步骤的丙酮酸产生可以结合途径中后来的丙酮酸消耗(参见以下的)。下一个步骤是缬氨酸的脱羧作用,这是在链霉菌中的valanimycin生物合成中发生的反应(Gang等,Mol.Microbiol.46(2):505-517(2002))。所得到的异丁胺可以在吡哆醛5’-磷酸-依赖性反应中转化成异丁醛,例如通过ω-转氨酶家族的酶。已经证明来自河流弧菌(Vibrio fluvialis)这样的酶具有异丁胺活性(Shin等,Biotechnol.Bioeng.65(2):206-211(1999))。已经克隆了另一种来自反硝化产碱菌的ω-转氨酶并且具有一定的丁胺活性(Yun等,Appl.Environ.Microbiol.70(4):2529-2534(2004))。在这种方向中,这些酶使用丙酮酸作为氨基受体,产生丙氨酸。如上所述,在途径的早期使用产生丙酮酸的转氨酶可以抵消对丙酮酸池的不利影响。然后异丁醛通过支链醇脱氢酶转化成异丁醇,如以上对于第一个途径所述的。
第四个异丁醇生物合成途径包括图1中步骤k,g,e所示的底物至产物的转化。已知各种生物体通过丁酰-CoA中间产物产生丁酸和/或丁醇(Dürre等,FEMS Microbiol.Rev.17(3)251-262(1995);Abbad-Andaloussi等,Microbiology 142(5):1149-1158(1996))。通过加入能够转化丁酰-CoA至异丁酰-CoA的突变酶在这些生物体中工程化异丁醇产生(图1,步骤k)。已经从链霉菌克隆了异丁酰-CoA突变酶(辅酶B12-依赖性酶)的两个亚基的基因(Ratnatilleke等,J.Biol.Chem.274(44):31679-31685(1999))。异丁酰-CoA转化成异丁醛(图1中的步骤g),其转化成异丁醇(图1中的步骤e)。
因此,在提供丙酮酸至异丁醇的多个重组途径中,存在各种选择来满足单个转化步骤,并且本领域技术人员将能够利用公众可获得的序列来构建相关的途径。以下的表2中列出了本领域已知的且异丁醇生物合成途径的构建中有用的代表性基因的列表。
表2
异丁醇生物合成途径基因的来源
基因 | GenBank引用 |
乙酰乳酸合酶 | Z99122,枯草芽孢杆菌完整基因组(21个片段中19个):从3608981至3809670gi|3248830|emb|Z99122.2|BSUB0019[32468830] |
M73842,肺炎克雷伯氏菌乙酰乳酸合酶(iluk)基因,完整cdsgi|1429210|gb|M73842.1|KPNILUK[149210] | |
L16975,乳酸乳球菌α-乙酰乳酸合酶(als)基因,完整cdsgi|473900|gb|L16975.1|LACALS[473900] | |
乙酰醇酸异构还原酶 | NC_000913,大肠杆菌K12,完整基因组gi|49175990|ref|NC_000913.2|[49175990] |
NC_001144,酿酒酵母染色体XII,完整的染色体序列gi|42742286|ref|NC_001144.3|[42742286] | |
BX957220,海藻甲烷球菌S2完整基因组;片段2/5gi|44920669|emb|BX957220.1|[44920669] | |
Z99118,枯草芽孢杆菌完整基因组(21个片段中15个):从2812801至3013507gi|32468802|emb|Z99118.2|BSUB0015[32468802] | |
乙酰醇酸脱水酶 | NC_000913,大肠杆菌K12,完整基因组gi|49175990|ref|NC_000913.2|[49175990] |
NC_001142,酿酒酵母染色体X,完整染色体序列gi|42742252|ref|NC_001142.5|[42742252] | |
BX957219,海藻甲烷球菌S2完整基因组;片段1/5gi|45047123|emb|BX957219.1|[45047123] | |
Z99115,枯草芽孢杆菌完整基因组(21个片段中12个):从2207806 |
至2409180gi|32468778|emb|Z99115.2|BSUB0012|[32468778] | |
支链α酮酸脱羧酶 | AY548760,乳酸乳球菌支链α酮酸脱羧酶(kdcA)基因,完整cdsgi|44921616|gb|AY548760.1|[44921616] |
AJ746364,α酮异戊酸脱羧酶的乳酸乳球菌subsp.lactis kivd基因,菌株IFPL730gi|51870501|emb|AJ746364.1|[51870501] | |
NC_003197,鼠伤寒沙门氏菌LT2,完整基因组gi|16763390|ref|NC_003197.1|[16763390] | |
NC_001988,丙醇丁酮梭菌ATCC 824质粒pSOL1,完整序列gi|15004705|ref|NC_001988.2|[15004705] | |
支链醇脱氢酶 | NC_001136,酿酒酵母染色体IV,完整染色体序列gi|50593138|ref|NC_001136.6|[50593138] |
NC_001145,酿酒酵母染色体XIII,完整染色体序列gi|44829554|ref|NC_001145.2|[44829554] | |
NC_000913,大肠杆菌K12,完整基因组gi|49175990|ref|NC_000913.2|[49175990] | |
NC_003030,丙醇丁酮梭菌ATCC824,完整基因组gi|15893298|ref|NC_003030.1|[15893298] | |
支链酮酸脱氢酶 | Z99116,枯草芽孢杆菌完整基因组(21个片段中13个):从2409151至2613687gi|32468787|emb|Z99116.2|BSUB0013[32468787] |
M57613,恶臭假单胞菌支链酮酸脱氢酶操纵子(bkdA1,bkdA1和bkdA2),转酰酶E2(bkdB),bkdR和硫辛酰胺脱氢酶(lpdV)基因,完整cdsgi|790512|gb|M57613.1|PSEBKDPPG2[790512] | |
酰化醛脱氢酶 | AF157306,拜氏梭菌菌株NRRL B593假定的蛋白,辅酶A酰化醛脱氢酶(ald),乙酰醋酸:丁酸/醋酸辅酶A转移酶(ctfA),乙酰醋酸:丁酸/醋酸辅酶A转移酶(ctfB),和乙酰醋酸脱羧酶(adc)基因,完整cdsgi|47422980|gb|AF157306.2|[47422980] |
NC_001988,丙醇丁酮梭菌ATCC824质粒pSOL1,完整序列 |
gi|15004705|ref|NC_001988.2|[15004705] | |
U13232,恶臭假单胞菌NCIB9816乙醛脱氢酶(nahO)和4-羟基-2-氧戊酸醛缩酶(nahM)基因,完整cds,和4-草酰巴豆酯脱羧酶(nahK)基因和2-氧代-4-戊酸甲酯水合酶(nahL)基因,部分cdsgi|595671|gb|U13232.1|PPU13232[595671] | |
转氨酶 | NC_000913,大肠杆菌K12,完整基因组gi|49175990|ref|NC_000913.2|[49175990] |
NC_006322,地衣芽孢杆菌ATCC14580,完整基因组gi|52783855|ref|NC_006322.1|[52783855] | |
NC_001142,酿酒酵母染色体X,完整染色体序列gi|42742252|ref|NC_001142.5|[42742252] | |
NC_000916,嗜热碱甲烷杆菌str.Delta H,完整基因组gi|15678031|ref|NC_000916.1|[15678031] | |
缬氨酸脱氢酶 | NC_003888,天蓝色链霉菌A3(2),完整基因组gi|32141095|ref|NC_003888.3|[321410945] |
Z99116,枯草芽孢杆菌完整基因组(21个片段中13个):从2409151至2613687gi|32468787|emb|Z99116.2|BSUB0013[32468787] | |
缬氨酸脱羧酶 | AY116644,生绿链霉菌氨基酸转氨酶基因,部分cds;酮酸还原异构酶,乙酰乳酸合酶小亚基,乙酰乳酸合酶大亚基,完整cds;氧化偶氮基抗生素valanimycin基因簇,完整序列;以及公认的转移酶,和公认的分泌的蛋白基因,完整cdsgi|27777548|gb|AY116644.1|[27777548] |
ω转氨酶 | AY330220,反硝化产碱菌ω氨基酸:丙酮酸转氨酶(aptA)基因,完整cdsgi|33086797|gb|AY330220.1|[33086797] |
NC_007347,真养雷氏菌JMP134染色体1,完整序列gi|73539706|ref|NC_007347.1|[73539706] | |
NC_004347,Shewanella oneidensis MR-1,完整基因组gi|24371600|ref|NC_004347.1|[24371600] | |
NZ_AAAG02000002,深红红螺菌(Rhodospirillum rubrum)Rrub02_2, |
全基因组鸟枪序列gi|48764549|ref|NZ_AAAG02000002.1|[48764549] | |
AE016776,恶臭假单胞菌KT2440,完整基因组的21个片段中的3个gi|26557019|gb|AE016776.1|[26557019] | |
异丁酰-CoA突变酶 | U67612,肉桂地链霉菌辅酶B12-依赖性异丁酰CoA突变酶(icm)基因,完整cdsgi|3002491|gb|U67612.1|SCU67612[3002491] |
AJ246005,异丁酰-CoA突变酶的肉桂地链霉菌icmB基因,小亚基gi|6137076|emb|AJ246005.1|SCI246005[6137076] | |
AL939123,天蓝色链霉菌A3(2)完整基因组;片段20/29gi|24430032|emb|AL939123.1|SCO939123[24430032] | |
AL9939121,天蓝色链霉菌A3(2)完整基因组;片段18/29gi|24429533|emb|AL939121.1|SCO939121[24429533] | |
NC_003155,除虫链霉菌MA-4680,完整基因组gi|57833846|ref|NC_003155.3|[57833846| |
用于异丁醇生产的微生物宿主
用于异丁醇生产的微生物宿主可以选自细菌,蓝细菌,丝状真菌和酵母。用于异丁醇生产的微生物宿主优选耐异丁醇,使得产量不受到丁醇毒性的限制。在高异丁醇滴定水平具有代谢活性的微生物不是本领域公知的。尽管已经从产溶剂(solventogenic)梭菌分离了耐丁醇的突变体,但是关于其他潜在有用菌株的丁醇耐受性的信息获得得很少。对于细菌醇耐受性比较的大部分研究表明丁醇比乙醇更毒(de Cavalho等,Microsc.Res.Tech.64:215-22(2004)和Kabelitz等,FEMS Microbiol.Lett.220:223-227(2003))。Tomas等(J.Bacteriol.186:2006-2018(2004))报道了丙醇丁酮梭菌发酵过程中的1-丁醇产量受到了1-丁醇毒性的限制。1-丁醇对丙醇丁酮梭菌的主要影响是膜功能的破坏(Hermann等,Appl.Environ.Microbiol.50:1238-1243(1985))。
选择用于异丁醇生产的微生物宿主优选耐异丁醇,并应当能够将碳水化合物转化成异丁醇。选择合适微生物宿主的标准包括以下的:内在的异丁醇耐受性,高葡萄糖利用率,用于基因操纵的遗传工具的可用性和产生稳定的染色体改变的能力。
可以通过基于菌株内在耐受性的筛选来鉴定具有异丁醇耐受性的合适宿主菌株。可以通过测定在最小培养基中生长时引起50%生长速率抑制(IC50)的异丁醇浓度来测量微生物对异丁醇的内在耐受性。可以使用本领域已知的方法来测定IC50值。例如,可以将目标微生物在各种含量的异丁醇存在下生长并通过测量600纳米处的光密度来监控生长速率。从生长曲线的对数部分计算倍增时间并用作生长速率的测量。可以从生长的百分比抑制对异丁醇浓度的图中测定出产生50%生长抑制的异丁醇浓度。优选,宿主菌株对于异丁醇应当具有高于约0.5%的IC50。
用于异丁醇生产的微生物宿主还应当以高速率来利用葡萄糖。大部分微生物能够利用碳水化合物。然而,特定的环境微生物不能高效地利用碳水化合物,并因此不是合适的宿主。
在遗传上修饰宿主的能力对于任何重组微生物的产生是必须的。基因转移技术的方式可以通过电穿孔,缀合,转导或天然转化。各种宿主可缀合质粒和抗药性标记是可获得的。基于在该宿主中抗生素抗性标记的性质,对宿主生物体定制克隆载体。
微生物宿主还必须得到操纵,以便通过删除各种基因使碳流的竞争途径失活。这需要利用指导失活的转座子或染色体整合载体。此外,生产宿主应当顺从化学诱变,使得可以获得提高内在异丁醇耐受性的突变。
基于上述的标准,用于异丁醇生产的合适微生物宿主包括,但不限于,梭菌属,发酵单胞菌属(Zymomonas),埃希氏菌属(Escherichia),沙门氏菌属,红球菌属(Rhodococcus),假单胞菌属,芽孢杆菌属,乳杆菌属(Lactobacillus),肠球菌属(Enterococcus),产碱杆菌属(Alcaligenes),克雷伯氏菌属,类芽孢杆菌属(Paenibacillus),节杆菌属(4rthrobacter),棒状杆菌属(Corynebacterium),短杆菌属(Brevibacterium),毕赤氏酵母属(Pichia),假丝酵母属(Candida),汉森酵母属(Hansenula)和酵母属(Saccharomyces)的成员。优选的宿主包括:大肠杆菌(Escherichia coli),真养产碱杆菌(Alcaligeneseutrophus),地衣芽孢杆菌,浸麻类芽孢杆菌(Paenibacillus macerans),红串红球菌(Rhodococcus erythropolis),恶臭假单胞菌,植物乳杆菌(Lactobacillus plantarum),屎肠球菌(Enterococcus faecium),鹑鸡肠球菌(Enterococcus gallinarium),粪肠球菌(Enterococcus faecalis),枯草芽孢杆菌和酿酒酵母。
生产宿主的构建
可以使用本领域公知的技术来构建含有必需基因的重组生物体,该基因编码可发酵碳底物转化成异丁醇的酶途径。在本发明中,可以从各种来源分离编码本发明异丁醇生物合成途径之一的酶,例如,乙酰乳酸合酶,乙酰醇酸异构还原酶,乙酰醇酸脱水酶,支链α-酮酸脱羧酶,如上所述。
从细菌基因组获得所需基因的方法是分子生物学领域中常见的和公知的。例如,如果基因的序列是已知的,可以通过限制性核酸内切酶分泌合适的基因组文库,并可以使用与所需基因序列互补的探针来筛选。一旦序列得到分离,使用标准引物定向扩增方法如聚合酶链式反应(U.S.4,683,202)扩增DNA来获得适于使用合适载体转化的适量DNA。用于在异源宿主中表达的密码子最佳化的工具是容易获得的。基于宿主生物体的GC含量可以使用一些用于密码子最佳化的工具。表3中给出了一些实例微生物宿主的GC含量。
表3
微生物宿主的GC含量
菌株 | %GC |
地衣芽孢杆菌 | 46 |
枯草芽孢杆菌 | 42 |
丙醇丁酮梭菌 | 37 |
大肠杆菌 | 50 |
恶臭假单胞菌 | 61 |
真养产碱杆菌 | 61 |
浸麻类芽孢杆菌 | 51 |
红串红球菌 | 62 |
短杆菌 | 50 |
多粘类芽孢杆菌(Paenibacilluspolymyxa) | 50 |
一旦鉴定并分离出相关的途径基因,可以通过本领域公知的方式将它们转化值合适的表达载体中。用于转化各种宿主细胞的载体或盒是常见的并从如下的公司可购得,(Madison,WI),Invitrogen Corp.(Carlsbad,CA),Stratagene(La Jolla,CA)和NewEngland Biolabs,Inc.(Beverly,MA)。通常,载体和盒含有指导相关基因的盒翻译的序列,选择标记和允许自主复制或染色体整合的序列。合适的载体包括带有转录启动控制的基因5’端和控制转录终止的DNA片段的3’端。两个控制片段可以源自与转化的宿主细胞同源的基因,尽管可以理解这样的控制片段也可以源自不是选为生产宿主的特定物种的天然基因。
用于驱动所需宿主细胞中相关途径编码片段的表达的启动控制片段或启动子是为数众多的并是本领域技术人员熟知的。实际上,能够驱动这些遗传序列的任何启动子都适用于本发明,包括但不限于,CYC1,HIS3,GAL1,GAL10,ADH1,PGK,PHO5,GAPDH,ADC1,TRP1,URA3,LEU2,ENO,TPI,CUP1,FBA,GPD和GPM(用于在酵母中的表达);AOX1(用于在毕赤氏酵母中的表达);和lac,ara,tet,trp,IPL,IPR,T7,tac和trc(用于大肠杆菌,产碱杆菌和假单胞菌中的表达);amy,apr,npr启动子和各种噬菌体启动子,用于枯草芽孢杆菌,地衣芽孢杆菌和浸麻类芽孢杆菌中的表达;nisA(用于革兰氏阳性细菌中的表达,Eichenbaum等,Appl.Environ.Microbiol.64(8):2763-2769(1998));和合成的P11启动子(用于植物乳杆菌中的表达,Rud等,Microbiology 152:1011-1019(2006))。
终止控制片段也可以源自优选宿主的各种天然基因。任选地,终止位点不是必需的,然而,如果包括,是最优选的。
特定的载体能够在大范围的宿主细菌中复制并可以通过缀合来转移。pRK4的完整和注释序列以及三个相关的载体-pRK437,pRK442和pRK442(H)是可获得的。已经证明这些衍生物对于革兰氏阴性细菌中的遗传操纵是有价值的工具(Scott等,Plasmid50(1):74-79(2003))。宽宿主范围的Inc P4质粒RSF1010的几个质粒衍生物也是可获得的,具有可以在各种革兰氏阴性细菌中起作用的启动子。质粒pAYC36和pAYC37,具有活性启动子和多个克隆位点,以允许异源基因在革兰氏阴性细菌中的表达。
还广泛使用了染色体基因替代工具。例如,已经修改宽宿主范围复制子pWV101的热敏感性变体来构建质粒pVE6002,其可以用于在各种革兰氏阳性细菌中实现基因替代(Maguin等,J.Bacteriol.174(17):5633-5638(1992))。此外,可利用体外transposome来形成来自商业来源如的各种基因组中的随机突变。
以下更详细地描述了各种优选微生物宿主中异丁醇生物合成途径的表达。
大肠杆菌中异丁醇生物合成途径的表达
用于大肠杆菌转化的载体或盒是常见的并从以上所列的公司可购得。例如,可以从各种来源分离异丁醇生物合成途径的基因,克隆至修饰的pUC9载体中并转化至大肠杆菌NM522中,如实施例6和7中所述的。
红串红球菌中异丁醇生物合成途径的表达
可以将一系列大肠杆菌-红球菌穿梭载体用于红串红球菌中的表达,包括但不限于,pRhBR17和pDA71(Kostichka等,Appl.Microbiol.Biotechnol.62:61-68(2003))。此外,可以将一系列启动子用于红串红球菌中的异源基因表达(参见例如Nakashima等,Appl.Environ.Microbiol.70:5557-5568(2004),和Tao等,Appl.Microbiol.Biotechnol.2005,DOI 10.1007/s00253-005-0064)。可以使用Tao等,上文和Brahs等(Appl.Environ.Microbiol.66:2029-2036(2000))所述的方法形成红串红球菌中染色体基因的靶向基因破坏。
如上所述,最初可以在pDA71或pRhBR71中克隆异丁醇生产需要的异源基因并转化至大肠杆菌中。然后通过电穿孔将载体转化至红串红球菌中,如Kostichka等所述的。可以将重组体生长于含有葡萄糖的培养基中并使用本领域已知的方法进行异丁醇的生产。
枯草芽孢杆菌中异丁醇生物合成途径的表达
用于枯草芽孢杆菌中的基因表达和突变形成的方法也是本领域公知的。例如,可以从各种来源分离异丁醇生物合成途径的基因,克隆至修饰的pUC19载体中并转化至枯草芽孢杆菌BE1010中,如实施例8中所述的。此外,可以将异丁醇生物合成途径的五个基因分成两个用于表达的操纵子,如实施例20中所述的。将途径的三个基因(bubB,ilvD和kivD)整合至枯草芽孢杆菌BE1010的染色体中(Payne和Jackson,J.Bacteriol.173:2278-2282(1991))。将剩余的两个基因(ilvD和bdhB)克隆至表达载体中并转化至带有整合的异丁醇基因的芽孢杆菌菌株中。
地衣芽孢杆菌中异丁醇生物合成途径的表达
在枯草芽孢杆菌中复制的大部分质粒和穿梭载体可以用于转化地衣芽孢杆菌,通过原生质体转化或电穿孔。在质粒pBE20或pBE60衍生物中克隆异丁醇生产需要的基因(Nagarajan等,Gene 114:121-126(1992))。转化地衣芽孢杆菌的方法是本领域已知的(例如,参见,Fleming等,Appl.Environ.Microbiol.61(11):3775-3780(1995))。可以将为了在枯草芽孢杆菌中表达而构建的质粒转化至地衣芽孢杆菌中来产生生产异丁醇的重组微生物宿主。
浸麻类芽孢杆菌中异丁醇生物合成途径的表达
可以如以上对枯草芽孢杆菌中的表达所述的来构建质粒并用于转化浸麻类芽孢杆菌,通过原生质体转化,以产生生产异丁醇的重组微生物宿主。
真养产碱杆菌(雷氏菌)中异丁醇生物合成途径的表达
用于真养产碱杆菌中的基因表达和突变形成的方法是本领域已知的(参见,例如,Taghavi等,Appl.Environ.Microbiol.,60(10):3585-3591(1994))。可以在上述的任一种宽宿主范围载体中克隆用于异丁醇生物合成途径的基因,并电穿孔来产生生产异丁醇的重组体。已经详细描述了产碱杆菌中的多(羟基丁酸)途径,各种修饰真养产碱杆菌的遗传技术是已知的,并且那些工具可以应用于工程化异丁醇生物合成途径。
恶臭假单胞菌中异丁醇生物合成途径的表达
用于恶臭假单胞菌中的基因表达的方法是本领域已知的(参见,例如,Ben-Bassat等,U.S.专利No.6,586,229,在此将其引入作为参考)。可以将丁醇途径基因插入pPUC18中并将该连接的DNA电穿孔至电感受态恶臭假单胞菌DOT-T1C5aAR1细胞中来产生生产异丁醇的重组体。
酿酒酵母中异丁醇生物合成途径的表达
用于酿酒酵母中的基因表达的方法是本领于已知的(参见,例如,Methods in Enzymology(酶学方法),194卷,Guide to Yeast Genetics andMolecular and Cell Biology(酵母遗传学以及分子和细胞生物学指南)(A部分,2004,Christine Guthrie and Gerald R.Fink(编辑),ElsevierAcademic Press,San Diego,CA)。酵母中基因的表达通常需要启动子,接着为目标基因,以及转录终止子。在构建用于编码异丁醇生物合成途径的基因的表达盒中可以使用各种酵母启动子,包括但不限于组成型启动子FBA,GPD,ADH1和GPM,和诱导型启动子GAL1,GAL10和CUP1。合适的转录终止子包括但不限于FBAt,GPDt,GMPt,ERG10t,GAL1t,CYC1和ADH1。例如,可以将异丁醇生物合成途径的合适启动子,转录终止子和基因克隆至大肠杆菌-酵母穿梭载体中,如实施例17中所述的。
植物乳杆菌中异丁醇生物合成途径的表达
乳杆菌属属于乳杆菌目(Lactobacillales)家族并且在枯草芽孢杆菌和链球菌的转化中所用的许多质粒和载体可以用于乳杆菌。合适载体的非限制性实例包括pAMβ1及其衍生物(Renault等,Gene 183:175-182(1996);和O’Sullivan等,Gene 137:227-231(1993));pMBB1和pHW800,pMBB1的衍生物(Wyckoff等,Appl.Environ.Microbiol.62:1481-1486(1996));pMG1,一种可缀合质粒(Tanimoto等,J.Bacteriol.184:5800-5804(2002));pNZ9520(Kleerebezem等,Appl.Environ.Microbiol.63:4581-4584(1997));pAM401(Fujimoto等,Appl.Environ.Microbiol.67:1262-1267(2001));和pAT392(Arthur等,Antimicrob.Agents Chemother.38:1899-1903(1994))。已经报道了几个来自植物乳杆菌的质粒(例如,van Kranenburg R,Golic N,Bongers R,Leer RJ,de Vos WM,Siezen RJ,Kleerebezem M,Appl.Environ.Microbiol.2005年3月;71(3):1223-1230)。例如,实施例21中描述了植物乳杆菌中异丁醇生物合成途径的表达。
屎肠球菌,鹑鸡肠球菌和粪肠球菌中异丁醇生物合成途径的表达
肠球菌属属于乳杆菌目家族并且在乳杆菌,枯草芽孢杆菌和链球菌的转化中所用的许多质粒和载体可以用于肠球菌。合适载体的非限制性实例包括pAMβ1及其衍生物(Renault等,Gene 183:175-182(1996);和O’Sullivan等,Gene 137:227-231(1993));pMBB1和pHW800,pMBB1的衍生物(Wyckoff等,Appl.Environ.Microbiol.62:1481-1486(1996));pMG1,一种可缀合质粒(Tanimoto等,J.Bacteriol.184:5800-5804(2002));pNZ9520(Kleerebezem等,Appl.Environ.Microbiol.63:4581-4584(1997));pAM401(Fujimoto等,Appl.Environ.Microbiol.67:1262-1267(2001));和pAT392(Arthur等,Antimicrob.Agents Chemother.38:1899-1903(1994))。使用来自乳球菌的nisA基因的用于粪肠球菌的表达载体也可使用(Eichenbaum等,Appl.Environ.Microbiol.64:2763-2769(1998)。此外,可以使用用于屎肠球菌染色体中基因替代的载体(Nallaapareddy等,Appl.Environ.Microbiol.72:334-345(2006))。例如,实施例22中描述了粪肠球菌中的异丁醇生物合成途径的表达。
发酵培养基
本发明中的发酵培养基必须含有合适的碳底物。合适的物质包括,但不限于,单糖,如葡萄糖和果糖,寡糖,如乳糖或蔗糖,多糖,如淀粉或纤维素或其混合物,以及来自可再生原料的未纯化混合物,如干酪乳清渗透物,玉米浆,甜菜糖蜜和麦芽。此外,碳底物还可以是单碳底物,如二氧化碳,或甲醇,对此已经证明了代谢转化成关键的生化中间产物。除了单碳和二碳底物,还知道甲醇(methylotrophic)生物体利用各种其他含碳化合物,如甲胺,葡糖胺和各种氨基酸,用于代谢活性。例如,已知甲醇酵母利用来自甲胺的碳来形成海藻糖或甘油(Bellion等,Microb.Growth C1 Compd.,[Int.Symp],7th(1993),415-32,编辑:Murrell,J.Collin;Kelly,Don P.,出版商:Intercept,Andover,UK)。相似地,假丝酵母的各种物种将代谢丙氨酸或油酸(Sulter等,Arch.Microbiol.153:485-489(1990))。因此,考虑了本发明中所用的碳源可以包括各种含碳底物并且只受生物体选择的限制。
尽管考虑了所有上述的碳底物及其混合物在本发明中是合适的,但优选的碳底物是葡萄糖,果糖和蔗糖。
除了合适的碳源,发酵培养基必须含有合适的矿物质,盐,辅因子,缓冲剂和其他成分,这些是本领域技术人员已知的,适于培养物的生长和促进异丁醇生产需要的酶途径。
培养条件
通常,细胞生长于合适培养基中约25℃至约40℃范围的温度。本发明中的合适生长培养基是通常的商业制备培养基,如Luria Bertani(LB)培养液,Sabouraud Dextrose(SD)培养液或酵母培养基(YM)培养液。还可以使用其他限定的或合成的生长培养基,并且用于特定微生物生长的合适培养基将是微生物学或发酵科学领域中技术人员已知的。还可以将已知直接或间接调节代谢产物抑制的试剂,例如,环腺苷2’:3’-单磷酸,掺入发酵培养基中。
用于发酵的合适pH范围为pH5.0至pH9.0,其中优选pH6.0至pH8.0作为起始条件。
可以在好氧或厌氧条件下进行发酵,其中优选厌氧或微好氧条件。
可以使用本领域已知的各种方法来测定发酵培养基中产生的异丁醇含量,例如,高性能液相色谱(HPLC)或气相色谱(GC)。
工业分批发酵和连续发酵
本发明使用分批发酵方法。传统的分批发酵是封闭的系统,其中在发酵开始时设定培养基的组成并且在发酵过程中没有接受人工改变。因此,在发酵开始时,用所需的微生物接种培养基,并且允许发酵发生而没有给系统添加任何东西。通常,然而,“分批”发酵对于碳源的添加是分批的,并且通常努力控制一些因素如pH和氧浓度。在分批系统中,系统的代谢产物和生物量组成恒定地改变直至停止发酵的时间。在分批培养内,细胞从静态的停滞阶段调节至高生长对数期,最后到达静止期,其中生长速率减小或停止。如果未处理,静止期中的细胞最终将死亡。对数期的细胞通常负责终产物或中间产物的大量产生。
对标准分批系统的改进是给料-分批系统。给料-分批发酵方法也适用于本发明并包括典型的分批系统,除了随着发酵进展增量添加底物。当代谢产物抑制倾向于抑制细胞代谢并且希望在培养基中含有有限量的底物时,给料-分批系统是有用的。给料-分批系统中实际底物浓度的测量是困难的,并因此基于测量因素的改变来估算,这些测量因素如pH,溶解氧和废气如CO2的分压。分批和给料-分批发酵是本领域常见的和公知的,并且可以在以下找到实例:Thomas D.Brock,在Biotechnology:A Textbook of Industrial Microbiology(生物技术:工业微生物学的教科书),第二版(1989)Sinauer Associates,Inc.,Sunderland,MA.,或Deshpande,Mukund V.,Appl.Biochem.Biotechnol.,36:227,(1992),在此引入作为参考。
尽管以分批模式进行本发明,但考虑了该方法也将适于连续发酵方法。连续发酵是开放的系统,其中将限定的发酵培养基连续加入生物反应器中,并且同时取出等量的调节过的培养基用于处理。连续发酵通常将培养物维持于恒定的高密度,其中细胞主要处于对数期生长。
连续发酵允许调节影响细胞生长或终产物浓度的一种因素或任何数量的因素。例如,一种方法以固定的速率维持限制的营养素如碳源或氮水平,并允许所有其他参数来调节。在其他系统中,可以连续改变影响生长的多种因素,而通过培养基浊度测量的细胞浓度保持恒定。连续系统为维持稳态生长条件而努力,因此由于抽取培养基引起的细胞损耗必须相对于发酵中的细胞生长率而平衡。调节连续发酵过程的营养素和生长因子的方法以及用于最大化产品发酵速率的技术是工业微生物领域中公知的,并且Brock,上文中详细描述了各种方法。
考虑了可以使用分批,给料-分批或连续过程来实践本发明,并且任何已知的发酵模式将是合适的。此外,考虑了可以将细胞固定于作为完整细胞催化剂的物质上并接受用于异丁醇生产的发酵条件。
从发酵培养基分离异丁醇的方法
可以使用本领域已知的方法从发酵培养基分离生物产生的异丁醇。例如,可以通过离心,过滤,滗析等从发酵培养基去除固体。然后,可以从如上所述已经处理过除去固体的发酵培养基分离异丁醇,使用如蒸馏,液-液提取或基于膜的分离的方法。因为异丁醇与水形成低沸点的共沸混合物,蒸馏只可以将混合物分离成共沸组合物。可以结合另一种分离方法使用蒸馏来获得绕过共沸组成分离。可以结合蒸馏使用来分离和纯化异丁醇的方法包括,但不限于,滗析,液-液提取,吸附和基于膜的技术。此外,可以使用共沸蒸馏来分离异丁醇,使用夹带剂(参见,例如,Doherty和Malone,Conceptual Design of DistillationSystem,McGraw Hill,New York,2001)。
异丁醇-水混合物形成异质的共沸混合物,使得可以结合滗析使用蒸馏来分离和纯化异丁醇。在该方法中,将含有异丁醇的发酵培养液蒸馏至接近共沸组成。然后,冷凝共沸混合物,并通过滗析从发酵培养基分离异丁醇。可以将滗析过的水相随着回流返回至第一个蒸馏柱。可以通过在第二个蒸馏柱中蒸馏来进一步纯化富含异丁醇的滗析过的有机相。
还可以使用液-液提取结合蒸馏从发酵培养基中分离异丁醇。在该方法中,使用合适的溶剂使用液-液提取从发酵培养液中提取异丁醇。然后蒸馏含有异丁醇的有机相以从溶剂分离异丁醇。
还可以使用蒸馏结合吸附以从发酵培养液中分离异丁醇。在该方法中,将含有异丁醇的发酵培养液蒸馏至接近非沸组成,然后使用吸附剂如分子筛除去剩余的水(Aden等,Lignocellulosic Biomass toEthanol Process Design and Economics Utilizing Co-Current Dilute AcidPrehydrolysis and Enzymatic Hydrolysis for Corn Stoveer,ReportNREL/TP-510-32438,National Renwable Energy Laboratory,2002年6月)。
此外,可以使用蒸馏结合全蒸发过程从发酵培养基分离和纯化异丁醇。在该方法中,将含有异丁醇的发酵培养液蒸馏至接近共沸组成,然后借由通过亲水性膜的全蒸发过程来除去剩余的水(Guo等,J.Member.Sci.245,199-210(2004))。
实施例
在以下的实施例中进一步限定了本发明。应当理解这些实施例只是通过说明的方式给出,尽管表示了本发明的优选实施方案。从以上的讨论和这些实施例,本领域技术人员可以确定本发明的必要特征,而没有脱离其精神和范围,可以进行本发明的各种改变和改进以使其适于各种用途和条件。
总的方法
实施例中所用的标准重组DNA和分子克隆技术上本领域公知的并描述于Sambrook,J.,Fritsch,E.F.和Maniatis,T.Molecular Cloning:A Laboratory Manual(分子克隆:实验室手册);Cold Spring HarborLaboratory Press:Cold Spring Harbor,NY(1989)(Maniatis)和T.J.Silhavy,M.L.Bennan,和L.W.Enquist,Experiments with Gene Fusions(使用基因融合的实验),Cold Spring Harbor Laboratory Press:ColdSpring Harbor,N.Y.(1984)和Ausubel,F.M.等,Current Protocols inMolecular Biology(分子生物学中的通用实验方案),由GreenePublishing Assoc.和Wiley-Interscience(1987)出版。
适用于细菌培养物的维持和生长的材料和方法是本领域公知的。适用于以下实施例的技术可以在以下所列的找到,Manual of Methodsfor General Bacteriology(一般细菌学的方法手册)(Phillipp Gerhardt,R.G.E.Murray,Ralph N.Costilow,Eugene W.Nester,Willis A.Wood,Noel R.Krieg和G.Briggs Phillips编辑),American Society forMicrobiology,Washington,DC.(1994))或Thomas D.Brock,在Biotechnology:A Textbook of Industrial Microbiology(生物技术:工业微生物学的教科书),第二版,Sinauer Associates,Inc.,Sunderland,MA(1989)。用于细菌细胞生长和维持的所有试剂,限制酶和材料从Aldrich Chemicals(Milwaukee,WI),BD Diagnostic Systems(Sparks,MD),Life Technologies(Rockville,MD),或Sigma Chemical Company(St.Louis,MO)获得,除非另外指出。
从美国典型培养物中心(ATCC),Manassas,VA获得微生物菌株,除非另外指出。
表4中给出了以下实施例中使用的寡核苷酸引物。通过Sigma-Genosys(Woodlands,TX)合成所有寡核苷酸引物。
表4
寡核苷酸克隆,测序和测序引物
名称 | 序列 | 描述 | SEQ IDNO: |
N80 | CACCATGGACAAACAGTATCCGGTACGCC | budB正向 | 11 |
N81 | CGAAGGGCGATAGCTTTACCAATCC | budB反向 | 12 |
N100 | CACCATGGCTAACTACTTCAATACACTGA | ilvC正向 | 13 |
N101 | CCAGGAGAAGGCCTTGAGTGTTTTCTCC | ilvC反向 | 14 |
N102 | CACCATGCCTAAGTACCGTTCCGCCACCA | ilvD正向 | 15 |
N103 | CGCAGCACTGCTCTTAAATATTCGGC | ilvD反向 | 16 |
N104 | CACCATGAACAACTTTAATCTGCACACCC | yqhD正向 | 17 |
N105 | GCTTAGCGGGCGGCTTCGTATATACGGC | yqhD反向 | 18 |
N110 | GCATGCCTTAAGAAAGGAGGGGGGTCACATGGACAAACAGTATCC | budB正向 | 19 |
N111 | ATGCATTTAATTAATTACAGAATCTGACTCAGATGCAGC | budB反向 | 20 |
N112 | GTCGACGCTAGCAAAGGAGGGAATCACCATGGCTAACTACTTCAA | ilvC正向 | 21 |
N113 | TCTAGATTAACCCGCAACAGCAATACGTTTC | ilvC反向 | 22 |
N114 | TCTAGAAAAGGAGGAATAAAGTATGCCTAAGTACCGTTC | ilvD正向 | 23 |
N115 | GGATCCTTATTAACCCCCCAGTTTCGATTTA | ilvD反向 | 24 |
N116 | GGATCCAAAGGAGGCTAGACATATGTATACTGTGGGGGA | kivD正向 | 25 |
N117 | GAGCTCTTAGCTTTTATTTTGCTCCGCAAAC | kivD反向 | 26 |
N118 | GAGCTCAAAGGAGGAGCAAGTAATGAACAACTTTAATCT | yqhD正向 | 27 |
N119 | GAATTCACTAGTCCTAGGTTAGCGGGCGGCTTCGTATATACGG | yqhD反向 | 28 |
BenNF | CAACATTAGCGATTTTCTTTTCTCT | Npr正向 | 29 |
BenASR | CATGAAGCTTACTAGTGGGCTTAAGTTTTGAAAATAATGAAAACT | Npr反向 | 30 |
N110.2 | GAGCTCACTAGTCAATTGTAAGTAAGTAAAAGGAGGTGGGTCACATGGACAAACAGTATCC | budB正向 | 31 |
N111.2 | GGATCCGATCGACTTAAGCCTCAGCTTACAGAATCTGACTCAGATGCAGC | budB反向 | 32 |
N112.2 | GAGCTCCTTAAGAAGGAGGTAATCACCATGGCTAACTACTTCAA | ilvC正向 | 33 |
N113.2 | GGATCCGATCGAGCTAGCGCGGCCGCTTAACCCGCAACAGCAATACGTTTC | ilvC反向 | 34 |
N114.2 | GAGCTCGCTAGCAAGGAGGTATAAAGTATGCCTAAGTACCGTTC | ilvD正向 | 35 |
N115.2 | GGATCCGATCGATTAATTAACCTAAGGTTATTAACCCCCCAGTTTCGATTTA | ilvD反向 | 36 |
N116.2 | GAGCTCTTAATTAAAAGGAGGTTAGACATATGTATACTGTGGGGGA | kivD正向 | 37 |
N117.2 | GGATCCAGATCTCCTAGGACATGTTTAGCTTTTATTTTGCTCCGCAAAC | kivD反向 | 38 |
N130SeqF1 | TGTTCCAACCTGATCACCG | 测序引物 | 40 |
N130SeqF2 | GGAAAACAGCAAGGCGCT | 测序引物 | 41 |
N130SeqF3 | CAGCTGAACCAGTTTGCC | 测序引物 | 42 |
N130SeqF4 | AAAATACCAGCGCCTGTCC | 测序引物 | 43 |
N130SeqR1 | TGAATGGCCACCATGTTG | 测序引物 | 44 |
N130SeqR2 | GAGGATCTCCGCCGCCTG | 测序引物 | 45 |
N130SeqR3 | AGGCCGAGCAGGAAGATC | 测序引物 | 46 |
N130SeqR4 | TGATCAGGTTGGAACAGCC | 测序引物 | 47 |
N131SeqF1 | AAGAACTGATCCCACAGGC | 测序引物 | 48 |
N131SeqF2 | ATCCTGTGCGGTATGTTGC | 测序引物 | 49 |
N131SeqF3 | ATTGCGATGGTGAAAGCG | 测序引物 | 50 |
N131SeqR1 | ATGGTGTTGGCAATCAGCG | 测序引物 | 51 |
N131SeqR2 | GTGCTTCGGTGATGGTTT | 测序引物 | 52 |
N131SeqR3 | TTGAAACCGTGCGAGTAGC | 测序引物 | 53 |
N132SeqF1 | TATTCACTGCCATCTCGCG | 测序引物 | 54 |
N132SeqF2 | CCGTAAGCAGCTGTTCCT | 测序引物 | 55 |
N132SeqF3 | GCTGGAACAATACGACGTTA | 测序引物 | 56 |
N132SeqF4 | TGCTCTACCCAACCAGCTTC | 测序引物 | 57 |
N132SeqR1 | ATGGAAAGACCAGAGGTGCC | 测序引物 | 58 |
N132SeqR2 | TGCCTGTGTGGTACGAAT | 测序引物 | 59 |
N132SeqR3 | TATTACGCGGCAGTGCACT | 测序引物 | 60 |
N132SeqR4 | GGTGATTTTGTCGCAGTTAGAG | 测序引物 | 61 |
N133SeqF1 | TCGAAATTGTTGGGTCGC | 测序引物 | 62 |
N133SeqF2 | GGTCACGCAGTTCATTTCTAAG | 测序引物 | 63 |
N133SeqF3 | TGTGGCAAGCCGTAGAAA | 测序引物 | 64 |
N133SeqF4 | AGGATCGCGTGGTGAGTAA | 测序引物 | 65 |
N133SeqR1 | GTAGCCGTCGTTATTGATGA | 测序引物 | 66 |
N133SeqR2 | GCAGCGAACTAATCAGAGATTC | 测序引物 | 67 |
N133SeqR3 | TGGTCCGATGTATTGGAGG | 测序引物 | 68 |
N133SeqR4 | TCTGCCATATAGCTCGCGT | 测序引物 | 69 |
Scr1 | CCTTTCTTTGTGAATCGG | 测序引物 | 72 |
Scr2 | AGAAACAGGGTGTGATCC | 测序引物 | 73 |
Scr3 | AGTGATCATCACCTGTTGCC | 测序引物 | 74 |
Scr4 | AGCACGGCGAGAGTCGACGG | 测序引物 | 75 |
T-budB(BamHI) | AGATAGATGGATCCGGAGGTGGGTCACATGGACAAACAGT | budB正向 | 144 |
B-kivD(BamHI) | CTCTAGAGGATCCAGACTCCTAGGACATG | kivD反向 | 145 |
T-groE(XhoI) | AGATAGATCTCGAGAGCTATTGTAACATAATCGGTACGGGGGTG | PgroE正向 | 147 |
B-groEL(SpeI,BamH1) | ATTATGTCAGGATCCACTAGTTTCCTCCTTTAATTGGGAATTGTTATCCGC | PgroE反向 | 148 |
T-groEL | AGCTATTGTAACATAATCGGTACGGGGGTG | PgroE正向 | 149 |
T-ilvCB.s.(BamHI) | ACATTGATGGATCCCATAACAAGGGAGAGATTGAAATGGTAAAAG | ilvC正向 | 150 |
B-ilvCB.s.(SpeIBamHI) | TAGACAACGGATCCACTAGTTTAATTTTGCGCAACGGAGACCACCGC | ilvC反向 | 151 |
T-BD64(DraIII) | TTACCGTGGACTCACCGAGTGGGTAACTAGCCTCGCCGGAAAGAGCG | pBD64正向 | 152 |
B-BD64(DraIII) | TCACAGTTAAGACACCTGGTGCCGTTAATGCGCCATGACAGCCATGAT | pBD64反向 | 153 |
T-lacIq(DraIII) | ACAGATAGATCACCAGGTGCAAGCTAATTCCGGTGGAAACGAGGTCATC | lacIq正向 | 154 |
B-lacIq(DraIII) | ACAGTACGATACACGGGGTGTCACTGCCCGCTTTCCAGTCGGGAAACC | lacIq反向 | 155 |
T-groE(DraIII) | TCGGATTACGCACCCCGTGAGCTATTGTAACATAATCGGTACGGGGGTG | PgroE正向 | 156 |
B-B.silvC(DraIII) | CTGCTGATCTCACACCGTGTGTTAATTTTGCGCAACGGAGACCACCGC | ilvC反向 | 157 |
T-bdhB(DraIII) | TCGATAGCATACACACGGTGGTTAACAAAGGAGGGGTTAAAATGGTTGATTTCG | bdhB正向 | 159 |
B-bdhB(rmBT1DraIII) | ATCTACGCACTCGGTGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATCTTACACAGATTTTTTGAATATTTGTAGGAC | bdhB反向 | 160 |
LDH EcoRV F | GACGTCATGACCACCCGCCGATCCCTTTT | ldhL正向 | 161 |
LDH AatIIR | GATATCCAACACCAGCGACCGACGTATTAC | ldhL反向 | 162 |
Cm F | ATTTAAATCTCGAGTAGAGGATCCCAACAAACGAAAATTGGATAAAG | Cm正向 | 163 |
Cm R | ACGCGTTATTATAAAAGCCAGTCATTAGG | Cm反向 | 164 |
P11 F-StuI | CCTAGCGCTATAGTTGTTGACAGAATGGACATACTATGATATATTGTTGCTATAGCGA | P11启动子正向 | 165 |
P11 R-SpeI | CTAGTCGCTATAGCAACAATATATCATAGTATGTCCATTCTGTCAACAACTATAGCGCTAGG | P11启动子反向 | 166 |
PldhL F-HindIII | AAGCTTGTCGACAAACCAACATTATGACGTGTCTGGGC | IdhL正向 | 167 |
PldhL R-BamHI | GGATCCTCATCCTCTCGTAGTGAAAATT | IdhL反向 | 168 |
F-bdhB-AvrII | TTCCTAGGAAGGAGGTGGTTAAAATGGTTGATTTCG | bdhB正向 | 169 |
R-bdhB-BamHI | TTGGATCCTTACACAGATTTTTTGAATAT | bdhB反向 | 170 |
F-ilvC(B.s.)-AfIII | AACTTAAGAAGGAGGTGATTGAAATGGTAAAAGTATATT | ilvC正向 | 171 |
R-ilvC(B.s.)-NotI | AAGCGGCCGCTTAATTTTGCGCAACGGAGACC | ivIC反向 | 172 |
F-PnisA(HindIII) | TTAAGCTTGACATACTTGAATGACCTAGTC | nisA启动子正向 | 173 |
R-PnisA(SpeIBamHI) | TTGGATCCAAACTAGTATAATTTATTTTGTAGTTCCTTC | nisA启动子反向 | 174 |
测定培养基中异丁醇浓度的方法
可以通过本领域已知的各种方法来测定培养基中的异丁醇浓度。例如,特异性高性能液相色谱(HPLC)方法使用了Shodex SH-1011柱和Shodex SH-G保护柱,两者都购自Waters Corporation(Milford,MA),具有折射率(RI)检测。使用0.01M H2SO4作为移动相获得色谱分离,使用0.5mL/分钟的流速和50℃的柱温。异丁醇在所用的条件下具有46.6分钟的停留时间。或者,可以用气相色谱(GC)方法。例如,特定的GC方法使用HP-INNOWax柱(30m×0.53mm,同前,1μm膜厚,Agilent Technologies,Wilmington,DE),使用火焰电离检测仪(FID)。载气是4.5mL/分钟流速的氦,在150℃测量,使用恒定的排除压力;注射器分裂是200℃1∶25;烘箱温度是45℃1分钟,以10℃/分钟45升至220℃,和220℃5分钟;并在240℃使用FID检测,使用26mL/分钟氦补充气体。异丁醇的停留时间是4.5分钟。
缩写的意思如下:“s”表示秒,“min”表示分钟,“h”表示小时,“psi”表示每平方英寸的磅数,“nm”表示纳米,“d”表示天,“μL”表示微升,“mL”表示毫升,“L”表示升,“mm”表示微米,“nm”表示纳米,“mM”表示毫摩尔,“μM”表示微摩尔,“M”表示摩尔,“mmol”表示毫摩尔,“μmol”表示微摩尔,“g”表示克,“μg”表示微克和“ng”表示纳克,“PCR”表示聚合酶链式反应,“OD”表示光密度,“OD600”表示在600nm波长测量的光密度,“kDa”表示千道尔顿,“g“表示重力常数,“bp”表示碱基对,“kbp”表示千碱基对,“%w/v”表示重量/体积百分比,“%v/v”表示体积/体积百分比,“IPTG”表示异丙基-B-D-硫代半乳糖吡喃糖苷,“RBS”表示核糖体结合位点,“HPLC”表示高性能液相色谱,和“GC”表示气相色谱。术语“摩尔选择性”是每摩尔消耗的糖底物产生的产物摩尔数,并记录为百分比。
实施例1
乙酰乳酸合酶的克隆和表达
该实施例的目的是从肺炎克雷伯氏菌克隆budB基因并在大肠杆菌BL21-AI中表达。使用PCR从肺炎克雷伯氏菌菌株ATCC 25955基因组DNA扩增budB基因,获得1.8kbp产物。
使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN;目录编号D-5000A)制备基因组DNA。通过PCR从肺炎克雷伯氏菌基因组DNA扩增budB基因,使用引物N80和N81(参见表2),各自如SEQ ID NO:11和12给出。在制造商的试剂盒中提供了其他PCR扩增试剂,例如,Finnzymes PhusionTM High-Fidelity PCR MasterMix(New England Biolabs Inc.,Beverly,MA;目录no.F-531)并根据制造商的实验方案来使用。在DNA Thermocycler GeneAmp 9700(PEApplied Biosystems,Foster city,CA)中进行扩增。
为了表达研究,使用Gateway克隆技术(Invitrogen Corp.,Carlsbad,CA)。入门载体pENTRSDD-TOPO允许定向的克隆并提供了目标基因的Shine-Dalgarno序列。目标载体pDEST14使用了T7启动子,用于没有标记物的基因的表达。正向引物就在转录起始密码子邻近引入四个碱基(CACC),以允许定向克隆至pENTRSDD-TOPO(Invitrogen)中来产生质粒pENTRSDD-TOPObudB。根据制造商的推荐将pENTR构建体转化至大肠杆菌Top10(Invitrogen)细胞中并涂布。将转化体生长过夜并根据制造商的推荐使用QIAprep Spin Miniprep试剂盒(Qiagen,Valencia,CA;目录no.27106)制备质粒DNA。将克隆测序来证实以正确方向插入了基因并证实序列。SEQ ID NO:1和SEQ IDNO:2各自给出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基酸序列。
为了形成表达克隆,通过重组将budB基因转移至pDEST14载体中来产生pDEST14budB。将pDEST14budB载体转化至大肠杆菌BL21-AI细胞中(Invitrogen)。将转化体接种至补充50μg/mL氨苄青霉素的Luria Bertani(LB)培养基中并生长过夜。并将等份的过夜培养物用来接种50mL补充50μg/mL氨苄青霉素的LB。将培养物在37℃振荡培养直至OD600达到0.6-0.8。将培养物分成两个25-mL培养物,并将阿拉伯糖加入一个烧瓶中至0.2%w/v的终浓度。阴性对照烧瓶没有用阿拉伯糖来诱导。将烧瓶在37℃振荡培养4h。通过离心收集细胞并将细胞沉淀物重悬浮于50mM MOPS中,pH 7.0缓冲液。通过超声波或通过French Pressure Cell破坏细胞。将全细胞裂解产物离心,产生无细胞提取物的上清液或不溶性部分。将等份的每个部分(全细胞裂解产物,无细胞提取物和不溶性部分)重悬浮于SDS(MES)装载缓冲液中(Invitrogen),加热至85℃10分钟,并接受SDS-PAGE分析(NuPAGE 4-12%Bis-Tris凝胶,目录no.NP0322Box,Invitrogen)。在诱导的培养物中存在预期约60kDa分子量的蛋白质,如从核酸序列推导的,但在未诱导的对照中不存在。
使用Bauerle等(Biochim.Biophys.Acta 92(1):142-149(1964))描述的方法来测量无细胞提取物中的乙酰乳酸合酶活性。
实施例2(预测的)
乙酰醇酸还原异构酶的克隆和表达
该预测实施例的目的是描述怎样从大肠杆菌K12克隆ilvC基因并在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增ilvC基因。
以实施例1中所述的budB基因相同的方式克隆和表达ilvC基因。使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN;目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增ilvC基因,使用引物N100和N101(参见表2),各自如SEQ ID NO:13和14给出,产生1.5kbp产物。正向引物就在转录起始密码子邻近引入四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen)中来产生质粒pENTRSDD-TOPOilvC。将克隆测序来证实以正确方向插入了基因并证实序列。SEQ ID NO:3和SEQ ID NO:4各自给出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基酸序列。
为了形成表达克隆,通过重组将ilvC基因转移至pDEST14(Invitrogen)载体中来产生pDEST 14ilvC。将pDEST 14ilvC载体转化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表达。在诱导的培养物中存在预期约54kDa分子量的蛋白质,如从核酸序列推导的,但在未诱导的对照中不存在。
使用Arfin和Umbarger(J.Biol.Chem.244(5):1118-1127(1969))所述的方法来测量无细胞提取物中的乙酰醇酸还原异构酶。
实施例3(预测的)
乙酰醇酸脱水酶的克隆和表达
该预测实施例的目的是描述怎样从大肠杆菌K12克隆ilvD基因并在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增ilvD基因。
以实施例1中所述的budB基因相同的方式克隆和表达ilvD基因。使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN;目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增ilvD基因,使用引物N102和N 103(参见表2),各自如SEQ ID NO:15和16给出,产生1.9kbp产物。正向引物就在转录起始密码子邻近引入四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen)中来产生质粒pENTRSDD-TOPOilvD。将克隆接受测序来证实以正确方向插入了基因并证实序列。SEQ ID NO:5和SEQ ID NO:6各自给出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基酸序列。
为了形成表达克隆,通过重组将ilvD基因转移至pDEST14(Invitrogen)载体中来产生pDEST14ilvD。将pDEST14ilvD载体转化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表达。在诱导的培养物中存在预期约66kDa分子量的蛋白质,如从核酸序列推导的,但在未诱导的对照中不存在。
使用Flint等(J.Biol.Chem.268(20):14732-14742(1993))描述的方法来测量无细胞提取物中的乙酰醇酸脱水酶活性。
实施例4(预测的)
支链酮酸脱羧酶的克隆和表达
该预测实施例的目的是描述怎样从乳酸乳球菌克隆kivD基因并在大肠杆菌BL21-AI中表达。
从GenScript(Piscataway,NJ)获得编码乳酸乳球菌的支链酮酸脱羧酶(kivD)的DNA序列。获得的序列对于在大肠杆菌和枯草芽孢杆菌中的表达是密码子最佳化的并克隆至pUC57中,来形成pUC57-kivD。SEQ ID NO:7和SEQ ID NO:8各自给出了用于该基因的开放阅读框(ORF)的密码子最佳化核苷酸序列和预测的酶氨基酸序列。
为了形成表达克隆,使用NdeI和BamHI限制酶位点将来自pUC57-kivD的1.7kbp kivD片段克隆至载体pET-3a中(Novagen,Madison,WI)。这形成了表达克隆pET-3a-kivD。将pET-3a-kivD载体转化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表达。在诱导的培养物中存在预期约61kDa分子量的蛋白质,如从核酸序列推导的,但在未诱导的对照中不存在。
使用Smit等(Appl.Microbiol.Biotechnol.64:396-402(2003))描述的方法来测量无细胞提取物中的支链酮酸脱羧酶活性。
实施例5(预测的)
支链醇脱氢酶的克隆和表达
该预测实施例的目的是描述怎样从大肠杆菌K12克隆yqhD基因并在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增yqhD基因。
以实施例1中所述的budB基因相同的方式克隆和表达yqhD基因。使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN;目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增yqhD基因,使用引物N104和N105(参见表2),各自如SEQ ID NO:17和18给出,产生1.2kbp产物。正向引物就在转录起始密码子邻近引入四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen)中来产生质粒pENTRSDD-TOPOyqhD。将克隆接受测序来证实以正确方向插入了基因并证实序列。SEQ ID NO:9和SEQ ID NO:10各自给出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基酸序列。
为了形成表达克隆,通过重组将yqhD基因转移至pDEST 14(Invitrogen)载体中来产生pDEST14yqhD。将pDEST14yqhD载体转化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表达。在诱导的培养物中存在预期约42kDa分子量的蛋白质,如从核酸序列推导的,但在未诱导的对照中不存在。
使用Sulzenbacher等(J.Biol.Chem.342(2):489-502(2004))描述的方法来测量无细胞提取物中的支链醇脱氢酶活性。
实施例6(预测的)
用于异丁醇生物合成途径中的基因的转化载体的构建
该预测实施例的目的是描述怎样构建转化载体,该载体包括编码异丁醇生物合成途径中五个步骤的基因。将所有基因置于单个启动子控制下的单个操纵子中。通过PCR扩增单个基因,使用引入限制位点的引物,用于之后的克隆,并且正向引物含有最佳化的大肠杆菌核糖体结合位点(AAAGGAGG)。将PCR产物TOPO克隆至pCR4Blunt-TOPO载体中并转化至大肠杆菌Top10细胞中(Invitrogen)。从TOPO克隆制备质粒DNA并证实基因的序列。根据制造商的推荐使用限制酶和T4DNA连接酶(New England Biolabs,Beverly,MA)。为了克隆实验,使用QIAquick凝胶提取试剂盒(Qiagen)将限制片段凝胶纯化。证实序列后,将基因亚克隆至作为克隆平台的修饰pUC19载体中。通过HindIII/SapI消化来修饰pUC19载体,形成pUC19dHS。该消化除去了邻接MCS(多克隆位点)的lac启动子,防止操纵子在载体中的转录。
通过PCR从肺炎克雷伯氏菌ATCC 25955基因组DNA扩增budB基因,使用引物对N110和N111(参见表2),各自如SEQ ID NO:19和20给出,形成1.8kbp产物。正向引物引入SphI和AfIII限制位点和核糖体结合位点(RBS)。反向引物引入PacI和NsiI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-budB。从TOPO克隆制备质粒DNA并证实基因的序列。
通过PCR从大肠杆菌K12基因组扩增ilvC基因,使用引物对N112和N113(参见表2),各自如SEQ ID NO:21和22给出,形成1.5kbp产物。正向引物引入SalI和NheI限制位点和RBS。反向引物引入XbaI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-ilvC。从TOPO克隆制备质粒DNA并证实基因的序列。
通过PCR从大肠杆菌K12基因组扩增ilvD基因,使用引物对N114和N115(参见表2),各自如SEQ ID NO:23和24给出,形成1.9kbp产物。正向引物引入XbaI限制位点和RBS。反向引物引入BamHI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-ilvD。从TOPO克隆制备质粒DNA并证实基因的序列。
通过PCR从pUC57-kivD(实施例4中所述的)扩增kivD基因,使用引物对N116和N117(参见表2),各自如SEQ ID NO:25和26给出,形成1.7bp产物。正向引物引入BamHI限制位点和RBS。反向引物引入SacI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-kivD。从TOPO克隆制备质粒DNA并证实基因的序列。
通过PCR从大肠杆菌K12基因组DNA扩增yqhD基因,使用引物对N118和N119(参见表2),各自如SEQ ID NO:27和28给出,形成1.2kbp产物。正向引物引入SacI限制位点。反向引物引入SpeI和EcoRI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-yqhD。从TOPO克隆制备质粒DNA并证实基因的序列。
为了构建异丁醇途径操纵子,用ScaI和EcoRI从pCR4Blunt-TOPO-yqhD切除yqhD基因,释放1.2kbp片段。将该片段连接之前已经用SacI和EcoRI消化过的pUC19dHS。通过限制消化来证实所得到的克隆,pUC19dHS-yqhD。接着,用SalI和XbaI从pCR4Blunt-TOPO-ilvC切除ilvC基因,释放1.5kbp片段。将该片段连接之前已经用SalI和XbaI消化过的pUC19dHS-yqhD。通过限制消化来证实所得到的克隆,pUC19dHS-ilvC-yqhD。然后用SphI和NsiI从pCR4Blunt-TOPO-budB切除budB基因,释放1.8kbp片段。用SphI和PstI消化pUC 19dHS-ilvC-yqhD并连接SphI/NsiI budB片段(NsiI和PstI产生相容性末端),形成pUC19dHS-budB-ilvC-yqhD。用XbaI和BamHI从pCR4Blunt-TOPO-ilvD切除1.9kbp含有ilvD基因的片段,并连接用这些相同的酶消化过的pUC19dHS-budB-ilvC-yqhD,形成pUC19dHS-budB-ilvC-ilvD-yqhD。最后,用BamHI和SacI从pCR4Blunt-TOPO-kivD切除kivD,释放1.7kbp片段。将该片段连接之前已经用BamHI和SacI消化过的pUC 19dHS-budB-ilvC-ilvD-yqhD,形成pUC19dHS-budB-ilvC-ilvD-kivD-yqhD。
用AfIII和SpeI消化pUC19dHS-budB-ilvC-ilvD-kivD-yqhD来释放克隆至pBenAS(大肠杆菌-枯草杆菌穿梭载体)中的8.2kbp操纵子片段。通过修饰pBE93载体来形成质粒,Nagarajan描述了这(WO93/24631,实施例4)。为了制备pBenAS,用pBE93的NcoI/HindIII消化除去解淀粉芽孢杆菌(Bacillus amyloliquefaciens)中性蛋白酶启动子(NPR),信号序列和phoA基因。从pBE93PCR扩增NPR启动子,使用引物BenNF和BenASR,各自如SEQ ID NO:29和30给出。引物BenASR在启动子下游引入AfIII,SpeI和HindIII位点。用NcoI和HindIII消化PCR产物,并将片段克隆至载体中的相应位点中,形成pBenAS。将操纵子片段亚克隆至pBenAS中的AfIII和SpeI位点中,形成pBen-budB-ilvC-ilvD-kivD-yqhD。
实施例7(预测的)
大肠杆菌中异丁醇生物合成途径的表达
该预测实施例的目的是描述怎样在大肠杆菌中表达异丁醇生物合成途径。
将按照实施例6中所述构造的质粒pBen-budB-ilvC-ilvD-kivD-yqhD转化至大肠杆菌NM522中(ATCCNo.47000),来获得大肠杆菌株NM522/pBen-budB-ilvC-ilvD-kivD-yqhD,并通过SDS-PAGE,酶测试和蛋白质印迹分析来监控操纵子中的基因表达。对于蛋白质印迹,通过Sigma-Genosys(The Woodlands,TX)引发对合成肽的抗体。
将大肠杆菌株NM522/pBen-budB-ilvC-ilvD-kivD-yqhD接种至含有50mL培养基的250mL摇瓶中并在250rpm和35℃振荡。培养基组成如下:葡萄糖(5g/L),MOPS(0.05M),硫酸铵(0.01M),磷酸钾,单价碱(0.005M),S10金属混合物(1%(v/v)),酵母提取物(0.1%(w/v)),酪蛋白氨基酸(0.1%(w/v)),硫胺素(0.1mg/L),脯氨酸(0.05mg/L),和生物素(0.002mg/L),并用KOH滴定至pH7.0。S10金属混合物含有:MgCl2(200mM),CaCl2(70mM),MnCl2(5mM),FeCl3(0.1mM),ZnCl2(0.1mM),盐酸硫胺素(0.2mM),CuSO4(172μM),CoCl2(253μM)和Na2MoCO4(242μM)。18h小时后,通过HPLC或GC分析来检测异丁醇,使用本领域公知的方法,例如,以上一般方法部分中所述的。
实施例8(预测的)
枯草芽孢杆菌中异丁醇生物合成途径的表达
该预测实施例的目的是描述怎样在枯草芽孢杆菌中表达异丁醇生物合成途径。使用实施例7中所述的相同方法。
使用按照实施例6中所述构造的质粒pBen-budB-ilvC-ilvD-kivD-yqhD。将该质粒转化至枯草芽孢杆菌BE1010中(J.Bacteriol.173:2278-2282(1991))来获得枯草芽孢杆菌株BE1010/pBen-budB-ilvC-ilvD-kivD-yqhD,并按照实施例7中所述的来监控每个操纵子中的基因表达。
将枯草芽孢杆菌株BE 1010/pBen-budB-ilvC-ilvD-kivD-yqhD接种至含有50mL培养基的250mL摇瓶中并在250rpm和35℃振荡18h。培养基组成如下:葡萄糖(5g/L),MOPS(0.05M),谷氨酸(0.02M),硫酸铵(0.01M),磷酸钾,单价碱缓冲剂(0.005M),S10金属混合物(和实施例11中所述的一样,1%(v/v)),酵母提取物(0.1%(w/v)),酪蛋白氨基酸(0.1%(w/v)),色氨酸(50mg/L),甲硫氨酸(50mg/L)和赖氨酸(50mg/L),并用KOH滴定至pH7.0。18h小时后,通过HPLC或GC分析来检测异丁醇,使用本领域公知的方法,例如,以上一般方法部分中所述的。
实施例9
乙酰乳酸合酶的克隆和表达
为了形成另一个乙酰乳酸合酶表达克隆,将budB基因克隆至载体pTrc99A中。首先从pENTRSDD-TOPObudB(实施例1中所述的)扩增budB基因,使用在5’端引入SacI,SpeI和MfeI位点和3’端引入BbvCI,AflII和BamHI位点的引物(N110.2和N111.2,各自如SEQ IDNO:31和32给出)。将所得到的1.75kbp PCR产物克隆至pCR4-BluntTOPO(Invitrogen)中,并证实了DNA序列(使用N130Seq测序引物F1-F4和R1-R4,各自如SEQ ID NO:40-47给出)。然后使用SacI和BamHI从该载体切除budB基因并克隆至pTrc99A中(Amann等,Gene69(2):301-315(1988)),产生pTrc99A::budB。将pTrc99A::budB载体转化至大肠杆菌TOP 10细胞中并将转化体接种至补充50μg/mL氨苄青霉素的LB培养基中并生长过夜。将等份的过夜培养物用来接种50mL补充50μg/mL氨苄青霉素的LB。将培养物在37℃振荡培养直至OD600达到0.6-0.8。然后通过加入0.4mM IPTG来诱导从Trc启动子的budB表达。还制备了没有用IPTG诱导的阴性对照摇瓶。将摇瓶在37℃振荡培养4h。按照实施例1中所述的制备无细胞培养物。
按照实施例1中所述的测量无细胞提取物中的乙酰乳酸合酶活性。用IPTG诱导后三个小时,检测到8单位/mg的乙酰乳酸合酶活性。只携带pTrc99A质粒的对照菌株呈现出0.03单位/mg的乙酰乳酸合酶活性。
实施例10
乙酰醇酸还原异构酶的克隆和表达
该实施例的目的是从大肠杆菌K12克隆ilvC基因并在大肠杆菌TOP10中表达。使用PCR从大肠杆菌菌株FM5(ATCC 53911)扩增ilvC基因。
以以上实施例2中对ilvC的克隆和表达所述的相同方式克隆和表达ilvC基因。使用PCR从大肠杆菌FM5基因组扩增ilvC,使用引物N112.2和N113.2(各自为SEQ ID NO:33和34)。引物在5’端形成SacI和AfllII位点以及最佳的RBS,在ilvC的3’端形成NotI,NheI和BamHI位点。根据制造商的实验方案(Invitrogen)将1.5kbp PCR产物克隆至pCR4Blunt TOPO中,产生pCR4Blunt TOPO::ilvC。使用测序引物(N131SeqF1-F3,和N131SeqR1-R3,各自如SEQ ID NO:48-53给出)证实了PCR产物的序列。为了形成表达克隆,使用SacI和BamHI从pCR4Blunt TOPO::ilvC切除ilvC基因并克隆至pTrc99A中。将pTrc99A::ilvC载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的制备无细胞提取物。
按照实施例2中所述的测量无细胞提取物中的乙酰醇酸还原异构酶活性。用IPTG诱导三小时后,检测到0.026单位/mg的乙酰醇酸还原异构酶活性。只携带pTrc99A质粒的对照菌株呈现出低于0.001单位/mg的乙酰醇酸还原异构酶活性。
实施例11
乙酰醇酸脱水酶的克隆和表达
该实施例的目的是从大肠杆菌K12克隆ilvD基因并在大肠杆菌TOP10中表达。使用PCR从大肠杆菌菌株FM5(ATCC 53911)扩增ilvD基因。
以实施例10中所述的ilvC基因相同的方式克隆和表达ilvD基因。使用PCR从大肠杆菌FM5基因组扩增ilvD,使用引物N114.2和N115.2(各自为SEQ ID NO:35和36)。引物在5’端形成SacI和NheI位点以及最佳的RBS,在ilvD的3’端形成Bsu36I,PacI和BamHI位点。根据制造商的实验方案(Invitrogen)将1.9kbp PCR产物克隆至pCR4Blunt TOPO中,产生pCR4Blunt TOPO::ilvD。证实了PCR产物的序列(测序引物N 132SeqF1-F4,和N132SeqR1-R4,各自如SEQ IDNO:54-61给出)。为了形成表达克隆,使用SacI和BamHI从质粒pCR4Blunt TOPO::ilvD切除ilvD基因并克隆至pTrc99A中。将pTrc99A::ilvD载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的制备无细胞提取物。
按照实施例3中所述的测量无细胞提取物中的乙酰醇酸脱水酶活性。用IPTG诱导三小时后,测量到46单位/mg的乙酰醇酸脱水酶活性。只携带pTrc99A质粒的对照菌株没有呈现出乙酰醇酸脱水酶活性。
实施例12
支链酮酸脱羧酶的克隆和表达
该实施例的目的是从乳酸乳球菌克隆kivD基因并在大肠杆菌TOP10中表达。
以以上实施例10中对ilvC所述的相同方式克隆和表达kivD基因。使用PCR从质粒pUC57-kivD(参见以上的实施例4)扩增kivD,使用引物N 116.2和N117.2(各自为SEQ ID NO:37和38)。引物在5’端形成SacI和PacI位点以及最佳的RBS,在kivD的3’端形成AvrII,BglII和BamHI位点。根据制造商的实验方案(Invitrogen)将1.7kbp PCR产物克隆至pCR4Blunt TOPO中,产生pCR4Blunt TOPO::kivD。使用引物N133SeqF1-F4,和N133SeqR1-R4(各自如SEQ ID NO:62-69给出)证实了PCR产物的序列。为了形成表达克隆,使用SacI和BamHI从质粒pCR4Blunt TOPO::kivD切除ilvD基因并克隆至pTrc99A中。将pTrc99A::kivD载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的制备无细胞提取物。
按照实施例4中所述的测量无细胞提取物中的支链酮酸脱羧酶活性,除了使用Purpald试剂(Al drich,目录No.162892)来检测和定量醛反应产物。用IPTG诱导三小时后,检测到高于3.7单位/mg的支链酮酸脱羧酶活性。只携带pTrc99A质粒的对照菌株没有呈现出支链酮酸脱羧酶活性。
实施例13
支链醇脱氢酶的表达
大肠杆菌含有鉴定为1,3-丙二醇脱氢酶的天然基因(yqhD)(U.S.专利No.6,514,733)。YqhD蛋白与来自梭菌的AdhB(由adhB编码)具有40%同一性,AdhB是推定的NADH-依赖性丁醇脱氢酶。使用λRed技术(Datsenko和Wanner,Proc.Natl.Acad.Sci.U.S.A.97:6640(2000))将yqhD基因置于大肠杆菌菌株MG1655 1.6yqhD::Cm(WO2004/033646)中的葡萄糖异构酶启动子1.6GI(SEQ ID NO.70)变体的组成型表达之下。MG1655 1.6yqhD::Cm含有FRT-CmR-FRT盒,使得可以除去抗生素标记。相似地,由1.5GI启动子(W2003/089621)(SEQ ID NO.71)替代天然启动子,形成菌株MG16551.5GI-yqhD::Cm,因此,用1.5GI启动子替代了MG1655 1.6yqhD::Cm的1.6GI启动子。
将菌株MG 1655 1.5GI-yqhD::Cm在LB培养基中生长至中-对数期并按照实施例1中所述的制备无细胞提取物。测定细胞提取物时,按照在pH7.5和35℃在340nm的吸收值降低,发现该菌株具有NAPDH-依赖性异丁醛还原酶活性。
为了产生第二个含有1.5GI yqhD::Cm的表达菌株,从MG16551.5GI yqhD::Cm制备P1裂解物,并通过转导将盒转移至BL21(DE3)(Invitrogen),形成BL21(DE3)1.5GI yqhD::Cm。
实施例14
用于异丁醇生物合成途径中头四个基因的转化载体的构建
该实施例的目的是构建包括异丁醇生物合成途径中头四个基因(即,budB,ilvC,ilvD和kivD)的转化载体。
为了构建转化载体,首先,通过用AflII和BamHI消化从pTrc99A::ilvC(实施例10中所述的)获得ilvC基因并克隆至pTrc99A::budB中(实施例9中所述的),将其用AflII和BamHI消化来产生质粒pTrc99A::budB-ilvC。接着,各自通过用NheI和PacI(ilvD)以及PacI和BamHI(kivD)从pTrc99A::ilvD(实施例11中所述的)和pTrc99A::kivD(实施例12中所述的)获得ilvD和kivD基因。通过三路(three-way)连接将这些基因引入首先用NheI和BamHI消化的pTrc99A::budB-ilvC中。通过PCR筛选和限制消化证实了最后的质粒pTrc99A::budB-ilvC-ilvD-kivD中的所有四个基因。
实施例15
生长于葡萄糖之上的大肠杆菌中异丁醇生物合成途径的表达
为了形成大肠杆菌异丁醇生产菌株,将pTrc99A::budB-ilvC-ilvD-kivD(实施例14中所述的)转化至大肠杆菌MG16551.5GI yqhD::Cm和大肠杆菌BL21(DE3)1.5GI yqhD::Cm(实施例13中所述的)中。最初将转化体生长在含有50μg/mL卡那霉素和100μg/mL羧苄青霉素的LB培养基中。将来自这些培养物的细胞用来接种摇瓶(大约175mL总体积),摇瓶含有50或170mL TM3a/葡萄糖培养基,各自表示高和低氧条件。TM3a/葡萄糖培养基含有(每升):葡萄糖(10g),KH2PO4(13.6g),柠檬酸一水合物(2.0g),(NH4)2SO4(3.0g),MgSO4·7H2O(2.0g),CaCl2·2H2O(0.2g),柠檬酸铁铵(0.33g),·HCl(1.0mg),酵母提取物(0.50g),和10mL微量元素溶液。用NH4OH将pH调节至6.8。微量元素溶液含有:柠檬酸·H2O(4.0g/L),MnSO4·H2O(3.0g/L),NaCl(1.0g),FeSO4·7H2O(0.10g/L),CoCl2.6H2O(0.10g/L),ZnSO4·7H2O(0.10g/L),CuSO4·5H2O(0.010g/L),H3BO3(0.010g/L),和Na2MoSO4·2H2O(0.010g/L)。
在≤0.01单位的起始OD600接种摇瓶并在34℃300rpm振荡培养。用0.2μm过滤器帽子密封含有50mL培养基的摇瓶;用密封的帽子封闭含有150mL培养基的摇瓶。当细胞达到OD600≥0.4单位时,加入IPTG至0.04mM的终浓度。大约培养18h后,通过HPLC(Shodex SugarSH1011柱(Showa Denko America,Inc.NY),使用折射率(RI)检测)和GC(Varian CP-WAX 58(FFAP)CB,0.25mm×0.2μm×25m(Varian,Inc.,Palo Alto,CA,使用火焰电离检测(FID))分析等份培养基的异丁醇含量,如一般方法部分中所述的。在只携带pTrc99A载体的对照菌株中没有检测到异丁醇(结果未显示)。表5中显示了通过携带pTrc99A::budB-ilvC-ilvD-kivD的菌株产生的异丁醇摩尔选择性和滴定度。在低氧条件下生长的培养物中获得显著较高的异丁醇滴定度。
表5
生长于葡萄糖之上的大肠杆菌菌株产生的异丁醇
菌株 | O2条件 | 异丁醇mM* | 摩尔选择性(%) |
MG1655 1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD | 高 | 0.4 | 4.2 |
MG1655 1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD | 低 | 9.9 | 39 |
BL21(DE3)1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD | 高 | 0.3 | 3.9 |
BL21(DE3)1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD | 低 | 1.2 | 12 |
*通过HPLC测定的
实施例16
生长于蔗糖之上的大肠杆菌中异丁醇生物合成途径的表达
因为实施例15中所述的菌株不能够生长于蔗糖之上,构建另外的质粒以允许利用蔗糖用于异丁醇生产。从源自ATCC菌株13281的利用蔗糖的大肠杆菌菌株的基因组DNA分离蔗糖利用基因簇cscBKA,如SED ID NO:39所给出的。蔗糖利用基因(cscA,cscK和cscB)编码蔗糖水解酶(CscA),如SEQ ID NO:139所给出的,D-果糖激酶(CscK),如SEQ ID NO:140所给出的,和蔗糖透性酶(CscB),如SEQ ID NO:141所给出的。没有包括蔗糖特异性阻遏基因cscR,使得三个基因cscBKA在大肠杆菌中从它们的天然启动子组成型表达。
用BamHI和EcoRI消化来自蔗糖利用大肠杆菌菌株的基因组DNA。从琼脂糖凝胶分离具有约4kbp平均大小的片段并连接用BamHI和EcoRI消化的质粒pLitmus28(New England Biolabs),并转化至超感受态大肠杆菌TOP10F’细胞中(Invitrogen)。将转化体在含有1%蔗糖和氨苄青霉素(100μg/mL)的MacConkey琼脂平板上划线并筛选紫色菌落的外观。从紫色转化体分离质粒DNA,并用M13正向和反向引物(Invitrogen)以及Scr1-4(各自如SEQ ID NO:72-75中所给出的)测序。将含有cscB,cscK和cscA(cscBKA)基因的质粒命名为pScr1。
为了形成与异丁醇途径质粒(实施例14)相容的蔗糖利用质粒,将来自pScr1的操纵子亚克隆至pBHR1中(MoBiTec,Goettingen,Germany)。通过用XhoI消化pScr1(接着用Klenow酶培养来产生钝末端),然后用AgeI消化来分离cscBKA基因。将所得到的4.2kbp片段连接已经用NaeI和AgeI消化的pBHR1中,形成9.3kbp质粒pBHR1::cscBKA。
通过电穿孔将蔗糖质粒pBHR1::cscBKA转化至大肠杆菌BL21(DE3)1.5yqhD/pTrc99A::budB-ilvC-ilvD-kivD和大肠杆菌MG16551.5yqhD/pTrc99A::budB-ilvC-ilvD-kivD中(实施例15中所述的)。首先在含有100μg/mL氨苄青霉素和50μg/mL卡那霉素的LB培养基上选择转化体,然后在MacConkey蔗糖(1%)平板上筛选来证实蔗糖操纵子的功能表达。为了异丁醇的生产,将菌株生长于含有替代葡萄糖的1%蔗糖的TM3a最小限定培养基中(实施例15中所述的),分析培养基中所产生的异丁醇含量,如实施例15中所述的,除了在培养14h后取样。再者,在只携带pTrc99A载体的对照菌株中没有检测到异丁醇(结果未显示)。表6中显示了通过携带pTrc99A::budB-ilvC-ilvD-kivD的MG1655 1.5yqhD产生的异丁醇的摩尔选择性和滴定度。
表6
通过生长于蔗糖之上的大肠杆菌菌株MG1655 1.5yqhD/
pTrc99A::budB-ilvC-ilvD-kivD/pBHR1::cScBKA的异丁醇产生
O2条件 | IPTG,mM | 异丁醇,mM* | 摩尔选择性,% |
高 | 0.04 | 0.17 | 2 |
高 | 0.4 | 1.59 | 21 |
低 | 0.04 | 4.03 | 26 |
低 | 0.4 | 3.95 | 29 |
*通过HPLC测定的
实施例17
酿酒酵母中异丁醇途径基因的表达
为了在酿酒酵母中表达异丁醇途径基因,构建多个大肠杆菌-酵母穿梭载体。使用PCR方法(Yu等,Fungal Genet.Biol.41:973-981(2004))来融合具有酵母启动子和终止子的基因。特别地,将GPD启动子(SEQ ID NO:76)和CYC 1终止子(SEQ ID NO:77)融合来自枯草芽孢杆菌的alsS基因(SEQ ID NO:78),将FBA启动子(SEQID NO:79)和CYC1终止子融合来自酿酒酵母的ILV5基因(SEQ IDNO:80),将ADH1启动子(SEQ ID NO:81)和ADH1终止子(SEQID NO:82)融合来自酿酒酵母的ILV3基因(SEQ ID NO:83),将GPM启动子(SEQ ID NO:84)和ADH1终止子融合来自乳酸乳球菌的kivD基因(SEQ ID NO:7)。将表7中给出的引物设计来包括限制位点,用于将启动子/基因/终止子产物克隆至来自pRS400系列的大肠杆菌-酵母穿梭载体中(Christianson等,Gene 110:119-122(1992)),并用于交换构建体之间的启动子。用于ILV5和ILV3的5’端的引物(各自为N138和N155,各自如SEQ ID NO:95和107给出的)产生新的起始密码子来消除这些酶的线粒体靶向。
首先通过TOPO克隆反应(Invitrogen)将所有融合的PCR产物克隆至pCR4-Blunt中,并证实了序列(使用M13正向和反向引物(Invitrogen)以及表7中提供的测序引物)。通过TOPO反应将另外两个启动子(CUP1和GAL1)克隆至pCR4-Blunt中,并通过测序来证实;表7中显示了引物序列。表8中描述了构建的质粒。将质粒转化至酿酒酵母BY4743(ATCC 201390)或YJR148w(ATCC 4036939)中,以使用实施例1-4和实施例9-12中所述的酶测试来测定酶特异性活性。为了测定酶活性,将培养物在缺少表达质粒选择需要的任何代谢物的合成完全培养基(Methods in Yeast Genetics,2005,Cold Spring HarborLaboratory Press,Cold Spring Harbor,NY,pp.201-202)中生长至1.0的OD600,通过离心收集(在4℃2600×g 8分钟),用缓冲液洗涤,再次离心,并冷冻于-80℃。将细胞融化,重悬浮于20mM Tris-HCl pH8.0中至2mL的终体积,然后使用1.2g玻璃珠(0.5mm大小)使用珠子搅拌器来破坏。将每个样品在高速处理总的3分钟时间(每分钟搅打后在冰上孵育)。通过离心将提取物清除细胞碎片(在4℃,20,000×g 10分钟)。
表7
用于酿酒酵母表达载体的克隆和测序的引物序列
名称 | 序列 | 描述 | SEQ ID NO: |
N98SeqF1 | CGTGTTAGTCACATCAGGAC | B.subtilis alsS测序引物 | 85 |
N98SeqF2 | GGCCATAGCAAAAATCCAAACAGC | B.subtilis alsS测序引物 | 86 |
N98SeqF3 | CCACGATCAATCATATCGAACACG | B.subtilis alsS测序引物 | 87 |
N98SeqF4 | GGTTTCTGTCTCTGGTGACG | B.subtilis alsS测序引物 | 88 |
N99SeqR1 | GTCTGGTGATTCTACGCGCAAG | B.subtilis alsS测序引物 | 89 |
N99SeqR2 | CATCGACTGCATTACGCAACTC | B.subtilis alsS测序引物 | 90 |
N99SeqR3 | CGATCGTCAGAACAACATCTGC | B.subtilis alsS测序引物 | 91 |
N99SeqR4 | CCTTCAGTGTTCGCTGTCAG | B.subtilis alsS测序引物 | 92 |
N136 | CCGCGGATAGATCTGAAATGAATAACAATACTGACA | 具有SacII/Bg1II位点的FBA启动子正向引物 | 93 |
N137 | TACCACCGAAGTTGATTTGCTTCAACATCCTCAGCTCTAGATTTGAATATGTATTACTTGGTTAT | 具有BbvCI位点和ILV5-退火区的FBA启动子反向引物 | 94 |
N138 | ATGTTGAAGCAAATCAACTTCGGTGGTA | ILV5正向引物(产生另一起始密码子) | 95 |
N139 | TTATTGGTTTTCTGGTCTCAAC | ILV5反向引物 | 96 |
N140 | AAGTTGAGACCAGAAAACCAATAATTATTAATCATGTAATAGTTATGTCACGCTT | 具有PacI位点和ILV5-退火区的CYC终止子正向引物 | 97 |
N141 | GCGGCCGCCCGCAAATTAAAGCCTTCGAGC | 具有NotI位点的CYC终止子反向引物 | 98 |
N142 | GGATCCGCATGCTTGCATTTAGTCGTGC | 具有BamHI位点的GPM启动子正向引物 | 99 |
N143 | CAGGTAATCCCCCACAGTATACATCCTCAGCTATTGTAATATGTGTGTTTGTTTGG | 具有BbvCI位点和kivD-退火区的GPM启动子反向引物 | 100 |
N144 | ATGTATACTGTGGGGGATTACC | kivD正向引物 | 101 |
N145 | TTAGCTTTTATTTTGCTCCGCA | kivD反向引物 | 102 |
N146 | TTTGCGGAGCAAAATAAAAGCTAATTAATTAAGAGTAAGCGAATTTCTTATGATTTA | ADH终止子正向引物具有PacI位点和kivD-退火区 | 103 |
N147 | ACTAGTACCACAGGTGTTGTCCTCTGAG | 具有SpeI位点的ADH终止子反向引物 | 104 |
N151 | CTAGAGAGCTTTCGTTTTCATG | alsS反向引物 | 105 |
N152 | CTCATGAAAACGAAAGCTCTCTAGTTAATTAATCATGTAATTAGTTATGTCACGCTT | 具有PacI位点和alsS-退火区的CYC终止子正向引物 | 106 |
N155 | ATGGCAAAGAAGCTCAACAAGTACT | ILV3正向引物(另一起始密码子) | 107 |
N156 | TCAAGCATCTAAAACACAACCG | ILV3反向引物 | 108 |
N157 | AACGGTTGTGTTTTAGATGCTTGATTAATTAAGAGTAAGCGAATTTCTTATGATTTA | 具有PacI位点和ILV3-退火区的ADH终止子正向引物 | 109 |
N158 | GGATCCTTTTCTGGCAACCAAACCCATA | 具有BamHI位点的ADH启动子正向引物 | 110 |
N159 | CGAGTACTTGTTGAGCTTCTTTGCCATCCTCAGCGAGATAGTTGATTGTATGCTTG | 具有BbvCI位点和ILV3-退火区的ADH启动子反向引物 | 111 |
N160SeqF1 | GAAAACGTGGCATCCTCTC | FBA::ILV5::CYC测序引物 | 112 |
N160SeqF2 | GCTGACTGGCCAAGAGAAA | FBA::ILV5::CYC测序引物 | 113 |
N160SeqF3 | TGTACTTCTCCCACGGTTTC | FBA::ILV5::CYC测序引物 | 114 |
N160SeqF4 | AGCTACCCAATCTCTATACCCA | FBA::ILV5::CYC测序引物 | 115 |
N160SeqF5 | CCTGAAGTCTAGGTCCCTATTT | FBA::ILV5::CYC测序引物 | 116 |
N 160SeqR1 | GCGTGAATGTAAGCGTGAC | FBA::ILV5::CYC测序引物 | 117 |
N160SeqR2 | CGTCGTATTGAGCCAAGAAC | FBA::ILV5::CYC测序引物 | 118 |
N160SeqR3 | GCATCGGACAACAAGTTCAT | FBA::ILV5::CYC测序引物 | 119 |
N160SeqR4 | TCGTTCTTGAAGTAGTCCAACA | FBA::ILV5::CYC测序引物 | 120 |
N160SeqR5 | TGAGCCCGAAAGAGAGGAT | FBA::ILV5::CYC测序引物 | 121 |
N161SeqF1 | ACGGTATACGGCCTTCCTT | ADH::ILV3::ADH测序引物 | 122 |
N161SeqF2 | GGGTTTGAAAGCTATGCAGT | ADH::ILV3::ADH测序引物 | 123 |
N161SeqF3 | GGTGGTATGTATACTGCCAACA | ADH::ILV3::ADH测序引物 | 124 |
N161SeqF4 | GGTGGTACCCAATCTGTGATTA | ADH::ILV3::ADH测序引物 | 125 |
N161SeqF5 | CGGTTTGGGTAAAGATGTTG | ADH::ILV3::ADH测序引物 | 126 |
N161SeqF6 | AAACGAAAATTCTTATTCTTGA | ADH::ILV3::ADH测序引物 | 127 |
N161SeqR1 | TCGTTTTAAAACCTAAGAGTCA | ADH::ILV3::ADH测序引物 | 128 |
N161SeqR2 | CCAAACCGTAACCCATCAG | ADH::ILV3::ADH测序引物 | 129 |
N161SeqR3 | CACAGATTGGGTACCACCA | ADH::ILV3::ADH测序引物 | 130 |
N161SeqR4 | ACCACAAGAACCAGGACCTG | ADH::ILV3::ADH测序引物 | 131 |
N161SeqR5 | CATAGCTTTCAAACCCGCT | ADH::ILV3::ADH测序引物 | 132 |
N161SeqR6 | CGTATACCGTTGCTCATTAGAG | ADH::ILV3::ADH测序引物 | 133 |
N162 | ATGTTGACAAAAGCAACAAAAGA | alsS正向引物 | 134 |
N189 | ATCCGCGGATAGATCTAGTTCGAGTTTATCATTATCAA | 具有SacII/BglII位点的GPD正向引物 | 135 |
N190.1 | TTCTTTTGTTGCTTTTGTCAACATCCTCAGCGTTTATGTGTGTTTATTCGAAA | 具有BbvCI位点和alsS-退火的区GPD启动子反向引物 | 136 |
N176 | ATCCGCGGATAGATCTATTAGAAGCCGCCGAGCGGGCG | 具有SacII/BglII位点的GAL1启动子正向引物 | 137 |
N177 | ATCCTCAGCTTTTCTCCTTGACGTTAAAGTA | 具有BbvCI位点的GAL1启动子反向 | 138 |
N191 | ATCCGCGGATAGATCTCCCATTACCGACATTTGGGCGC | 具有SacII/BgIII位点的CUP1启动子正向引物 | 175 |
N192 | ATCCTCAGCGATGATTGATTGATTGATTGTA | 具有BbvCI位点的CUP1启动子反向 | 176 |
表8
携带异丁醇途径基因的大肠杆菌-酵母穿梭载体
质粒名称 | 构建 |
pRS426[ATCC No.77107],URA3选择 | - |
pRS426::GPD::alsS::CYC | 用SacII/NotI消化的GPD::alsS::CYC PCR产物克隆至用相同酶消化的pRS426中 |
pRS426::FBA::ILV5::CYC | 用SacII/NotI消化的FBA::ILV5::CYC PCR产物克隆至用相同酶消化的pRS426中 |
pRS425[ATCC No.77106],LEU2选择 | - |
pRS425::ADH::ILV3::ADH | 用BamHI/SpeI消化的ADH::ILV3::ADH PCR产物克隆至用相同酶消化的pRS425中 |
pRS425::GPM::kivD::ADH | 用BamHI/SpeI消化的GPM::kivD::ADH PCR产物克隆至用相同酶消化的pRS425中 |
pRS426::CUPI::alsS | 来自pRS426::GPD::alsS::CYC的7.7kbpSacII/BbvCI片段连接SacII/BbvCI CUPI片段 |
pRS426::GAL1::ILV5 | 来自pRS426::FBA::ILV5::CYC的7kbpSacII/BbvCI片段连接SacII/BbvCI GAL1片段 |
pRS425::FBA::ILV3 | 来自pRS425::ADH::ILV3::ADH的8.9kbpBamHI/BbvCI 片段连接来自pRS426::FBA::ILV5::CYC的0.65kbpBglII/BbvCI FBA片段 |
pRS425::CPU1-alsS+FBA-ILV3 | 来自pRS426::CPU1::alsS的2.4kbp SacII/NotI片段克隆至用SacII/NotI 切割的pRS425::FBA::ILV3中 |
pRS426::FBA-ILV5+GPM-kivD | 来自pRS425::GMP::kivD::ADH的2.7kbpBamHI/SpeI片段克隆至用BamHI/SpeI切割的pRS426::FBA::ILV5::CYC中 |
pRS426::GAL1-FBA+GPM-kivD | 来自pRS426::FBA-ILV5+GPM-kivD的8.5kbpSacII/NotI片段连接来自pRS426::GAL1::ILV5的1.8kbp SacII/NotI片段 |
pSR423[ATCC No.77104],HIS3选择 | - |
pSR423::CUP1-alsS+FBA-ILV3 | 来自pRS425::CUP1-alsS+FBA-ILV3的5.2kbpSacI/SalI片段连接用SacI/SalI切割的pSR423 |
pHR81[ATCC No.87541],URA3和leu2-d选择 | - |
pHR81::FBA-ILV5+GPM-kivD | 来自pRS426::FBA-ILV5+GPM-kivD的4.7kbpSacI/BamH片段连接用SacI/BamHI切割的pHR81 |
实施例18
通过重组酿酒酵母生产异丁醇
将质粒pSR423::CUP1-alsS+FBA-ILV3和pHR81::FBA-ILV5+GPM-kivD(实施例17中所述的)转化至酿酒酵母YJR148w中来产生菌株YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD。通过将载体pSR423和pHR81(实施例17中所述的)转化至酿酒酵母YJR148w中制备对照菌株(YJR148w/pSR423/pHR81)。将菌株维持于含有2%葡萄糖或蔗糖但缺少尿嘧啶和组氨酸的标准酿酒酵母合成完全培养基上(Methods in Yeast Genetics,2005,Cold Spring Harbor LaboratoryPress,Cold Spring Harbor,NY,pp.201-202),以确保质粒的维持。
为了异丁醇生产,将细胞转移至缺少尿嘧啶,组氨酸和亮氨酸的合成完全培养基中。由于leu2-d等位基因的转录差,从培养基除去亮氨酸用来引发pHR81基质粒拷贝数的增加(Erhart和Hollenberg,J.Bacteriol.156:625-635(1983))。将好氧培养物生长于含有50mL培养基的175mL容量瓶中,在30℃和200rpm的Innova400培养箱中(NewBrunswick Scientific,Edison,NJ)。在接种和在30℃保持后,通过将45mL培养基加入用有波纹的帽子密封的60mL血清瓶中来制备低氧培养物。按照需要,无菌注射器用来取样和加入诱导剂。接种后大约24h,将诱导剂CuSO4加入至0.03M终浓度。还制备了每个菌株没有添加CuSO4的对照培养物。通过GC和HPLC分析CuSO4加入后18或19h和35h的培养物上清液的异丁醇含量,如以上实施例15中所述的。表9中呈现了生长于葡萄糖之上的酿酒酵母YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD的结果。对于表9中给出的结果,在35h从好氧培养物取样,在19h从低氧培养物取样,并通过HPLC测量。
表10中呈现了生长于蔗糖之上的酿酒酵母YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD的结果。用18h取的样并通过HPLC测量来获得该表中的结果。
表9
通过生长于葡萄糖之上的酿酒酵母
YJR148w/pSR423::CUJP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv
D的异丁醇生产
菌株 | O2水平 | 异丁醇,mM | 摩尔选择性% |
YJR148w/pRS423/pHR81(对照) | 好氧 | 0.12 | 0.04 |
YJR148w/pRS423/pHR81(对照) | 好氧 | 0.11 | 0.04 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD a | 好氧 | 0.97 | 0.34 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD b | 好氧 | 0.93 | 0.33 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD c | 好氧 | 0.85 | 0.30 |
YJR148w/pRS423/pHR81(对照) | 低 | 0.11 | 0.1 |
YJR148w/pRS423/pHR81(对照) | 低 | 0.08 | 0.1 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD a | 低 | 0.28 | 0.5 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD b | 低 | 0.20 | 0.3 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiVD c | 低 | 0.33 | 0.6 |
表10
通过生长于蔗糖之上的酿酒酵母
YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv
D的异丁醇生产
菌株 | O2水平 | 异丁醇,mM | 摩尔选择性,% |
YJR148w/pRS423/pHR81(对照) | 好氧 | 0.32 | 0.6 |
YJR148w/pRS423/pHR81(对照) | 好氧 | 0.17 | 0.3 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD a | 好氧 | 0.68 | 1.7 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD b | 好氧 | 0.54 | 1.2 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD c | 好氧 | 0.92 | 2.0 |
YJR148w/pRS423/pHR81(对照) | 低 | 0.18 | 0.3 |
YJR148w/pRS423/pHR81(对照) | 低 | 0.15 | 0.3 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD a | 低 | 0.27 | 1.2 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD b | 低 | 0.30 | 1.1 |
YJR148w/pRS423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD c | 低 | 0.21 | 0.8 |
菌株后缀“a”,“b”和“c”表示分开的分离物。
结果显示了,在好氧和低氧条件下在葡萄糖或蔗糖上生长,菌株YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD一致地生产出比对照菌株更高水平的异丁醇。
实施例19
通过重组酿酒酵母的异丁醇生产
将质粒pRS425::CPU1-alsS+FBA-ILV3和pRS426::GAL1-ILV5+GPM-kivD(实施例17中所述的)转化至酿酒酵母YJR148w中来产生菌株YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD。通过将载体pSR425和pRS426(实施例17中所述的)转化至酿酒酵母YJR148w中制备对照菌株(YJR148w/pSR425/pRS426)。将菌株维持于合成完全培养基上,如实施例18中所述的。
为了异丁醇生产,将细胞转移至含有2%半乳糖和1%棉子糖而缺少尿嘧啶和亮氨酸的合成完全培养基中。按照实施例18中所述的制备好氧和低氧培养物。接种后大约12h,将诱导剂CuSO4加入高达至0.5mM的终浓度。还制备了每个菌株没有添加CuSO4的对照培养物。在CuSO4加入后23h将培养物上清液取样,如实施例18中所述的。表11中呈现了结果。由于观察到的最终光密度非常不同并与定量残留的碳源相关,表中提供了每OD600单位的异丁醇浓度(替代摩尔选择性),以允许将含有异丁醇生物合成途径基因的菌株与对照相比较。
表11
通过生长于半乳糖和棉子糖之上的酿酒酵母YJR148w/
pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD的异丁
醇生产
菌株 | O2水平 | CuSO4,mM | 异丁醇mM | 每OD单位的mM异丁醇 |
YJR148w/pSR425/pRS426(对照) | 好氧 | 0.1 | 0.12 | 0.01 |
YJR148w/pSR425/pRS426(对照) | 好氧 | 0.5 | 0.13 | 0.01 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD a | 好氧 | 0 | 0.20 | 0.03 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-1LV5+GPM-kivD b | 好氧 | 0.03 | 0.82 | 0.09 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD c | 好氧 | 0.1 | 0.81 | 0.09 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD d | 好氧 | 0.5 | 0.16 | 0.04 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD e | 好氧 | 0.5 | 0.18 | 0.01 |
YJR148w/pSR425/pRS426(对照) | 低 | 0.1 | 0.042 | 0.007 |
YJR148w/pSR425/pRS426(对照) | 低 | 0.5 | 0.023 | 0.006 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD a | 低 | 0 | 0.1 | 0.04 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD b | 低 | 0.03 | 0.024 | 0.02 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD c | 低 | 0.1 | 0.030 | 0.04 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD d | 低 | 0.5 | 0.008 | 0.02 |
YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD e | 低 | 0.5 | 0.008 | 0.004 |
菌株后缀“a”,“b”,“c”,“d”和“e”表示分开的分离物。
结果表示总的说来,在好氧和低氧条件下,与对照菌株相比较,YJR148w/pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD菌株产生的每光密度单位的异丁醇水平更高。
实施例20
枯草芽孢杆菌中异丁醇生物合成途径的表达
该实施例的目的是在枯草芽孢杆菌中表达异丁醇生物合成途径。将异丁醇途径的五个基因(图1中的途径步骤(a)至(e))分成两个用于表达的操纵子。将各自编码乙酰乳酸合酶,乙酰醇酸脱水酶和支链酮酸脱羧酶的三个基因budB,ilvD和kivD整合至枯草芽孢杆菌BE1010的染色体中(Payne和Jackson,J.Bacteriol.173:2278-2282(1991))。将各自编码乙酰醇酸异构还原酶和丁醇脱氢酶的两个基因ilvC和bdhB克隆至表达载体中并转化至携带整合的异丁醇基因的芽孢杆菌菌株中。
三个基因budB,ilvD和kivD整合至枯草芽孢杆菌BE1010的染色 体中。芽孢杆菌整合载体pFP988DssPspac和pFP988DssPgroE用于三个基因budB(SEQ ID NO:1),ilvD(SEQ ID NO:5)和kivD(SEQ IDNO:7)的染色体整合。两个质粒都含有来自pBR322的大肠杆菌复制子,用于在大肠杆菌中选择的氨苄青霉素抗生素标记和两个与枯草芽孢杆菌染色体中的sacB基因同源的指导载体整合的片段,以及通过同源重组的插入序列。在sacB同源片段之间是pFP988DssPspac上的spac启动子(PgroE)或pFP988DssPgroE上的groEL启动子(PgroE),和用于芽孢杆菌的选择标记,红霉素。启动子片段还含有lacO序列,用于调节lacI阻遏蛋白的表达。pFP988DssPspac(6,341bp)和pFP988DssPgroE(6,221bp)的序列各自如SEQ ID NO:142和SEQ IDNO:143给出的。
通过从质粒pTrc99a budB-ilvC-ilvD-kivD删除ilvC基因来构建具有三个基因bubB-ilvD-kivD的盒。实施例14中描述了质粒pTrc99A::budB-ilvC-ilvD-kivD的构建。用AflII和NheI消化质粒pTrc99A::budB-ilvC-ilvD-kivD,用DNA聚合酶的Klenow片段处理来制备钝末端,并凝胶纯化含有pTrc99a载体,budB,ilvD和kivD的所得到的9.4kbp片段。将9.4kbp载体片段自我连接来形成pTrc99A::budB-ilvD-kivD,并转化至DH5α感受态细胞(Invitrogen)中。通过限制性作图证实pTrc99a::budB-ilvD-kivD克隆的ilvC基因删除。用SacI消化所得到的质粒pTrc99A::budB-ilvD-kivD并用DNA聚合酶的Klenow片段处理来制备钝末端。然后用BamHI消化质粒并将所得到的5,297bp budB-ilvD-kivD片段凝胶纯化。将5,297bp budB-ilvD-kivD片段连接整合载体pFP988DssPspac的SmaI和BamHI位点。将连接混合物转化至DH5α感受态细胞中。通过5.3kbp budB-ilvD-kivD片段的PCR扩增来筛选转化体,使用引物T-budB(BamHI)(SEQ ID NO:144)和B-kivD(BamHI)(SEQ ID NO:145)。将正确的克隆称为pFP988DssPspac-budB-ilvD-kivD。
从大肠杆菌转化体制备质粒pFP988DssPspac-budB-ilvD-kivD,并转化至按照Doyle等所述制备的枯草芽孢杆菌BE1010感受态细胞中(J.Bacteriol.144:957(1980))。通过离心收集感受态细胞并将细胞沉淀物重悬浮于小体积的上清液中。将两体积的SPII-EGTA培养基加入一体积的感受态细胞中(Methods for General and Molecular Bacteriology,P.Gerhardt等,编辑,American Society for Microbiology,Washington,DC(1994))。将等份(0.3mL)细胞分散于试管中,然后将2至3μg质粒pFP988DssPspac-budB-ilvD-kivD加入试管中。将试管在37℃振荡培养30分钟,此后将0.1mL 10%酵母提取物加入每个试管中,然后进一步培养60分钟。将转化体生长于用于选择的含有红霉素(1.0μg/mL)的LB平板上,使用双重琼脂覆盖方法(Methods for General andMolecular Bacteriology,上文)。通过PCR扩增筛选转化体,对于budB使用引物N 130SeqF1(SEQ ID NO:40)和N 130SeqR1(SEQ ID NO:44),对于kivD使用引物N 133SeqF1(SEQ ID NO:62)和N133SeqR1(SEQ ID NO:66)。阳性整合体显示出预期的1.7kbp budB和1.7kbpkivD PCR产物。鉴定了两个阳性整合体并命名为枯草芽孢杆菌BE1010ΔsacB::Pspac-budB-ilvD-kivD #2-3-2和枯草芽孢杆菌BE1010ΔsacB::Pspac-budB-ilvD-kivD#6-12-7。
整合体枯草芽孢杆菌BE 1010ΔsacB::Pspac-budB-ilvD-kivD#2-3-2和枯草芽孢杆菌BE1010ΔsacB::Pspac-budB-ilvD-kivD#6-12-7中的酶活性测定表明BudB,IlvD和KivD在spac启动子(Pspac)的控制下是低的。为了提高功能性酶的表达,由来自质粒pHT01(MoBitec,Goettingen,Germany)的PgroE启动子替代Pspac启动子。
通过XhoI和BamHI限制性消化从不相关的质粒切除6,039bppFP988Dss载体片段,如SEQ ID NO:146给出的,并凝胶纯化。从质粒pHT01PCR扩增PgroE启动子,使用引物T-groE(XhoI)(SEQ IDNO:147)和B-groEL(SpeI,BamH1)(SEQ ID NO:148)。用XhoI和BamHI消化PCR产物,连接6,039bp pFP988Dss载体片段,并转化至DH5α感受态细胞中。通过PCR扩增筛选转化体,使用引物T-groE(XhoI)和B-groEL(SpeI,BamH1)。阳性克隆显示出预期的174bpPgroE PCR产物并称为pFP988DssPgroE。还通过DNA测序证实了质粒pFP988DssPgroE。
用SpeI和PmeI消化质粒pFP988DssPspac-budB-ilvD-kivD并将所得到的5,313bp budB-ilvD-kivD片段凝胶纯化。将budB-ilvD-kivD片段连接pFP988DssPgroE的SpeI和PmeI位点并转化至DH5α感受态细胞中。通过PCR扩增筛选阳性克隆的1,690bp PCR产物,使用引物T-groEL(SEQ ID NO:149)和N 111(SEQ ID NO:20)。将阳性克隆称为pFP988DssPgroE-budB-ilvD-kivD。
从大肠杆菌转化体制备质粒pFP988DssPgroE-budB-ilvD-kivD,并转化至枯草芽孢杆菌BE1010感受态细胞中,如上所述。通过PCR扩增筛选转化体,对于budB使用引物N130SeqF1(SEQ ID NO:40)和N130SeqR1(SEQ ID NO:44),对于kivD使用引物N133SeqF1(SEQID NO:62)和N 133SeqR1(SEQ ID NO:66)。阳性整合体显示出预期的1.7kbpbudB和1.7kbpkivD PCR产物。分离了两个阳性整合体并命名为枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7和枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#8-16。
ilvC和bdhB基因的质粒表达。从质粒表达两个剩余的异丁醇基因,ilvC和bdhB。将质粒pHT01(MoBitec),芽孢杆菌-大肠杆菌穿梭载体,用于将来自枯草芽孢杆菌的ilvC基因与PgroE启动子融合,使得ilvC基因从含有lacO序列的PgroE启动子表达。从枯草芽孢杆菌BR151(ATCC 33677)基因组DNA PCR扩增ilvC基因,如SEQ ID NO:186给出的,使用引物T-ilvCB.s.(BamHI)(SEQ ID NO:150)和B-ilvCB.s.(SpeI BamHI)(SEQ ID NO:151)。用BamHI消化1,067bp ilvC PCR产物并连接pHT01的BamHI位点。将连接混合物转化至DH5α感受态细胞中。通过PCR扩增筛选阳性克隆的1,188bp PCR产物,使用引物T-groEL和B-ilvB.s(SpeI BamHI)。将阳性克隆称为pHT01-ilvC(B.s)。将质粒pHT01-ilvC(B.s)用作PgroE-ilvC融合片段的PCR扩增的模板。
质粒pBD64(Minton等,Nucleic Acids Res.18:1651(1990))是相当稳定的载体,用于在枯草芽孢杆菌中表达外源基因,并含有repB基因以及氯霉素和卡那霉素抗性基因,用于在枯草芽孢杆菌中的选择。该质粒用于ilvC和bdhB在PgroE启动子控制下的表达。为了将PgroE-ilvC,bdhB和lacI阻遏基因克隆至质粒pBD64中,使用一步装配方法(Tsuge等,Nucleic Acids Res.31:e133(2003))。从pBD64 PCR扩增3,588bp pBD64片段,该片段含有repB基因,其包括复制功能,和卡那霉素抗生素标记,使用引物T-BD64(DraIII)(SEQ ID NO:152),其引入DraIII序列(CACCGAGTG),和B-BD64(DraIII)(SEQ ID NO:153),其引入DraIII序列(CACCTGGTG)。从pMUTIN4PCR扩增1,327bp lacI阻遏基因(Vagner等,Microbiol.144:3097-3104(1988)),使用T-lacIq(DraIII)(SEQ ID NO:154),其引入了DraIII序列(CACCAGGTG),和B-lacIq(DraIII)(SEQ ID NO:155),其引入了DraIII序列(CACGGGGTG)。从pHT01-ilvC(B.s)PCR扩增1,224bpPgroE-ilvC融合的盒,使用T-groE(DraIII)(SEQ ID NO:156),其引入了DraIII序列(CACCCCGTG),和B-B.s.ilvC(DraIII)(SEQ ID NO:157),其引入了DraIII序列(CACCGTGTG)。从丙醇丁酮梭菌(ATCC824)基因组DNA PCR扩增1.2kbp bdhB基因(SEQ ID NO:158),使用引物T-bdhB(DraIII)(SEQ ID NO:159),其引入了DraIII序列(CACACGGTG),和B-bdhB(rrnBT1DraIII)(SEQ ID NO:160),其引入了DraIII序列(CACTCGGTG)。DraIII识别序列的可变区中的三个下划线字母是为了特异性碱基配对而设计的,以pBD64-lacI-PgroEilvC-bdhB的次序装配四个片段。用DraIII分开消化两个末端的每个具有DraIII的PCR产物,将所得到的DraIII片段,3,588bp pBD64,lacI,PgroEilvC和bdhB凝胶纯化,使用QIAGEN凝胶提取试剂盒(QIAGEN)。制备含有等摩尔浓度的每个片段的混合物,用于连接,总DNA浓度为30至50μg/100μL。然后将连接溶液在16℃孵育过夜。连接产生高分子量串联重复DNA。将连接的长线性DNA混合物直接转化至感受态枯草芽孢杆菌BE1010中,按照以上所述的来制备。枯草芽孢杆菌优选采用长的重复的线性DNA形式,而不是环状DNA来建立质粒。转化后,将培养物涂布在含有10μg/mL卡那霉素的LB平板上,用于选择。通过DraIII消化筛选阳性重组质粒,获得四个具有预期大小3,588bp(pBD64),1,327bp(lacI),1,244bp(PgoE-ilvC)和1,194bp(bdhB)的片段。将阳性质粒称为pBDPgroE-ilvC(B.s.)-bdhB。
通过枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD/pBDPgroE-ilvC (B.s.)-bdhB从葡萄糖或 蔗糖生产异丁醇的证明。为了构建表达异丁醇生物合成途径的五个基因的重组枯草芽孢杆菌,如上所述制备两个整合体枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7和枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#8-16的感受态细胞,并用质粒pBDPgroE-ilvC(B.s.)-bdhB 转化,产生枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB和枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#8-16/pBDPgroE-ilvC(B.s.)-bdhB。
将两个重组菌株在125mL烧瓶中的25mL或100mL含有卡那霉素(10μg/mL)的葡萄糖培养基中培养,来各自模拟高氧和低氧条件,并在37℃200rpm振荡好氧生长。培养基由10mM(NH4)2SO4,5mM磷酸钾缓冲液(pH7.0),100mM MOPS/KOH缓冲液(pH7.0),20mM谷氨酸/KOH(pH7.0),2%S10金属混合物,1%葡萄糖,0.01%酵母提取物,0.01%酪蛋白氨基酸以及各自50μg/mL的L-色氨酸,L-甲硫氨酸和L-赖氨酸组成。S10金属混合物由200mM MgCl2,70mM CaCl2,5mMMnCl2,0.1mM FeCl3,0.1mM ZnCl2,0.2mM盐酸硫胺素,0.172mMCuSO4,0.253mM CoCl2和0.242mM Na2MoCO4组成。在早对数期(大约0.2的OD600)用1.0mM异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导细胞。接种后24h,通过HPLC(Shodex Sugar SH1011柱)使用折射率(RI)检测分析等份培养液的异丁醇含量,如一般方法部分中所述。表12中显示了HPLC结果。
表12
通过枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD
/pBDPgroE-ilvC(B.s.)-bdhB菌株从葡萄糖生产异丁醇
菌株 | O2水平 | 异丁醇,mM | 摩尔选择性,% |
枯草芽孢杆菌a(诱导的) | 高 | 1.00 | 1.8 |
枯草芽孢杆菌b(诱导的) | 高 | 0.87 | 1.6 |
枯草芽孢杆菌a(诱导的) | 低 | 0.06 | 0.1 |
枯草芽孢杆菌b(诱导的) | 低 | 0.14 | 0.3 |
枯草芽孢杆菌a是枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB
枯草芽孢杆菌b是枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#8-16/pBDPgroE-ilvC(B.s.)-bdhB
还检测了枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s)-bdhB分离物从蔗糖的异丁醇生产,基本上如上所述。将重组菌株在125mL烧瓶中的25mL或75mL含有卡那霉素(10μg/mL)的蔗糖培养基中培养,来模拟高氧和中等氧水平,并在37℃200rpm振荡生长。除了用10g/L蔗糖替代葡萄糖(10g/L),蔗糖培养基与葡萄糖培养基相同。细胞没有诱导,或在早对数期(大约0.2的OD600)用1.0mM异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导。接种后24h,通过HPLC(Shodex Sugar SH1011柱)使用折射率(RI)检测分析等份培养液的异丁醇含量,如一般方法部分中所述。表13中显示了HPLC结果。
表13
通过枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD
/pBDPgroE-ilvC(B.s.)-bdhB菌株从蔗糖生产异丁醇
菌株 | O2水平 | 异丁醇,mM | 摩尔选择性,% |
枯草芽孢杆菌a(未诱导) | 高 | 未测出 | 未测出 |
枯草芽孢杆菌a(诱导的) | 高 | 0.44 | 4.9 |
枯草芽孢杆菌a(诱导的) | 中等 | 0.83 | 8.6 |
枯草芽孢杆菌a是枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB
实施例21(预测的)
植物乳杆菌中异丁醇生物合成途径的表达
该预测实施例的目的是描述怎样在植物乳杆菌中表达异丁醇生物合成途径。将异丁醇途径的编码五个酶活性的五个基因分至用于表达的两个操纵子中。通过同源重组将各自编码乙酰乳酸合酶,乙酰醇酸脱水酶和支链α-酮酸脱羧酶的基因budB,ilvD和kivD整合至植物乳杆菌的染色体中,使用Hols等所述的方法(Appl.Environ.Microbiol.60:1401-1413(1994))。将剩余的两个基因(ilvC和bdhB,各自编码乙酰醇酸还原异构酶和丁醇脱氢酶)克隆至表达质粒中并转化至携带整合异丁醇基因的乳杆菌菌株中。将植物乳杆菌生长于37℃MRS培养基(Difco Laboratories,Detroit,MI)中,并分离染色体DNA,如Moreira等所述的(BMC Microbiol.5:15(2005))。
整合。通过同源重组将合成P11启动子(Rud等,Microbiology 152:1011-1019(2006))控制下的budB-ilvD-kivD盒在IdhL1基因座整合至植物乳杆菌ATCC BAA-793(NCIMB 8826)的染色体中。为了构建IdhL整合靶向载体,将来自植物乳杆菌的具有IdhL同源性的DNA片段(Genbank NC_004567)PCR扩增,使用引物IDH EcoRV F(SEQ ID NO:161)和IDH AatIIR(SEQ ID NO:162)。将1986bp PCR片段克隆至pCR4Blunt-TOPO 中并测序。用EcoRV和AatII消化pCR4Blunt-TOPO-IdhL1克隆,释放1982bp IdhL1片段,将其凝胶纯化。用HindIII消化整合载体pFR988,如SEQ ID NO:177给出的,用KlenowDNA聚合酶处理来钝化末端。然后用AatII消化线性化的质粒,并将2931bp载体片段凝胶纯化。将EcoRV/AatII IdhL1片段连接pFP988载体片段并转化至大肠杆菌Top10细胞中。在含有氨苄青霉素(100μg/mL)的LB琼脂平板上选择转化体并通过菌落PCR筛选来证实pFP988-IdhL的构建。
为了将选择标记添加至整合DNA,从pC194(GenBankNC_002013,SEQ ID NO:267)PCR扩增具有启动子的Cm基因,使用引物Cm F(SEQ ID NO:163)和Cm R(SEQ ID NO:164),扩增了836bpPCR产物。将该PCR产物克隆至pCR4Blunt-TOPO中并转化至大肠杆菌Top10细胞中,形成pCR4Blunt-TOPO-Cm。测序后证实了通过PCR没有引入错误,从pCR4Blunt-TOPO-Cm将Cm盒消化为828bpMluI/SwaI片段并凝胶纯化。用MluI和SwaI消化含有IdhL同源性的整合载体pFP988-IdhL,并将4740bp载体片段凝胶纯化。将Cm盒片段连接pFP988-IdhL载体,形成pFP988-DIdhL::Cm。
最后,用合成的P11启动子替代淀粉酶启动子来修饰实施例20中所述的来自pFP988DssPspac-budB-ilvD-kivD的budB-ilvD-kivD盒。然后,将整个操纵子转移至pFP988-DIdhL::Cm中。通过用引物P11F-StuI(SEQ ID NO:165)和P11R-SpeI(SEQ ID NO:166)退火寡核苷酸来构建P11启动子。将退火的寡核苷酸在6%Ultra PAGE凝胶上凝胶纯化(Embi Tec,San Diego,CA)。用StuI和SpeI消化含有淀粉酶启动子的质粒pFP988DssPspac-budB-ilvD-kivD,并将所得到的10.9kbp载体片段凝胶纯化。将分离的P11片段连接消化的pFP988DssPspac-budB-ilvD-kivD形成pFP988-P11-budB-ilvD-kivD。然后用StuI和BamHI消化质粒pFP988DssPspac-budB-ilvD-kivD,并将所得到的5.4kbp P11-budB-ilvD-kivD片段凝胶纯化。用HpaI和BamHI消化pFP988-DIdhL::Cm并分离5.5kbp载体片段。将budB-ilvD-kivD操纵子连接整合载体pFP988-DIdhL::Cm形成pFP988-DIdhL-P11-budB-ilvD-kivD::Cm。
将pFP988-DIdhL-P11-budB-ilvD-kivD::Cm整合至植物乳杆菌 BAA-793中形成包括外源budB,ilvD,kivD基因的植物乳杆菌 ΔIdhL1::budB-ilvD-kivD::Cm。按照Aukrust,T.W.等所述的制备植物乳杆菌的电感受态细胞(在:Electroporation Protocols for Microorganisms;Nickoloff,J.A编辑;Methods in Molecular Biology,Vol.47;HumanaPress,Inc.,Totowa,NJ,1995,pp201-208)。电穿孔后,按照上文中Aukrust等所述的将细胞在MRSSM培养基中(补充0.5M蔗糖和0.1MMgCl2的MRS培养基)37℃生长2h,没有振荡)。将电穿孔的细胞涂布于用于选择的含有氯霉素(10μg/mL)的MRS平板上并在37℃培养。最初通过菌落PCR扩增来筛选转化体以证实整合,然后通过PCR使用一组引物来更严格地筛选最初的阳性克隆。
ilvC和bdhB基因的质粒表达。在植物乳杆菌IdhL启动子的控制下(Ferain等,J.Bacteriol.176:596-601(1994))从质粒pTRKH3(O’Sullivan DJ和Klaenhammer TR,Gene 137:227-231(1993))表达剩余的两个异丁醇基因。使用引物PIdhL F-HindIII(SEQ ID NO:167)和PIdhL R-BamHI(SEQ ID NO:168)从植物乳杆菌ATCC BAA-793的基因组PCR扩增IdhL启动子。将411bp PCR产物克隆至pCR4Blunt-TOPO中并测序。用HindIII和BamHI消化所得到的质粒pCR4Blunt-TOPO-PIdhL,释放PIdhL片段。
用HindIII和SphI消化质粒pTRKH3,并将凝胶纯化的载体片段以三通方式连接PIdhL片段和来自芽孢杆菌表达质粒pBDPgroE-ilvC(B.s.)-bdhB的凝胶纯化的含有ilvC(B.s.)-bdhB的2.4kbpBamHI/SphI片段(实施例20)。将连接混合物转化至大肠杆菌Top 10细胞中并将转化体生长于含有红霉素(150mg/L)的脑心浸液(BHI,Difco Laboratories,Detroit,MI)平板上。通过PCR筛选转化体来证实构建。通过电穿孔将所得到的表达质粒pTRKH3-ilvC(B.s.)-bdhB转化至植物乳杆菌ΔIdhL1::budB-ilvD-kivD::Cm中,如上所述。
将含有pTRKH3-ilvC(B.s.)-bdhB的植物乳杆菌ΔIdhL1::budB-ilvD-kivD::Cm接种至含有50mL添加红霉素(10μg/mL)的MRS培养基的250mL烧瓶中并在37℃生长18至24h,没有振荡,此后,通过HPLC或GC分析检测异丁醇,如一般方法部分所述的。
实施例22(预测的)
粪肠球菌中异丁醇生物合成途径的表达
该预测实施例的目的是描述怎样在粪肠球菌中表达异丁醇生物合成途径。已经公开了粪肠球菌菌株V583的完整基因组序列(Paulsen等,Science 299:2071-2074(2003)),将其用作该实施例中表达异丁醇生物合成途径的宿主菌株。将大肠杆菌革兰氏阳性穿梭载体,质粒pTRKH3(O’Sullivan DJ和Klaenhammer TR,Gene 137:227-231(1993)),用于异丁醇途径的五个基因(bubB,ilvC,ilvD,kivD,bdhB)在一个操纵子中的表达。pTRKH3含有大肠杆菌质粒p15A复制起点,pAMβ1复制子,以及用于四环素和红霉素的两个抗生素抗性选择标记。四环素抗性只在大肠杆菌中得到表达,而红霉素抗性在大肠杆菌和革兰氏阳性细菌中都得到表达。质粒pAMβ1衍生物可以在粪肠球菌中复制(Poyart等,FEMS Microbiol.Lett.156:193-198(1997))。将诱导型nisA启动子(PnisA)用于控制编码异丁醇生物合成途径的酶的五个所需基因的表达,该启动子已经用于基因表达的有效控制,通过各种革兰氏阳性细菌包括粪肠球菌中的乳酸链球菌肽(Eichenbaum等,Appl.Environ.Microbiol.64:2763-2769(1998))。
将含有异丁醇途径操纵子的质粒pTrc99A::budB-ilvC-ilvD-kivD(实施例14中所述的)进行修饰,用枯草芽孢杆菌ilvC基因(SEQ IDNO:??)替代大肠杆菌ilvC基因(SEQ ID NO:3)。此外,将来自丙醇丁酮梭菌的bdhB基因(SEQ ID NO:158)添加至操纵子的末端。首先,使用引物F-bdhB-AvrII(SEQ ID NO:169)和R-bdhB-BamHI(SEQ ID NO:170)扩增来自pBDPgroE-ilvC(B.s.)-bdhB(实施例20中所述的)的bdh基因,然后TOPO克隆并测序。通过用AvrII和BamHI消化接着凝胶纯化分离了1194bp bdhB片段。将bdhB片段连接之前已经用AvrII和BamHI消化的pTrc99A::budB-ilvC-ilvD-kivD,并将所得到的片段凝胶纯化。通过电穿孔将连接混合物转化至大肠杆菌Top10细胞中并在含有氨苄青霉素(100μg/mL)的LB琼脂平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化体以证实含有pTrc99A::budB-ilvC-ilvD-kivD-bdhB的正确克隆。
接着,使用引物F-ilvC(B.s.)-AflII(SEQ ID NO:171)和R-ilvC(B.s.)-NotI(SEQ ID NO:172)扩增来自pBDPgroE-ilvC(B.s.)-bdhB(实施例20中所述的)的ilvC(B.s)。将PCR产物克隆并测序。通过用AflII和NotI消化接着凝胶纯化分离了1051bp ilvC(B.s.)片段。将该片段连接已经用AflII和NotI切割释放了大肠杆菌ilvC(在连接ilvC(B.s.)之前凝胶纯化了10.7kbp载体条带)的pTrc99A::budB-ilvC-ilvD-kivD-bdhB。通过电穿孔将连接混合物转化至大肠杆菌Top10细胞中并在含有氨苄青霉素(100μg/mL)的LB琼脂平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化体以证实含有pTrc99A::budB-ilvC(B.s.)-ilvD-kivD-bdhB的正确克隆。
为了提供用于大肠杆菌革兰氏阳性穿梭载体pTRKH3的启动子,使用引物F-PnisA(HindIII)(SEQ ID NO:173)和R-PnisA(SpeIBamHI)(SEQ ID NO:174)从乳酸乳球菌基因组DNA PCR扩增nisA启动子(Chandrapati等,Mol.Microbiol.46(2):467-477(2002)),然后TOPO克隆。测序后,通过用HindIII和BamHI消化接着凝胶纯化分离了213bp nisA启动子片段。用HindIII和BamHI消化质粒pTRKH3并将载体片段凝胶纯化。将线性化pTRKH3连接PnisA片段并通过电穿孔转化至大肠杆菌Top10细胞中。在含有红霉素(25μg/mL)的LB琼脂平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化体以证实pTRKH3-PnisA的正确克隆。
用SpeI和BamHI消化质粒pTRKH3-PnisA,并将载体凝胶纯化。用SpeI和BamHI消化上述的质粒pTrc99A::budB-ilvC(B.s.)-ilvD-kivD-bdhB,并将7.5kbp片段凝胶纯化。将7.5kbp budB-ilvC(B.s.)-ilvD-kivD-bdhB片段在SpeI和BamHI位点连接pTRKH3-PnisA。通过电穿孔将连接混合物转化至大肠杆菌Top10细胞中并在含有红霉素(25μg/mL)的LB琼脂平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化体。将所得到的质粒称为pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB。从大肠杆菌转化体制备该质粒并使用本领域已知的方法转化至电感受态粪肠球菌V583细胞中(Aukrust,T.W.等,在:Electroporation Protocols for Microorganisms(微生物的电穿孔方案);Nickoloff,J.A.编辑;Methods in MolecularBiology(分子生物学方法),Vol.47;Humana Press,Inc.,Totowa,NJ.,1995,pp.217-226),得到粪肠球菌V583/pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB。
通过电穿孔将含有nisA调控基因,nisR和nisK,add9壮观霉素抗性基因和pSH71复制起点的第二个质粒转化至粪肠球菌V583/pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB中。含有pSH71复制起点的质粒与粪肠球菌中的pAMβ1衍生物相容(Eichenbaum等,上文)。在37℃生长的含有红霉素(25μg/mL)和壮观霉素(100μg/mL)的LB琼脂平板上选择双药物抗性转化体。
将所得到的带有两个质粒(即表达质粒(pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB)和调控质粒(pSH71-nisRK))的粪肠球菌株V583B接种于含有50mL补充酵母提取物(0.2%)(Fischetti等,J.Exp.Med.161:1384-1401(1985)),乳酸链球菌肽(20μg/mL)(Eichenbaum等,上文),红霉素(25μg/mL)和壮观霉素(100μg/mL)的Todd-Hewitt培养液的250mL烧瓶中。将烧瓶在37℃培养18-24h,没有振荡,该时间后,通过HPLC或GC分析测量异丁醇产生,如一般方法部分中所述的。
序列表
<110>E.I.du Pont de Nemours and Co.
<120>四碳醇的发酵生产
<130>CL3243 PCT
<160>267
<170>PatentIn version 3.3
<210>1
<211>1680
<212>DNA
<213>肺炎克雷伯氏菌
<400>1
atggacaaac agtatccggt acgccagtgg gcgcacggcg ccgatctcgt cgtcagtcag 60
ctggaagctc agggagtacg ccaggtgttc ggcatccccg gcgccaaaat cgacaaggtc 120
tttgattcac tgctggattc ctccattcgc attattccgg tacgccacga agccaacgcc 180
gcatttatgg ccgccgccgt cggacgcatt accggcaaag cgggcgtggc gctggtcacc 240
tccggtccgg gctgttccaa cctgatcacc ggcatggcca ccgcgaacag cgaaggcgac 300
ccggtggtgg ccctgggcgg cgcggtaaaa cgcgccgata aagcgaagca ggtccaccag 360
agtatggata cggtggcgat gttcagcccg gtcaccaaat acgccatcga ggtgacggcg 420
ccggatgcgc tggcggaagt ggtctccaac gccttccgcg ccgccgagca gggccggccg 480
ggcagcgcgt tcgttagcct gccgcaggat gtggtcgatg gcccggtcag cggcaaagtg 540
ctgccggcca gcggggcccc gcagatgggc gccgcgccgg atgatgccat cgaccaggtg 600
gcgaagctta tcgcccaggc gaagaacccg atcttcctgc tcggcctgat ggccagccag 660
ccggaaaaca gcaaggcgct gcgccgtttg ctggagacca gccatattcc agtcaccagc 720
acctatcagg ccgccggagc ggtgaatcag gataacttct ctcgcttcgc cggccgggtt 780
gggctgttta acaaccaggc cggggaccgt ctgctgcagc tcgccgacct ggtgatctgc 840
atcggctaca gcccggtgga atacgaaccg gcgatgtgga acagcggcaa cgcgacgctg 900
gtgcacatcg acgtgctgcc cgcctatgaa gagcgcaact acaccccgga tgtcgagctg 960
gtgggcgata tcgccggcac tctcaacaag ctggcgcaaa atatcgatca tcggctggtg 1020
ctctccccgc aggcggcgga gatcctccgc gaccgccagc accagcgcga gctgctggac 1080
cgccgcggcg cgcagctcaa ccagtttgcc ctgcatcccc tgcgcatcgt tcgcgccatg 1140
caggatatcg tcaacagcga cgtcacgttg accgtggaca tgggcagctt ccatatctgg 1200
attgcccgct acctgtacac gttccgcgcc cgtcaggtga tgatctccaa cggccagcag 1260
accatgggcg tcgccctgcc ctgggctatc ggcgcctggc tggtcaatcc tgagcgcaaa 1320
gtggtctccg tctccggcga cggcggcttc ctgcagtcga gcatggagct ggagaccgcc 1380
gtccgcctga aagccaacgt gctgcatctt atctgggtcg ataacggcta caacatggtc 1440
gctatccagg aagagaaaaa atatcagcgc ctgtccggcg tcgagtttgg gccgatggat 1500
tttaaagcct atgccgaatc cttcggcgcg aaagggtttg ccgtggaaag cgccgaggcg 1560
ctggagccga ccctgcgcgc ggcgatggac gtcgacggcc cggcggtagt ggccatcccg 1620
gtggattatc gcgataaccc gctgctgatg ggccagctgc atctgagtca gattctgtaa 1680
<210>2
<211>559
<212>PRT
<213>肺炎克雷伯氏菌
<400>2
Met Asp Lys Gln Tyr Pro Val Arg Gln Trp Ala His Gly Ala Asp Leu
1 5 10 15
Val Val Ser Gln Leu Glu Ala Gln Gly Val Arg Gln Val Phe Gly Ile
20 25 30
Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Ser Leu Leu Asp Ser Ser
35 40 45
Ile Arg Ile Ile Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala
50 55 60
Ala Ala Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr
65 70 75 80
Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn
85 90 95
Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys Arg Ala
100 105 110
Asp Lys Ala Lys Gln Val His Gln Ser Met Asp Thr Val Ala Met Phe
115 120 125
Ser Pro Val Thr Lys Tyr Ala Ile Glu Val Thr Ala Pro Asp Ala Leu
130 135 140
Ala Glu Val Val Ser Asn Ala Phe Arg Ala Ala Glu Gln Gly Arg Pro
145 150 155 160
Gly Ser Ala Phe Val Ser Leu Pro Gln Asp Val Val Asp Gly Pro Val
165 170 175
Ser Gly Lys Val Leu Pro Ala Ser Gly Ala Pro Gln Met Gly Ala Ala
180 185 190
Pro Asp Asp Ala Ile Asp Gln Val Ala Lys Leu Ile Ala Gln Ala Lys
195 200 205
Asn Pro Ile Phe Leu Leu Gly Leu Met Ala Ser Gln Pro Glu Asn Ser
210 215 220
Lys Ala Leu Arg Arg Leu Leu Glu Thr Ser His Ile Pro Val Thr Ser
225 230 235 240
Thr Tyr Gln Ala Ala Gly Ala Val Asn Gln Asp Asn Phe Ser Arg Phe
245 250 255
Ala Gly Arg Val Gly Leu Phe Asn Asn Gln Ala Gly Asp Arg Leu Leu
260 265 270
Gln Leu Ala Asp Leu Val Ile Cys Ile Gly Tyr Ser Pro Val Glu Tyr
275 280 285
Glu Pro Ala Met Trp Asn Ser Gly Asn Ala Thr Leu Val His Ile Asp
290 295 300
Val Leu Pro Ala Tyr Glu Glu Arg Asn Tyr Thr Pro Asp Val Glu Leu
305 310 315 320
Val Gly Asp Ile Ala Gly Thr Leu Asn Lys Leu Ala Gln Asn Ile Asp
325 330 335
His Arg Leu Val Leu Ser Pro Gln Ala Ala Glu Ile Leu Arg Asp Arg
340 345 350
Gln His Gln Arg Glu Leu Leu Asp Arg Arg Gly Ala Gln Leu Asn Gln
355 360 365
Phe Ala Leu His Pro Leu Arg Ile Val Arg Ala Met Gln Asp Ile Val
370 375 380
Asn Ser Asp Val Thr Leu Thr Val Asp Met Gly Ser Phe His Ile Trp
385 390 395 400
Ile Ala Arg Tyr Leu Tyr Thr Phe Arg Ala Arg Gln Val Met Ile Ser
405 410 415
Asn Gly Gln Gln Thr Met Gly Val Ala Leu Pro Trp Ala Ile Gly Ala
420 425 430
Trp Leu Val Asn Pro Glu Arg Lys Val Val Ser Val Ser Gly Asp Gly
435 440 445
Gly Phe Leu Gln Ser Ser Met Glu Leu Glu Thr Ala Val Arg Leu Lys
450 455 460
Ala Asn Val Leu His Leu Ile Trp Val Asp Asn Gly Tyr Asn Met Val
465 470 475 480
Ala Ile Gln Glu Glu Lys Lys Tyr Gln Arg Leu Ser Gly Val Glu Phe
485 490 495
Gly Pro Met Asp Phe Lys Ala Tyr Ala Glu Ser Phe Gly Ala Lys Gly
500 505 510
Phe Ala Val Glu Ser Ala Glu Ala Leu Glu Pro Thr Leu Arg Ala Ala
515 520 525
Met Asp Val Asp Gly Pro Ala Val Val Ala Ile Pro Val Asp Tyr Arg
530 535 540
Asp Asn Pro Leu Leu Met Gly Gln Leu His Leu Ser Gln Ile Leu
545 550 555
<210>3
<211>1476
<212>DNA
<213>大肠杆菌
<400>3
atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60
cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120
gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180
ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240
aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300
ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360
ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420
gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480
gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540
aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600
caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660
gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720
gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780
atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840
gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900
cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960
gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020
accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080
atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140
atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200
atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260
aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320
ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380
gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440
atgacagata tgaaacgtat tgctgttgcg ggttaa 1476
<210>4
<211>491
<212>PRT
<213>大肠杆菌
<400>4
Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln
1 5 10 15
Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala
20 25 30
Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln
35 40 45
Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser
50 55 60
Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg
65 70 75 80
Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile
85 90 95
Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser
100 105 110
Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu
115 120 125
Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg
130 135 140
Lys Asp Ile Thr Val Val Met Val Ala Pro Lys Cys Pro Gly Thr Glu
145 150 155 160
Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala
165 170 175
Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys
180 185 190
Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser
195 200 205
Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile
210 215 220
Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu
225 230 235 240
Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe
245 250 255
Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys Gln Gly Gly Ile Thr Leu
260 265 270
Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu
275 280 285
Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met
290 295 300
Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp
305 310 315 320
Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys
325 330 335
Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln
340 345 350
Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly
355 360 365
Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu
370 375 380
Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr
385 390 395 400
Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr
405 410 415
Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu
420 425 430
Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile
435 440 445
Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala
450 455 460
Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr
465 470 475 480
Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly
485 490
<210>5
<211>1851
<212>DNA
<213>大肠杆菌
<400>5
atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60
ctgtggcgcg ccaccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120
aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180
gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240
gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cgaactgatc 300
gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360
aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420
atctttgttt ccggcggccc gatggaggcc gggaaaacca aactttccga tcagatcatc 480
aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540
agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600
gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660
ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcattgtt 720
gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780
agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840
accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900
atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960
taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020
cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080
ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 1140
ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200
gacgacgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260
ggcctggcgg tgctctacgg taactttgcg gaaaacggct gcatcgtgaa aacggcaggc 1320
gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380
gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440
gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500
tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560
tctggtcttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620
attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680
agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaagcctgg 1740
acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800
accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 1851
<210>6
<211>616
<212>PRT
<213>大肠杆菌
<400>6
Met Pro Lys Tyr Arg Ser Ala Thr Thr Thr His Gly Arg Asn Met Ala
1 5 10 15
Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe
20 25 30
Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro
35 40 45
Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile
50 55 60
Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp
65 70 75 80
Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser
85 90 95
Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys
100 105 110
Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly
115 120 125
Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser
130 135 140
Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile
145 150 155 160
Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val
165 170 175
Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys
180 185 190
Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr
195 200 205
Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr
210 215 220
His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val
225 230 235 240
Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro
245 250 255
Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp
260 265 270
Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala
275 280 285
Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu
290 295 300
Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys
305 310 315 320
Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu
325 330 335
Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val
340 345 350
Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu
355 360 365
Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly
370 375 380
Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu
385 390 395 400
Asp Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr
405 410 415
Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn
420 425 430
Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe
435 440 445
Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala
450 455 460
Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Val Ile Arg Tyr
465 470 475 480
Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr
485 490 495
Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr
500 505 510
Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val
515 520 525
Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly
530 535 540
Asp Leu Ile Ala Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val
545 550 555 560
Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly
565 570 575
Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala
580 585 590
Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val
595 600 605
Arg Asp Lys Ser Lys Leu Gly Gly
610 615
<210>7
<211>1662
<212>DNA
<213>乳酸乳球菌
<400>7
tctagacata tgtatactgt gggggattac ctgctggatc gcctgcacga actggggatt 60
gaagaaattt tcggtgtgcc aggcgattat aacctgcagt tcctggacca gattatctcg 120
cacaaagata tgaagtgggt cggtaacgcc aacgaactga acgcgagcta tatggcagat 180
ggttatgccc gtaccaaaaa agctgctgcg tttctgacga cctttggcgt tggcgaactg 240
agcgccgtca acggactggc aggaagctac gccgagaacc tgccagttgt cgaaattgtt 300
gggtcgccta cttctaaggt tcagaatgaa ggcaaatttg tgcaccatac tctggctgat 360
ggggatttta aacattttat gaaaatgcat gaaccggtta ctgcggcccg cacgctgctg 420
acagcagaga atgctacggt tgagatcgac cgcgtcctgt ctgcgctgct gaaagagcgc 480
aagccggtat atatcaatct gcctgtcgat gttgccgcag cgaaagccga aaagccgtcg 540
ctgccactga aaaaagaaaa cagcacctcc aatacatcgg accaggaaat tctgaataaa 600
atccaggaat cactgaagaa tgcgaagaaa ccgatcgtca tcaccggaca tgagatcatc 660
tcttttggcc tggaaaaaac ggtcacgcag ttcatttcta agaccaaact gcctatcacc 720
accctgaact tcggcaaatc tagcgtcgat gaagcgctgc cgagttttct gggtatctat 780
aatggtaccc tgtccgaacc gaacctgaaa gaattcgtcg aaagcgcgga ctttatcctg 840
atgctgggcg tgaaactgac ggatagctcc acaggcgcat ttacccacca tctgaacgag 900
aataaaatga tttccctgaa tatcgacgaa ggcaaaatct ttaacgagcg catccagaac 960
ttcgattttg aatctctgat tagttcgctg ctggatctgt ccgaaattga gtataaaggt 1020
aaatatattg ataaaaaaca ggaggatttt gtgccgtcta atgcgctgct gagtcaggat 1080
cgtctgtggc aagccgtaga aaacctgaca cagtctaatg aaacgattgt tgcggaacag 1140
ggaacttcat ttttcggcgc ctcatccatt tttctgaaat ccaaaagcca tttcattggc 1200
caaccgctgt gggggagtat tggttatacc tttccggcgg cgctgggttc acagattgca 1260
gataaggaat cacgccatct gctgtttatt ggtgacggca gcctgcagct gactgtccag 1320
gaactggggc tggcgatccg tgaaaaaatc aatccgattt gctttatcat caataacgac 1380
ggctacaccg tcgaacgcga aattcatgga ccgaatcaaa gttacaatga catcccgatg 1440
tggaactata gcaaactgcc ggaatccttt ggcgcgacag aggatcgcgt ggtgagtaaa 1500
attgtgcgta cggaaaacga atttgtgtcg gttatgaaag aagcgcaggc tgacccgaat 1560
cgcatgtatt ggattgaact gatcctggca aaagaaggcg caccgaaagt tctgaaaaag 1620
atggggaaac tgtttgcgga gcaaaataaa agctaaggat cc 1662
<210>8
<211>548
<212>PRT
<213>乳酸乳球菌
<400>8
Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly
1 5 10 15
Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu
20 25 30
Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn
35 40 45
Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys
50 55 60
Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val
65 70 75 80
Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile
85 90 95
Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His
100 105 110
His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu
115 120 125
Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val
130 135 140
Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val
145 150 155 160
Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro
165 170 175
Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln
180 185 190
Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro
195 200 205
Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr
210 215 220
Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn
225 230 235 240
Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile
245 250 255
Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser
260 265 270
Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr
275 280 285
Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn
290 295 300
Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe
305 310 315 320
Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys
325 330 335
Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala
340 345 350
Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln
355 360 365
Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380
Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu
385 390 395 400
Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile
405 410 415
Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430
Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn
435 440 445
Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu
450 455 460
Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr
465 470 475 480
Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser
485 490 495
Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala
500 505 510
Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys
515 520 525
Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu
530 535 540
Gln Asn Lys Ser
545
<210>9
<211>1164
<212>DNA
<213>大肠杆菌
<400>9
atgaacaactt taatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60
ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120
gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180
gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240
gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300
accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360
caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420
gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480
caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540
tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600
gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660
ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720
cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780
ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840
cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900
cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960
gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020
acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080
gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140
cgtatatacg aagccgcccg ctaa 1164
<210>10
<211>387
<212>PRT
<213>大肠杆菌
<400>10
Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
1 5 10 15
Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210>11
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物
<400>11
caccatggac aaacagtatc cggtacgcc 29
<210>12
<211>25
<212>DNA
<213>人造序列
<220>
<223>引物
<400>12
cgaagggcga tagctttacc aatcc 25
<210>13
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物
<400>13
caccatggct aactacttca atacactga 29
<210>14
<211>28
<212>DNA
<213>人造序列
<220>
<223>引物
<400>14
ccaggagaag gccttgagtg ttttctcc 28
<210>15
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物
<400>15
caccatgcct aagtaccgtt ccgccacca 29
<210>16
<211>26
<212>DNA
<213>人造序列
<220>
<223>引物
<400>16
cgcagcactg ctcttaaata ttcggc 26
<210>17
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物
<400>17
caccatgaac aactttaatc tgcacaccc 29
<210>18
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物
<400>18
caccatgaac aactttaatc tgcacaccc 29
<210>19
<211>45
<212>DNA
<213>人造序列
<220>
<223>引物
<400>19
gcatgcctta agaaaggagg ggggtcacat ggacaaacag tatcc 45
<210>20
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物
<400>20
atgcatttaa ttaattacag aatctgactc agatgcagc 39
<210>21
<211>45
<212>DNA
<213>人造序列
<220>
<223>引物
<400>21
gtcgacgcta gcaaaggagg gaatcaccat ggctaactac ttcaa 45
<210>22
<211>31
<212>DNA
<213>人造序列
<220>
<223>引物
<400>22
tctagattaa cccgcaacag caatacgttt c 31
<210>23
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物
<400>23
tctagaaaag gaggaataaa gtatgcctaa gtaccgttc 39
<210>24
<211>31
<212>DNA
<213>人造序列
<220>
<223>引物
<400>24
ggatccttat taacccccca gtttcgattt a 31
<210>25
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物
<400>25
ggatccaaag gaggctagac atatgtatac tgtggggga 39
<210>26
<211>31
<212>DNA
<213>人造序列
<220>
<223>引物
<400>26
gagctcttag cttttatttt gctccgcaaa c 31
<210>27
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物
<400>27
gagctcaaag gaggagcaag taatgaacaa ctttaatct 39
<210>28
<211>43
<212>DNA
<213>人造序列
<220>
<223>引物
<400>28
gaattcacta gtcctaggtt agcgggcggc ttcgtatata cgg 43
<210>29
<211>25
<212>DNA
<213>人造序列
<220>
<223>引物
<400>29
caacattagc gattttcttt tctct 25
<210>30
<211>45
<212>DNA
<213>人造序列
<220>
<223>引物
<400>30
catgaagctt actagtgggc ttaagttttg aaaataatga aaact 45
<210>31
<211>61
<212>DNA
<213>人造序列
<220>
<223>引物N110.2
<400>31
gagctcacta gtcaattgta agtaagtaaa aggaggtggg tcacatggac aaacagtatc 60
c 61
<210>32
<211>50
<212>DNA
<213>人造序列
<220>
<223>引物N111.2
<400>32
ggatccgatc gacttaagcc tcagcttaca gaatctgact cagatgcagc 50
<210>33
<211>44
<212>DNA
<213>人造序列
<220>
<223>引物N112.2
<400>33
gagctcctta agaaggaggt aatcaccatg gctaactact tcaa 44
<210>34
<211>51
<212>DNA
<213>人造序列
<220>
<223>引物N113.2
<400>34
ggatccgatc gagctagcgc ggccgcttaa cccgcaacag caatacgttt c 51
<210>35
<211>44
<212>DNA
<213>人造序列
<220>
<223>引物N114.2
<400>35
gagctcgcta gcaaggaggt ataaagtatg cctaagtacc gttc 44
<210>36
<211>52
<212>DNA
<213>人造序列
<220>
<223>引物N115.2
<400>36
ggatccgatc gattaattaa cctaaggtta ttaacccccc agtttcgatt ta 52
<210>37
<211>46
<212>DNA
<213>人造序列
<220>
<223>引物N116.2
<400>37
gagctcttaa ttaaaaggag gttagacata tgtatactgt ggggga 46
<210>38
<211>49
<212>DNA
<213>人造序列
<220>
<223>引物117.2
<400>38
ggatccagat ctcctaggac atgtttagct tttattttgc tccgcaaac 49
<210>39
<211>3883
<212>DNA
<213>大肠杆菌
<400>39
ctatattgct gaaggtacag gcgtttccat aactatttgc tcgcgttttt tactcaagaa 60
gaaaatgcca aatagcaaca tcaggcagac aatacccgaa attgcgaaga aaactgtctg 120
gtagcctgcg tggtcaaaga gtatcccagt cggcgttgaa agcagcacaa tcccaagcga 180
actggcaatt tgaaaaccaa tcagaaagat cgtcgacgac aggcgcttat caaagtttgc 240
cacgctgtat ttgaagacgg atatgacaca aagtggaacc tcaatggcat gtaacaactt 300
cactaatgaa ataatccagg ggttaacgaa cagcgcgcag gaaaggatac gcaacgccat 360
aatcacaact ccgataagta atgcattttt tggccctacc cgattcacaa agaaaggaat 420
aatcgccatg cacagcgctt cgagtaccac ctggaatgag ttgagataac catacaggcg 480
cgttcctaca tcgtgtgatt cgaataaacc tgaataaaag acaggaaaaa gttgttgatc 540
aaaaatgtta tagaaagacc acgtccccac aataaatatg acgaaaaccc agaagtttcg 600
atccttgaaa actgcgataa aatcctcttt ttttacccct cccgcatctg ccgctacgca 660
ctggtgatcc ttatctttaa aacgcatgtt gatcatcata aatacagcgc caaatagcga 720
gaccaaccag aagttgatat ggggactgat actaaaaaat atgccggcaa agaacgcgcc 780
aatagcatag ccaaaagatc cccaggcgcg cgctgttcca tattcgaaat gaaaatttcg 840
cgccattttt tcggtgaagc tatcaagcaa accgcatccc gccagatacc ccaagccaaa 900
aaatagcgcc cccagaatta gacctacaga aaaattgctt tgcagtaacg gttcataaac 960
gtaaatcata aacggtccgg tcaagaccag gatgaaactc atacaccaga tgagcggttt 1020
cttcagaccg agtttatcct gaacgatgcc gtagaacatc ataaatagaa tgctggtaaa 1080
ctggttgacc gaataaagtg tacctaattc cgtccctgtc aaccctagat gtcctttcag 1140
ccaaatagcg tataacgacc accacagcga ccaggaaata aaaaagagaa atgagtaact 1200
ggatgcaaaa cgatagtacg catttctgaa tggaatattc agtgccataa ttacctgcct 1260
gtcgttaaaa aattcacgtc ctatttagag ataagagcga cttcgccgtt tacttctcac 1320
tattccagtt cttgtcgaca tggcagcgct gtcattgccc ctttcgccgt tactgcaagc 1380
gctccgcaac gttgagcgag atcgataatt cgtcgcattt ctctctcatc tgtagataat 1440
cccgtagagg acagacctgt gagtaacccg gcaacgaacg catctcccgc ccccgtgcta 1500
tcgacacaat tcacagacat tccagcaaaa tggtgaactt gtcctcgata acagaccacc 1560
accccttctg cacctttagt caccaacagc atggcgatct catactcttt tgccagggcg 1620
catatatcct gatcgttctg tgtttttcca ctgataagtc gccattcttc ttccgagagc 1680
ttgacgacat ccgccagttg tagcgcctgc cgcaaacaca agcggagcaa atgctcgtct 1740
tgccatagat cttcacgaat attaggatcg aagctgacaa aacctccggc atgccggatc 1800
gccgtcatcg cagtaaatgc gctggtacgc gaaggctcgg cagacaacgc aattgaacag 1860
agatgtaacc attcgccatg tcgccagcag ggcaagtctg tcgtctctaa aaaaagatcg 1920
gcactggggc ggaccataaa cgtaaatgaa cgttcccctt gatcgttcag atcgacaagc 1980
accgtggatg tccggtgcca ttcatcttgc ttcagatacg tgatatcgac tccctcagtt 2040
agcagcgttc tttgcattaa cgcaccaaaa ggatcatccc ccacccgacc tataaaccca 2100
cttgttccgc ctaatctggc gattcccacc gcaacgttag ctggcgcgcc gccaggacaa 2160
ggcagtaggc gcccgtctga ttctggcaag agatctacga ccgcatcccc taaaacccat 2220
actttggctg acattttttt cccttaaatt catctgagtt acgcatagtg ataaacctct 2280
ttttcgcaaa atcgtcatgg atttactaaa acatgcatat tcgatcacaa aacgtcatag 2340
ttaacgttaa catttgtgat attcatcgca tttatgaaag taagggactt tatttttata 2400
aaagttaacg ttaacaattc accaaatttg cttaaccagg atgattaaaa tgacgcaatc 2460
tcgattgcat gcggcgcaaa acgccctagc aaaacttcat gagcaccggg gtaacacttt 2520
ctatccccat tttcacctcg cgcctcctgc cgggtggatg aacgatccaa acggcctgat 2580
ctggtttaac gatcgttatc acgcgtttta tcaacatcat ccgatgagcg aacactgggg 2640
gccaatgcac tggggacatg ccaccagcga cgatatgatc cactggcagc atgagcctat 2700
tgcgctagcg ccaggagacg ataatgacaa agacgggtgt ttttcaggta gtgctgtcga 2760
tgacaatggt gtcctctcac ttatctacac cggacacgtc tggctcgatg gtgcaggtaa 2820
tgacgatgca attcgcgaag tacaatgtct ggctaccagt cgggatggta ttcatttcga 2880
gaaacagggt gtgatcctca ctccaccaga aggaatcatg cacttccgcg atcctaaagt 2940
gtggcgtgaa gccgacacat ggtggatggt agtcggggcg aaagatccag gcaacacggg 3000
gcagatcctg ctttatcgcg gcagttcgtt gcgtgaatgg accttcgatc gcgtactggc 3060
ccacgctgat gcgggtgaaa gctatatgtg ggaatgtccg gactttttca gccttggcga 3120
tcagcattat ctgatgtttt ccccgcaggg aatgaatgcc gagggataca gttaccgaaa 3180
tcgctttcaa agtggcgtaa tacccggaat gtggtcgcca ggacgacttt ttgcacaatc 3240
cgggcatttt actgaacttg ataacgggca tgacttttat gcaccacaaa gctttttagc 3300
gaaggatggt cggcgtattg ttatcggctg gatggatatg tgggaatcgc caatgccctc 3360
aaaacgtgaa ggatgggcag gctgcatgac gctggcgcgc gagctatcag agagcaatgg 3420
caaacttcta caacgcccgg tacacgaagc tgagtcgtta cgccagcagc atcaatctgt 3480
ctctccccgc acaatcagca ataaatatgt tttgcaggaa aacgcgcaag cagttgagat 3540
tcagttgcag tgggcgctga agaacagtga tgccgaacat tacggattac agctcggcac 3600
tggaatgcgg ctgtatattg ataaccaatc tgagcgactt gttttgtggc ggtattaccc 3660
acacgagaat ttagacggct accgtagtat tcccctcccg cagcgtgaca cgctcgccct 3720
aaggatattt atcgatacat catccgtgga agtatttatt aacgacgggg aagcggtgat 3780
gagtagtcga atctatccgc agccagaaga acgggaactg tcgctttatg cctcccacgg 3840
agtggctgtg ctgcaacatg gagcactctg gctactgggt taa 3883
<210>40
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqF1
<400>40
tgttccaacc tgatcaccg 19
<210>41
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqF2
<400>41
ggaaaacagc aaggcgct 18
<210>42
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqF3
<400>42
cagctgaacc agtttgcc 18
<210>43
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqF4
<400>43
aaaataccag cgcctgtcc 19
<210>44
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqR1
<400>44
tgaatggcca ccatgttg 18
<210>45
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqR2
<400>45
gaggatctcc gccgcctg 18
<210>46
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqR3
<400>46
aggccgagca ggaagatc 18
<210>47
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N130SeqR4
<400>47
tgatcaggtt ggaacagcc 19
<210>48
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N131SeqF1
<400>48
aagaactgat cccacaggc 19
<210>49
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N131SeqF2
<400>49
atcctgtgcg gtatgttgc 19
<210>50
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N131Seqf3
<400>50
attgcgatgg tgaaagcg 18
<210>51
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N131SeqR1
<400>51
atggtgttgg caatcagcg 19
<210>52
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N131SeqR2
<400>52
gtgcttcggt gatggttt 18
<210>53
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N131SeqR3
<400>53
ttgaaaccgt gcgagtagc 19
<210>54
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqF1
<400>54
tattcactgc catctcgcg 19
<210>55
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqF2
<400>55
ccgtaagcag ctgttcct 18
<210>56
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqF3
<400>56
gctggaacaa tacgacgtta 20
<210>57
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqF4
<400>57
tgctctaccc aaccagcttc 20
<210>58
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqR1
<400>58
atggaaagac cagaggtgcc 20
<210>59
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqR2
<400>59
tgcctgtgtg gtacgaat 18
<210>60
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqR3
<400>60
tattacgcgg cagtgcact 19
<210>61
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N132SeqR4
<400>61
ggtgattttg tcgcagttag ag 22
<210>62
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqF1
<400>62
tcgaaattgt tgggtcgc 18
<210>63
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqF2
<400>63
ggtcacgcag ttcatttcta ag 22
<210>64
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqF3
<400>64
tgtggcaagc cgtagaaa 18
<210>65
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqF4
<400>65
aggatcgcgt ggtgagtaa 19
<210>66
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqR1
<400>66
gtagccgtcg ttattgatga 20
<210>67
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqR2
<400>67
gcagcgaact aatcagagat tc 22
<210>68
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqR3
<400>68
tggtccgatg tattggagg 19
<210>69
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N133SeqR4
<400>69
tctgccatat agctcgcgt 19
<210>70
<211>42
<212>DNA
<213>人造序列
<220>
<223>启动子1.6GI变体
<400>70
gcccttgaca atgccacatc ctgagcaaat aattcaacca ct 42
<210>71
<211>42
<212>DNA
<213>人造序列
<220>
<223>启动子1.5GI
<400>71
gcccttgact atgccacatc ctgagcaaat aattcaacca ct 42
<210>72
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物Scr1
<400>72
cctttctttg tgaatcgg 18
<210>73
<211>18
<212>DNA
<213>人造序列
<220>
<223>引物Scr2
<400>73
agaaacaggg tgtgatcc 18
<210>74
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物Scr3
<400>74
agtgatcatc acctgttgcc 20
<210>75
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物Scr4
<400>75
agcacggcga gagtcgacgg 20
<210>76
<211>672
<212>DNA
<213>酿酒酵母
<400>76
agttcgagtt tatcattatc aatactgcca tttcaaagaa tacgtaaata attaatagta 60
gtgattttcc taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta 120
catgcccaaa atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa 180
cagtttattc ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga 240
aaaaaaaaga atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc 300
cattctctta gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc 360
tcaatggagt gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca 420
tgtatctatc tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa 480
gctgaaaaaa aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta 540
tataaagacg gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt 600
ctacttttat agttagtctt ttttttagtt ttaaaacacc aagaacttag tttcgaataa 660
acacacataa ac 672
<210>77
<211>270
<212>DNA
<213>酿酒酵母
<400>77
gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 60
ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 120
tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 180
gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 240
cgaaggcttt aatttgcggc cggtacccaa 270
<210>78
<211>1716
<212>DNA
<213>枯草芽孢杆菌
<400>78
atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60
gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120
attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180
gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240
gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300
actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360
cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420
gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480
gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540
aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600
atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660
aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720
ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780
tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840
gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900
ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960
cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020
gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080
catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140
gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200
cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260
aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320
ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380
ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440
tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500
ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560
tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620
atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680
gaattcgggg aactcatgaa aacgaaagct ctctag 1716
<210>79
<211>643
<212>DNA
<213>酿酒酵母
<400>79
gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60
atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120
aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180
tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240
ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300
aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360
tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420
caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480
catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540
ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600
gtcatatata accataacca agtaatacat attcaaatct aga 643
<210>80
<211>1188
<212>DNA
<213>酿酒酵母
<400>80
atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60
acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120
ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180
tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240
gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300
ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360
ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420
gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480
ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540
actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600
agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660
aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720
ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780
ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840
aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900
tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960
agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020
gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080
caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140
tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 1188
<210>81
<211>760
<212>DNA
<213>酿酒酵母
<400>81
tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt tgtttccggg 60
tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat 120
accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata 180
ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg 240
gtacataacg aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc 300
actacccttt ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc 360
tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa 420
tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt 480
tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc 540
acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat 600
aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg 660
tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca 720
agctatacca agcatacaat caactatctc atatacaatg 760
<210>82
<211>316
<212>DNA
<213>酿酒酵母
<400>82
gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60
agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120
aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180
cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240
tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300
ggacaacacc tgtggt 316
<210>83
<211>1758
<212>DNA
<213>酿酒酵母
<400>83
atgggcttgt taacgaaagt tgctacatct agacaattct ctacaacgag atgcgttgca 60
aagaagctca acaagtactc gtatatcatc actgaaccta agggccaagg tgcgtcccag 120
gccatgcttt atgccaccgg tttcaagaag gaagatttca agaagcctca agtcggggtt 180
ggttcctgtt ggtggtccgg taacccatgt aacatgcatc tattggactt gaataacaga 240
tgttctcaat ccattgaaaa agcgggtttg aaagctatgc agttcaacac catcggtgtt 300
tcagacggta tctctatggg tactaaaggt atgagatact cgttacaaag tagagaaatc 360
attgcagact cctttgaaac catcatgatg gcacaacact acgatgctaa catcgccatc 420
ccatcatgtg acaaaaacat gcccggtgtc atgatggcca tgggtagaca taacagacct 480
tccatcatgg tatatggtgg tactatcttg cccggtcatc caacatgtgg ttcttcgaag 540
atctctaaaa acatcgatat cgtctctgcg ttccaatcct acggtgaata tatttccaag 600
caattcactg aagaagaaag agaagatgtt gtggaacatg catgcccagg tcctggttct 660
tgtggtggta tgtatactgc caacacaatg gcttctgccg ctgaagtgct aggtttgacc 720
attccaaact cctcttcctt cccagccgtt tccaaggaga agttagctga gtgtgacaac 780
attggtgaat acatcaagaa gacaatggaa ttgggtattt tacctcgtga tatcctcaca 840
aaagaggctt ttgaaaacgc cattacttat gtcgttgcaa ccggtgggtc cactaatgct 900
gttttgcatt tggtggctgt tgctcactct gcgggtgtca agttgtcacc agatgatttc 960
caaagaatca gtgatactac accattgatc ggtgacttca aaccttctgg taaatacgtc 1020
atggccgatt tgattaacgt tggtggtacc caatctgtga ttaagtatct atatgaaaac 1080
aacatgttgc acggtaacac aatgactgtt accggtgaca ctttggcaga acgtgcaaag 1140
aaagcaccaa gcctacctga aggacaagag attattaagc cactctccca cccaatcaag 1200
gccaacggtc acttgcaaat tctgtacggt tcattggcac caggtggagc tgtgggtaaa 1260
attaccggta aggaaggtac ttacttcaag ggtagagcac gtgtgttcga agaggaaggt 1320
gcctttattg aagccttgga aagaggtgaa atcaagaagg gtgaaaaaac cgttgttgtt 1380
atcagatatg aaggtccaag aggtgcacca ggtatgcctg aaatgctaaa gccttcctct 1440
gctctgatgg gttacggttt gggtaaagat gttgcattgt tgactgatgg tagattctct 1500
ggtggttctc acgggttctt aatcggccac attgttcccg aagccgctga aggtggtcct 1560
atcgggttgg tcagagacgg cgatgagatt atcattgatg ctgataataa caagattgac 1620
ctattagtct ctgataagga aatggctcaa cgtaaacaaa gttgggttgc acctccacct 1680
cgttacacaa gaggtactct atccaagtat gctaagttgg tttccaacgc ttccaacggt 1740
tgtgttttag atgcttga 1758
<210>84
<211>753
<212>DNA
<213>酿酒酵母
<400>84
gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60
gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120
acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180
caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240
aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300
gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360
cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420
ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480
agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540
aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600
ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660
gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720
ttaataatcc aaacaaacac acatattaca ata 753
<210>85
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N98SeqF1
<400>85
cgtgttagtc acatcaggac 20
<210>86
<211>24
<212>DNA
<213>人造序列
<220>
<223>引物N98SeqF2
<400>86
ggccatagca aaaatccaaa cagc 24
<210>87
<211>24
<212>DNA
<213>人造序列
<220>
<223>引物N98SeqF3
<400>87
ccacgatcaa tcatatcgaa cacg 24
<210>88
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N98SeqF4
<400>88
ggtttctgtc tctggtgacg 20
<210>89
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N99SeqR1
<400>89
gtctggtgat tctacgcgca ag 22
<210>90
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N99SeqR2
<400>90
catcgactgc attacgcaac tc 22
<210>91
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N99SeqR3
<400>91
cgatcgtcag aacaacatct gc 22
<210>92
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N99SeqR4
<400>92
ccttcagtgt tcgctgtcag 20
<210>93
<211>36
<212>DNA
<213>人造序列
<220>
<223>引物N136
<400>93
ccgcggatag atctgaaatg aataacaata ctgaca 36
<210>94
<211>65
<212>DNA
<213>人造序列
<220>
<223>引物N137
<400>94
taccaccgaa gttgatttgc ttcaacatcc tcagctctag atttgaatat gtattacttg 60
gttat 65
<210>95
<211>28
<212>DNA
<213>人造序列
<220>
<223>引物N138
<400>95
atgttgaagc aaatcaactt cggtggta 28
<210>96
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N139
<400>96
ttattggttt tctggtctca ac 22
<210>97
<211>57
<212>DNA
<213>人造序列
<220>
<223>引物N140
<400>97
aagttgagac cagaaaacca ataattaatt aatcatgtaa ttagttatgt cacgctt 57
<210>98
<211>30
<212>DNA
<213>人造序列
<220>
<223>引物N141
<400>98
gcggccgccc gcaaattaaa gccttcgagc 30
<210>99
<211>28
<212>DNA
<313>人造序列
<220>
<223>引物N142
<400>99
ggatccgcat gcttgcattt agtcgtgc 28
<210>100
<211>56
<212>DNA
<213>人造序列
<220>
<223>N143
<400>100
caggtaatcc cccacagtat acatcctcag ctattgtaat atgtgtgttt gtttgg 56
<210>101
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N144
<400>101
atgtatactg tgggggatta cc 22
<210>102
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N145
<400>102
ttagctttta ttttgctccg ca 22
<210>103
<211>57
<212>DNA
<213>人造序列
<220>
<223>引物N146
<400>103
tttgcggagc aaaataaaag ctaattaatt aagagtaagc gaatttctta tgattta 57
<210>104
<211>28
<212>DNA
<213>人造序列
<220>
<223>引物N147
<400>104
actagtacca caggtgttgt cctctgag 28
<210>105
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N151
<400>105
ctagagagct ttcgttttca tg 22
<210>106
<211>57
<212>DNA
<213>人造序列
<220>
<223>引物N152
<400>106
ctcatgaaaa cgaaagctct ctagttaatt aatcatgtaa ttagttatgt cacgctt 57
<210>107
<211>25
<212>DNA
<213>人造序列
<220>
<223>引物N155
<400>107
atggcaaaga agctcaacaa gtact 25
<210>108
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N156
<400>108
tcaagcatct aaaacacaac cg 22
<210>109
<211>57
<212>DNA
<213>人造序列
<220>
<223>引物N157
<400>109
aacggttgtg ttttagatgc ttgattaatt aagagtaagc gaatttctta tgattta 57
<210>110
<211>28
<212>DNA
<213>人造序列
<220>
<223>引物N158
<400>110
ggatcctttt ctggcaacca aacccata 28
<210>111
<211>56
<212>DNA
<213>人造序列
<220>
<223>引物N159
<400>111
cgagtacttg ttgagcttct ttgccatcct cagcgagata gttgattgta tgcttg 56
<210>112
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqF1
<400>112
gaaaacgtgg catcctctc 19
<210>113
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqF2
<400>113
gctgactggc caagagaaa 19
<210>114
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqF3
<400>114
tgtacttctc ccacggtttc 20
<210>115
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqF4
<400>115
agctacccaa tctctatacc ca 22
<210>116
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqF5
<400>116
cctgaagtct aggtccctat tt 22
<210>117
<211>19
<212>DNA
<213>人造序列
<220>
<223>N160SeqR1
<400>117
gcgtgaatgt aagcgtgac 19
<210>118
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqR2
<400>118
cgtcgtattg agccaagaac 20
<210>119
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqR3
<400>119
gcatcggaca acaagttcat 20
<210>120
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqR4
<400>120
tcgttcttga agtagtccaa ca 22
<210>121
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N160SeqR5
<400>121
tgagcccgaa agagaggat 19
<210>122
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF1
<400>122
acggtatacg gccttcctt 19
<210>123
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF2
<400>123
gggtttgaaa gctatgcagt 20
<210>124
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF3
<400>124
ggtggtatgt atactgccaa ca 22
<210>125
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF4
<400>125
ggtggtaccc aatctgtgat ta 22
<210>126
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF5
<400>126
cggtttgggt aaagatgttg 20
<210>127
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqF6
<400>127
aaacgaaaat tcttattctt ga 22
<210>128
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqR1
<400>128
tcgttttaaa acctaagagt ca 22
<210>129
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqR2
<400>129
ccaaaccgta acccatcag 19
<210>130
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqR3
<400>130
cacagattgg gtaccacca 19
<210>131
<211>20
<212>DNA
<213>人造序列
<220>
<223>引物N161Seqr4
<400>131
accacaagaa ccaggacctg 20
<210>132
<211>19
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqR5
<400>132
catagctttc aaacccgct 19
<210>133
<211>22
<212>DNA
<213>人造序列
<220>
<223>引物N161SeqR6
<400>133
cgtataccgt tgctcattagag 22
<210>134
<211>23
<212>DNA
<213>人造序列
<220>
<223>引物N162
<400>134
atgttgacaa aagcaacaaa aga 23
<210>135
<211>38
<212>DNA
<213>人造序列
<220>
<223>引物N189
<400>135
atccgcggat agatctagtt cgagtttatc attatcaa 38
<210>136
<211>53
<212>DNA
<213>人造序列
<220>
<223>引物N190.1
<400>136
ttcttttgtt gcttttgtca acatcctcag cgtttatgtg tgtttattcg aaa 53
<210>137
<211>38
<212>DNA
<213>人造序列
<220>
<223>引物N176
<400>137
atccgcggat agatctatta gaagccgccg agcgggcg 38
<210>138
<211>31
<212>DNA
<213>人造序列
<220>
<223>引物N177
<400>138
atcctcagct tttctccttg acgttaaagt a 31
<210>139
<211>477
<212>PRT
<213>大肠杆菌
<400>139
Met Thr Gln Ser Arg Leu His Ala Ala Gln Asn Ala Leu Ala Lys Leu
1 5 10 15
His Glu His Arg Gly Asn Thr Phe Tyr Pro His Phe His Leu Ala Pro
20 25 30
Pro Ala Gly Trp Met Asn Asp Pro Asn Gly Leu Ile Trp Phe Asn Asp
35 40 45
Arg Tyr His Ala Phe Tyr Gln His His Pro Met Ser Glu His Trp Gly
50 55 60
Pro Met His Trp Gly His Ala Thr Ser Asp Asp Met Ile His Trp Gln
65 70 75 80
His Glu Pro Ile Ala Leu Ala Pro Gly Asp Asp Asn Asp Lys Asp Gly
85 90 95
Cys Phe Ser Gly Ser Ala Val Asp Asp Asn Gly Val Leu Ser Leu Ile
100 105 110
Tyr Thr Gly His Val Trp Leu Asp Gly Ala Gly Asn Asp Asp Ala Ile
115 120 125
Arg Glu Val Gln Cys Leu Ala Thr Ser Arg Asp Gly Ile His Phe Glu
130 135 140
Lys Gln Gly Val Ile Leu Thr Pro Pro Glu Gly Ile Met His Phe Arg
145 150 155 160
Asp Pro Lys Val Trp Arg Glu Ala Asp Thr Trp Trp Met Val Val Gly
165 170 175
Ala Lys Asp Pro Gly Asn Thr Gly Gln Ile Leu Leu Tyr Arg Gly Ser
180 185 190
Ser Leu Arg Glu Trp Thr Phe Asp Arg Val Leu Ala His Ala Asp Ala
195 200 205
Gly Glu Ser Tyr Met Trp Glu Cys Pro Asp Phe Phe Ser Leu Gly Asp
210 215 220
Gln His Tyr Leu Met Phe Ser Pro Gln Gly Met Asn Ala Glu Gly Tyr
225 230 235 240
Ser Tyr Arg Asn Arg Phe Gln Ser Gly Val Ile Pro Gly Met Trp Ser
245 250 255
Pro Gly Arg Leu Phe Ala Gln Ser Gly His Phe Thr Glu Leu Asp Asn
260 265 270
Gly His Asp Phe Tyr Ala Pro Gln Ser Phe Leu Ala Lys Asp Gly Arg
275 280 285
Arg Ile Val Ile Gly Trp Met Asp Met Trp Glu Ser Pro Met Pro Ser
290 295 300
Lys Arg Glu Gly Trp Ala Gly Cys Met Thr Leu Ala Arg Glu Leu Ser
305 310 315 320
Glu Ser Asn Gly Lys Leu Leu Gln Arg Pro Val His Glu Ala Glu Ser
325 330 335
Leu Arg Gln Gln His Gln Ser Val Ser Pro Arg Thr Ile Ser Asn Lys
340 345 350
Tyr Val Leu Gln Glu Asn Ala Gln Ala Val Glu Ile Gln Leu Gln Trp
355 360 365
Ala Leu Lys Asn Ser Asp Ala Glu His Tyr Gly Leu Gln Leu Gly Thr
370 375 380
Gly Met Arg Leu Tyr Ile Asp Asn Gln Ser Glu Arg Leu Val Leu Trp
385 390 395 400
Arg Tyr Tyr Pro His Glu Asn Leu Asp Gly Tyr Arg Ser Ile Pro Leu
405 410 415
Pro Gln Arg Asp Thr Leu Ala Leu Arg Ile Phe Ile Asp Thr Ser Ser
420 425 430
Val Glu Val Phe Ile Asn Asp Gly Glu Ala Val Met Ser Ser Arg Ile
435 440 445
Tyr Pro Gln Pro Glu Glu Arg Glu Leu Ser Leu Tyr Ala Ser His Gly
450 455 460
Val Ala Val Leu Gln His Gly Ala Leu Trp Leu Leu Gly
465 470 475
<210>140
<211>304
<212>PRT
<213>大肠杆菌
<400>140
Met Ser Ala Lys Val Trp Val Leu Gly Asp Ala Val Val Asp Leu Leu
1 5 10 15
Pro Glu Ser Asp Gly Arg Leu Leu Pro Cys Pro Gly Gly Ala Pro Ala
20 25 30
Asn Val Ala Val Gly Ile Ala Arg Leu Gly Gly Thr Ser Gly Phe Ile
35 40 45
Gly Arg Val Gly Asp Asp Pro Phe Gly Ala Leu Met Gln Arg Thr Leu
50 55 60
Leu Thr Glu Gly Val Asp Ile Thr Tyr Leu Lys Gln Asp Glu Trp His
65 70 75 80
Arg Thr Ser Thr Val Leu Val Asp Leu Asn Asp Gln Gly Glu Arg Ser
85 90 95
Phe Thr Phe Met Val Arg Pro Ser Ala Asp Leu Phe Leu Glu Thr Thr
100 105 110
Asp Leu Pro Cys Trp Arg His Gly Glu Trp Leu His Leu Cys Ser Ile
115 120 125
Ala Leu Ser Ala Glu Pro Ser Arg Thr Ser Ala Phe Thr Ala Met Thr
130 135 140
Ala Ile Arg His Ala Gly Gly Phe Val Ser Phe Asp Pro Asn Ile Arg
145 150 155 160
Glu Asp Leu Trp Gln Asp Glu His Leu Leu Arg Leu Cys Leu Arg Gln
165 170 175
Ala Leu Gln Leu Ala Asp Val Val Lys Leu Ser Glu Glu Glu Trp Arg
180 185 190
Leu Ile Ser Gly Lys Thr Gln Asn Asp Gln Asp Ile Cys Ala Leu Ala
195 200 205
Lys Glu Tyr Glu Ile Ala Met Leu Leu Val Thr Lys Gly Ala Glu Gly
210 215 220
Val Val Val Cys Tyr Arg Gly Gln Val His His Phe Ala Gly Met Ser
225 230 235 240
Val Asn Cys Val Asp Ser Thr Gly Ala Gly Asp Ala Phe Val Ala Gly
245 250 255
Leu Leu Thr Gly Leu Ser Ser Thr Gly Leu Ser Thr Asp Glu Arg Glu
260 265 270
Met Arg Arg Ile Ile Asp Leu Ala Gln Arg Cys Gly Ala Leu Ala Val
275 280 285
Thr Ala Lys Gly Ala Met Thr Ala Leu Pro Cys Arg Gln Glu Leu Glu
290 295 300
<210>141
<211>415
<212>PRT
<213>大肠杆菌
<400>141
Met Ala Leu Asn Ile Pro Phe Arg Asn Ala Tyr Tyr Arg Phe Ala Ser
1 5 10 15
Ser Tyr Ser Phe Leu Phe Phe Ile Ser Trp Ser Leu Trp Trp Ser Leu
20 25 30
Tyr Ala Ile Trp Leu Lys Gly His Leu Gly Leu Thr Gly Thr Glu Leu
35 40 45
Gly Thr Leu Tyr Ser Val Asn Gln Phe Thr Ser Ile Leu Phe Met Met
50 55 60
Phe Tyr Gly Ile Val Gln Asp Lys Leu Gly Leu Lys Lys Pro Leu Ile
65 70 75 80
Trp Cys Met Ser Phe Ile Leu Val Leu Thr Gly Pro Phe Met Ile Tyr
85 90 95
Val Tyr Glu Pro Leu Leu Gln Ser Asn Phe Ser Val Gly Leu Ile Leu
100 105 110
Gly Ala Leu Phe Phe Gly Leu Gly Tyr Leu Ala Gly Cys Gly Leu Leu
115 120 125
Asp Ser Phe Thr Glu Lys Met Ala Arg Asn Phe His Phe Glu Tyr Gly
130 135 140
Thr Ala Arg Ala Trp Gly Ser Phe Gly Tyr Ala Ile Gly Ala Phe Phe
145 150 155 160
Ala Gly Ile Phe Phe Ser Ile Ser Pro His Ile Asn Phe Trp Leu Val
165 170 175
Ser Leu Phe Gly Ala Val Phe Met Met Ile Asn Met Arg Phe Lys Asp
180 185 190
Lys Asp His Gln Cys Val Ala Ala Asp Ala Gly Gly Val Lys Lys Glu
195 200 205
Asp Phe Ile Ala Val Phe Lys Asp Arg Asn Phe Trp Val Phe Val Ile
210 215 220
Phe Ile Val Gly Thr Trp Ser Phe Tyr Asn Ile Phe Asp Gln Gln Leu
225 230 235 240
Phe Pro Val Phe Tyr Ser Gly Leu Phe Glu Ser His Asp Val Gly Thr
245 250 255
Arg Leu Tyr Gly Tyr Leu Asn Ser Phe Gln Val Val Leu Glu Ala Leu
260 265 270
Cys Met Ala Ile Ile Pro Phe Phe Val Asn Arg Val Gly Pro Lys Asn
275 280 285
Ala Leu Leu Ile Gly Val Val Ile Met Ala Leu Arg Ile Leu Ser Cys
290 295 300
Ala Leu Phe Val Asn Pro Trp Ile Ile Ser Leu Val Lys Leu Leu His
305 310 315 320
Ala Ile Glu Val Pro Leu Cys Val Ile Ser Val Phe Lys Tyr Ser Val
325 330 335
Ala Asn Phe Asp Lys Arg Leu Ser Ser Thr Ile Phe Leu Ile Gly Phe
340 345 350
Gln Ile Ala Ser Ser Leu Gly Ile Val Leu Leu Ser Thr Pro Thr Gly
355 360 365
Ile Leu Phe Asp His Ala Gly Tyr Gln Thr Val Phe Phe Ala Ile Ser
370 375 380
Gly Ile Val Cys Leu Met Leu Leu Phe Gly Ile Phe Phe Leu Ser Lys
385 390 395 400
Lys Arg Glu Gln Ile Val Met Glu Thr Pro Val Pro Ser Ala Ile
405 410 415
<210>142
<211>6341
<212>DNA
<213>人造序列
<220>
<223>质粒pFP988DssPspac
<400>142
gatccaagtt taaactgtac actagatatt tcttctccgc ttaaatcatc aaagaaatct 60
ttatcacttg taaccagtcc gtccacatgt cgaattgcat ctgaccgaat tttacgtttc 120
cctgaataat tctcatcaat cgtttcatca attttatctt tatactttat attttgtgcg 180
ttaatcaaat cataattttt atatgtttcc tcatgattta tgtctttatt attatagttt 240
ttattctctc tttgattatg tctttgtatc ccgtttgtat tacttgatcc tttaactctg 300
gcaaccctca aaattgaatg agacatgcta cacctccgga taataaatat atataaacgt 360
atatagattt cataaagtct aacacactag acttatttac ttcgtaatta agtcgttaaa 420
ccgtgtgctc tacgaccaaa actataaaac ctttaagaac tttctttttt tacaagaaaa 480
aagaaattag ataaatctct catatctttt attcaataat cgcatccgat tgcagtataa 540
atttaacgat cactcatcat gttcatattt atcagagctc gtgctataat tatactaatt 600
ttataaggag gaaaaaatat gggcattttt agtatttttg taatcagcac agttcattat 660
caaccaaaca aaaaataagt ggttataatg aatcgttaat aagcaaaatt catataacca 720
aattaaagag ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt 780
caaaacataa tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct 840
ttgaaatcgg ctcaggaaaa ggccatttta cccttgaatt agtaaagagg tgtaatttcg 900
taactgccat tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc 960
acgataattt ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat 1020
cctataaaat atatggtaat ataccttata acataagtac ggatataata cgcaaaattg 1080
tttttgatag tatagctaat gagatttatt taatcgtgga atacgggttt gctaaaagat 1140
tattaaatac aaaacgctca ttggcattac ttttaatggc agaagttgat atttctatat 1200
taagtatggt tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca 1260
gattaagtag aaaaaaatca agaatatcac acaaagataa acaaaagtat aattatttcg 1320
ttatgaaatg ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt 1380
ccttaaaaca tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc 1440
ttttcaatag ctataaatta tttaataagt aagttaaggg atgcagttca tcgatgaagg 1500
caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg 1560
ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga 1620
atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca 1680
aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat 1740
gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa 1800
cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct 1860
gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta 1920
catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg 1980
ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc 2040
tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt 2100
ctacgcagac aaacaatcaa cgtttgcgcc aagcttgcat gcgagagtag ggaactgcca 2160
ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 2220
tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga 2280
agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta 2340
agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tttgtttatt 2400
tttctaaata cattcaaata tgtatccgct catgctccgg atctgcatcg caggatgctg 2460
ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt 2520
gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag 2580
taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg 2640
tatcattacc cccatgaaca gaaattcccc cttacacgga ggcatcaagt gaccaaacag 2700
gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag 2760
aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac 2820
gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 2880
cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2940
gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 3000
cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 3060
gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 3120
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3180
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3240
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3300
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3360
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3420
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3480
cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3540
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3600
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 3660
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3720
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 3780
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 3840
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3900
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3960
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4020
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4080
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4140
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4200
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4260
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4320
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4380
ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 4440
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 4500
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 4560
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4620
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4680
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 4740
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 4800
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 4860
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4920
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 4980
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 5040
cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 5100
ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5160
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 5220
aagcccgtca gggcgcgtca gcgggtgttc atgtgcgtaa ctaacttgcc atcttcaaac 5280
aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg 5340
aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca 5400
ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac 5460
ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa 5520
aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc 5580
ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac 5640
ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt 5700
tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc 5760
gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa 5820
gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat 5880
ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca 5940
tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac 6000
ggaaaaacgt atcaaaatgt acagaattcg agctctcgag taattctaca cagcccagtc 6060
cagactattc ggcactgaaa ttatgggtga agtggtcaag acctcactag gcaccttaaa 6120
aatagcgcac cctgaagaag atttatttga ggtagccctt gcctacctag cttccaagaa 6180
agatatccta acagcacaag agcggaaaga tgttttgttc tacatccaga acaacctctg 6240
ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt atctacaagg tgtggcataa 6300
tgtgtggaat tgtgagcgct cacaattaag cttgaattcc c 6341
<210>143
<211>6221
<212>DNA
<213>人造序列
<220>
<223>质粒pFP988DssPgroE
<400>143
tcgagagcta ttgtaacata atcggtacgg gggtgaaaaa gctaacggaa aagggagcgg 60
aaaagaatga tgtaagcgtg aaaaattttt tatcttatca cttgaaattg gaagggagat 120
tctttattat aagaattgtg gaattgtgag cggataacaa ttcccaatta aaggaggaaa 180
ctagtggatc caagtttaaa ctgtacacta gatatttctt ctccgcttaa atcatcaaag 240
aaatctttat cacttgtaac cagtccgtcc acatgtcgaa ttgcatctga ccgaatttta 300
cgtttccctg aataattctc atcaatcgtt tcatcaattt tatctttata ctttatattt 360
tgtgcgttaa tcaaatcata atttttatat gtttcctcat gatttatgtc tttattatta 420
tagtttttat tctctctttg attatgtctt tgtatcccgt ttgtattact tgatccttta 480
actctggcaa ccctcaaaat tgaatgagac atgctacacc tccggataat aaatatatat 540
aaacgtatat agatttcata aagtctaaca cactagactt atttacttcg taattaagtc 600
gttaaaccgt gtgctctacg accaaaacta taaaaccttt aagaactttc tttttttaca 660
agaaaaaaga aattagataa atctctcata tcttttattc aataatcgca tccgattgca 720
gtataaattt aacgatcact catcatgttc atatttatca gagctcgtgc tataattata 780
ctaattttat aaggaggaaa aaatatgggc atttttagta tttttgtaat cagcacagtt 840
cattatcaac caaacaaaaa ataagtggtt ataatgaatc gttaataagc aaaattcata 900
taaccaaatt aaagagggtt ataatgaacg agaaaaatat aaaacacagt caaaacttta 960
ttacttcaaa acataatata gataaaataa tgacaaatat aagattaaat gaacatgata 1020
atatctttga aatcggctca ggaaaaggcc attttaccct tgaattagta aagaggtgta 1080
atttcgtaac tgccattgaa atagaccata aattatgcaa aactacagaa aataaacttg 1140
ttgatcacga taatttccaa gttttaaaca aggatatatt gcagtttaaa tttcctaaaa 1200
accaatccta taaaatatat ggtaatatac cttataacat aagtacggat ataatacgca 1260
aaattgtttt tgatagtata gctaatgaga tttatttaat cgtggaatac gggtttgcta 1320
aaagattatt aaatacaaaa cgctcattgg cattactttt aatggcagaa gttgatattt 1380
ctatattaag tatggttcca agagaatatt ttcatcctaa acctaaagtg aatagctcac 1440
ttatcagatt aagtagaaaa aaatcaagaa tatcacacaa agataaacaa aagtataatt 1500
atttcgttat gaaatgggtt aacaaagaat acaagaaaat atttacaaaa aatcaattta 1560
acaattcctt aaaacatgca ggaattgacg atttaaacaa tattagcttt gaacaattct 1620
tatctctttt caatagctat aaattattta ataagtaagt taagggatgc agttcatcga 1680
tgaaggcaac tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga 1740
taaaggccac aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg 1800
cgaagaatct ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga 1860
aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct 1920
cggtatgatt gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc 1980
atctaacaca gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg 2040
gtacctgttc actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga 2100
tatttacatg cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa 2160
aactggcctt gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca 2220
cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag 2280
aggattctac gcagacaaac aatcaacgtt tgcgccaagc ttgcatgcga gagtagggaa 2340
ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 2400
gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg 2460
ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc 2520
aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actctttttg 2580
tttatttttc taaatacatt caaatatgta tccgctcatg ctccggatct gcatcgcagg 2640
atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc 2700
ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg 2760
ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt 2820
catcggtatc attaccccca tgaacagaaa ttccccctta cacggaggca tcaagtgacc 2880
aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt 2940
ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac 3000
gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac 3060
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 3120
agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 3180
cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 3240
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3300
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3360
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3420
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3480
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3540
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3600
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3660
tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt 3720
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3780
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3840
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3900
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3960
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4020
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4080
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4140
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4200
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 4260
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 4320
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4380
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4440
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4500
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4560
ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4620
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4680
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4740
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 4800
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 4860
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 4920
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4980
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 5040
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 5100
gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 5160
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 5220
catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 5280
ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa 5340
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 5400
gcagacaagc ccgtcagggc gcgtcagcgg gtgttcatgt gcgtaactaa cttgccatct 5460
tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa ggagacatga 5520
acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac taccgcactg 5580
ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc atataaggaa 5640
acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga acagcaaaaa 5700
aatgaaaaat atcaagttcc tgaattcgat tcgtccacaa ttaaaaatat ctcttctgca 5760
aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac 5820
tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc ggatgacaca 5880
tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct 5940
ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct aaaagaccaa 6000
acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg tttattctac 6060
actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca agttaacgta 6120
tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc aatctttgac 6180
ggtgacggaa aaacgtatca aaatgtacag aattcgagct c 6221
<210>144
<211>40
<212>DNA
<213>人造序列
<220>
<223>引物T-budB(BamHI)
<400>144
agatagatgg atccggaggt gggtcacatg gacaaacagt 40
<210>145
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物B-kivD(BamHI)
<400>145
ctctagagga tccagactcc taggacatg 29
<210>146
<211>6039
<212>DNA
<213>人造序列
<220>
<223>载体片段pFP988Dss
<400>146
gatccaagtt taaactgtac actagatatt tcttctccgc ttaaatcatc aaagaaatct 60
ttatcacttg taaccagtcc gtccacatgt cgaattgcat ctgaccgaat tttacgtttc 120
cctgaataat tctcatcaat cgtttcatca attttatctt tatactttat attttgtgcg 180
ttaatcaaat cataattttt atatgtttcc tcatgattta tgtctttatt attatagttt 240
ttattctctc tttgattatg tctttgtatc ccgtttgtat tacttgatcc tttaactctg 300
gcaaccctca aaattgaatg agacatgcta cacctccgga taataaatat atataaacgt 360
atatagattt cataaagtct aacacactag acttatttac ttcgtaatta agtcgttaaa 420
ccgtgtgctc tacgaccaaa actataaaac ctttaagaac tttctttttt tacaagaaaa 480
aagaaattag ataaatctct catatctttt attcaataat cgcatccgat tgcagtataa 540
atttaacgat cactcatcat gttcatattt atcagagctc gtgctataat tatactaatt 600
ttataaggag gaaaaaatat gggcattttt agtatttttg taatcagcac agttcattat 660
caaccaaaca aaaaataagt ggttataatg aatcgttaat aagcaaaatt catataacca 720
aattaaagag ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt 780
caaaacataa tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct 840
ttgaaatcgg ctcaggaaaa ggccatttta cccttgaatt agtaaagagg tgtaatttcg 900
taactgccat tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc 960
acgataattt ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat 1020
cctataaaat atatggtaat ataccttata acataagtac ggatataata cgcaaaattg 1080
tttttgatag tatagctaat gagatttatt taatcgtgga atacgggttt gctaaaagat 1140
tattaaatac aaaacgctca ttggcattac ttttaatggc agaagttgat atttctatat 1200
taagtatggt tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca 1260
gattaagtag aaaaaaatca agaatatcac acaaagataa acaaaagtat aattatttcg 1320
ttatgaaatg ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt 1380
ccttaaaaca tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc 1440
ttttcaatag ctataaatta tttaataagt aagttaaggg atgcagttca tcgatgaagg 1500
caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg 1560
ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga 1620
atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca 1680
aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat 1740
gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa 1800
cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct 1860
gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta 1920
catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg 1980
ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc 2040
tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt 2100
ctacgcagac aaacaatcaa cgtttgcgcc aagcttgcat gcgagagtag ggaactgcca 2160
ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 2220
tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga 2280
agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta 2340
agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tttgtttatt 2400
tttctaaata cattcaaata tgtatccgct catgctccgg atctgcatcg caggatgctg 2460
ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt 2520
gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag 2580
taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg 2640
tatcattacc cccatgaaca gaaattcccc cttacacgga ggcatcaagt gaccaaacag 2700
gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag 2760
aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac 2820
gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 2880
cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2940
gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 3000
cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 3060
gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 3120
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3180
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3240
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3300
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3360
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3420
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3480
cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3540
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3600
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 3660
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3720
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 3780
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 3840
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3900
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3960
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4020
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4080
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4140
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4200
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4260
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4320
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4380
ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 4440
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 4500
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 4560
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4620
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4680
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 4740
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 4800
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 4860
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4920
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 4980
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 5040
cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 5100
ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5160
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 5220
aagcccgtca gggcgcgtca gcgggtgttc atgtgcgtaa ctaacttgcc atcttcaaac 5280
aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg 5340
aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca 5400
ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac 5460
ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa 5520
aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc 5580
ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac 5640
ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt 5700
tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc 5760
gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa 5820
gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat 5880
ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca 5940
tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac 6000
ggaaaaacgt atcaaaatgt acagaattcg agctctcga 6039
<210>147
<211>44
<212>DNA
<213>人造序列
<220>
<223>引物T-grcE(XhoI)
<400>147
agatagatct cgagagctat tgtaacataa tcggtacggg ggtg 44
<210>148
<211>51
<212>DNA
<213>人造序列
<220>
<223>引物B-groEL(SpeI BamH1)
<400>148
attatgtcag gatccactag tttcctcctt taattgggaa ttgttatccg c 51
<210>149
<211>30
<212>DNA
<213>人造序列
<220>
<223>引物T-groEL
<400>149
agctattgta acataatcgg tacgggggtg 30
<210>150
<211>45
<212>DNA
<213>人造序列
<220>
<223>引物T-ilvCB.s.(BamHI)
<400>150
acattgatgg atcccataac aagggagaga ttgaaatggt aaaag 45
<210>151
<211>47
<212>DNA
<213>人造序列
<220>
<223>引物B-ilvCB.s.(SpeIBamHI)
<400>151
tagacaacgg atccactagt ttaattttgc gcaacggaga ccaccgc 47
<210>152
<211>47
<212>DNA
<213>人造序列
<220>
<223>引物T-BD64(DraIII)
<400>152
ttaccgtgga ctcaccgagt gggtaactag cctcgccgga aagagcg 47
<210>153
<211>48
<212>DNA
<213>人造序列
<220>
<223>引物B-BD64(DraIII)
<400>153
tcacagttaa gacacctggt gccgttaatg cgccatgaca gccatgat 48
<210>154
<211>49
<212>DNA
<213>人造序列
<220>
<223>引物T-lacIq(DraIII)
<400>154
acagatagat caccaggtgc aagctaattc cggtggaaac gaggtcatc 49
<210>155
<211>48
<212>DNA
<213>人造序列
<220>
<223>引物B-lacIq(DraIII)
<400>155
acagtacgat acacggggtg tcactgcccg ctttccagtc gggaaacc 48
<210>156
<211>49
<212>DNA
<213>人造序列
<220>
<223>引物T-groE(DraIII)
<400>156
tcggattacg caccccgtga gctattgtaa cataatcggt acgggggtg 49
<210>157
<211>48
<212>DNA
<213>人造序列
<220>
<223>引物B-B.s.ilvC(DraIII)
<400>157
ctgctgatct cacaccgtgt gttaattttg cgcaacggag accaccgc 48
<210>158
<211>1221
<212>DNA
<213>丙醇丁酮梭菌
<400>158
cacacggtgt aaataataat ctaaacagga ggggttaaaa tggttgattt cgaatattca 60
ataccaacta gaattttttt cggtaaagat aagataaatg tacttggaag agagcttaaa 120
aaatatggtt ctaaagtgct tatagtttat ggtggaggaa gtataaagag aaatggaata 180
tatgataaag ctgtaagtat acttgaaaaa aacagtatta aattttatga acttgcagga 240
gtagagccaa atccaagagt aactacagtt gaaaaaggag ttaaaatatg tagagaaaat 300
ggagttgaag tagtactagc tataggtgga ggaagtgcaa tagattgcgc aaaggttata 360
gcagcagcat gtgaatatga tggaaatcca tgggatattg tgttagatgg ctcaaaaata 420
aaaagggtgc ttcctatagc tagtatatta accattgctg caacaggatc agaaatggat 480
acgtgggcag taataaataa tatggataca aacgaaaaac taattgcggc acatccagat 540
atggctccta agttttctat attagatcca acgtatacgt ataccgtacc taccaatcaa 600
acagcagcag gaacagctga tattatgagt catatatttg aggtgtattt tagtaataca 660
aaaacagcat atttgcagga tagaatggca gaagcgttat taagaacttg tattaaatat 720
ggaggaatag ctcttgagaa gccggatgat tatgaggcaa gagccaatct aatgtgggct 780
tcaagtcttg cgataaatgg acttttaaca tatggtaaag acactaattg gagtgtacac 840
ttaatggaac atgaattaag tgcttattac gacataacac acggcgtagg gcttgcaatt 900
ttaacaccta attggatgga gtatatttta aataatgata cagtgtacaa gtttgttgaa 960
tatggtgtaa atgtttgggg aatagacaaa gaaaaaaatc actatgacat agcacatcaa 1020
gcaatacaaa aaacaagaga ttactttgta aatgtactag gtttaccatc tagactgaga 1080
gatgttggaa ttgaagaaga aaaattggac ataatggcaa aggaatcagt aaagcttaca 1140
ggaggaacca taggaaacct aagaccagta aacgcctccg aagtcctaca aatattcaaa 1200
aaatctgtgt aacaccgagt g 1221
<210>159
<211>54
<212>DNA
<213>人造序列
<220>
<223>引物T-bdhB(DraIII)
<400>159
tcgatagcat acacacggtg gttaacaaag gaggggttaa aatggttgat ttcg 54
<210>160
<211>91
<212>DNA
<213>人造序列
<220>
<223>引物B-bdhB(rrnBT1DraIII)
<400>160
atctacgcac tcggtgataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat 60
cttacacaga ttttttgaat atttgtagga c 91
<210>161
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物LDH EcoRV F
<400>161
gacgtcatga ccacccgccg atccctttt 29
<210>162
<211>30
<212>DNA
<213>人造序列
<220>
<223>引物LDH AatIIR
<400>162
gatatccaac accagcgacc gacgtattac 30
<210>163
<211>47
<212>DNA
<213>人造序列
<220>
<223>引物Cm F
<400>163
atttaaatct cgagtagagg atcccaacaa acgaaaattg gataaag 47
<210>164
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物Cm R
<400>164
acgcgttatt ataaaagcca gtcattagg 29
<210>165
<211>58
<212>DNA
<213>人造序列
<220>
<223>引物P11 F-StuI
<400>165
cctagcgcta tagttgttga cagaatggac atactatgat atattgttgc tatagcga 58
<210>166
<211>62
<212>DNA
<213>人造序列
<220>
<223>引物P11 R-SpeI
<400>166
ctagtcgcta tagcaacaat atatcatagt atgtccattc tgtcaacaac tatagcgcta 60
gg 62
<210>167
<211>38
<212>DNA
<213>人造序列
<220>
<223>引物PldhL F-HindIII
<400>167
aagcttgtcg acaaaccaac attatgacgt gtctgggc 38
<210>168
<211>28
<212>DNA
<213>人造序列
<220>
<223>引物PldhL R-BamHI
<400>168
ggatcctcat cctctcgtag tgaaaatt 28
<210>169
<211>36
<212>DNA
<213>人造序列
<220>
<223>引物F-bdhB-AvrII
<400>169
ttcctaggaa ggaggtggtt aaaatggttg atttcg 36
<210>170
<211>29
<212>DNA
<213>人造序列
<220>
<223>引物R-bdhB-BamHI
<400>170
ttggatcctt acacagattt tttgaatat 29
<210>171
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物F-ilvC(B.s.)-AflII
<400>171
aacttaagaa ggaggtgatt gaaatggtaa aagtatatt 39
<210>172
<211>32
<212>DNA
<213>人造序列
<220>
<223>引物R-ilvC(B.s.)-NotI
<400>172
aagcggccgc ttaattttgc gcaacggaga cc 32
<210>173
<211>30
<212>DNA
<213>人造序列
<220>
<223>引物F-PnisA(HindIII)
<400>173
ttaagcttga catacttgaa tgacctagtc 30
<210>174
<211>39
<212>DNA
<213>人造序列
<220>
<223>引物R-PnisA(SpeI BamHI)
<400>174
ttggatccaa actagtataa tttattttgt agttccttc 39
<210>175
<211>38
<212>DNA
<213>人造序列
<220>
<223>引物N191
<400>175
atccgcggat agatctccca ttaccgacat ttgggcgc 38
<210>176
<211>31
<212>DNA
<213>人造序列
<220>
<223>引物N192
<400>176
atcctcagcg atgattgatt gattgattgt a 31
<210>177
<211>6509
<212>DNA
<213>人造序列
<220>
<223>载体pFP988
<400>177
tcgaggcccc gcacatacga aaagactggc tgaaaacatt gagcctttga tgactgatga 60
tttggctgaa gaagtggatc gattgtttga gaaaagaaga agaccataaa aataccttgt 120
ctgtcatcag acagggtatt ttttatgctg tccagactgt ccgctgtgta aaaaatagga 180
ataaaggggg gttgttatta ttttactgat atgtaaaata taatttgtat aaggaattgt 240
gagcggataa caattcctac gaaaatgaga gggagaggaa acatgattca aaaacgaaag 300
cggacagttt cgttcagact tgtgcttatg tgcacgctgt tatttgtcag tttgccgatt 360
acaaaaacat cagccggatc ccaccatcac catcaccatt aagaattcct agaaactcca 420
agctatcttt aaaaaatcta gtaaatgcac gagcaacatc ttttgttgct cagtgcattt 480
tttattttgt acactagata tttcttctcc gcttaaatca tcaaagaaat ctttatcact 540
tgtaaccagt ccgtccacat gtcgaattgc atctgaccga attttacgtt tccctgaata 600
attctcatca atcgtttcat caattttatc tttatacttt atattttgtg cgttaatcaa 660
atcataattt ttatatgttt cctcatgatt tatgtcttta ttattatagt ttttattctc 720
tctttgatta tgtctttgta tcccgtttgt attacttgat cctttaactc tggcaaccct 780
caaaattgaa tgagacatgc tacacctccg gataataaat atatataaac gtatatagat 840
ttcataaagt ctaacacact agacttattt acttcgtaat taagtcgtta aaccgtgtgc 900
tctacgacca aaactataaa acctttaaga actttctttt tttacaagaa aaaagaaatt 960
agataaatct ctcatatctt ttattcaata atcgcatccg attgcagtat aaatttaacg 1020
atcactcatc atgttcatat ttatcagagc tcgtgctata attatactaa ttttataagg 1080
aggaaaaaat atgggcattt ttagtatttt tgtaatcagc acagttcatt atcaaccaaa 1140
caaaaaataa gtggttataa tgaatcgtta ataagcaaaa ttcatataac caaattaaag 1200
agggttataa tgaacgagaa aaatataaaa cacagtcaaa actttattac ttcaaaacat 1260
aatatagata aaataatgac aaatataaga ttaaatgaac atgataatat ctttgaaatc 1320
ggctcaggaa aaggccattt tacccttgaa ttagtaaaga ggtgtaattt cgtaactgcc 1380
attgaaatag accataaatt atgcaaaact acagaaaata aacttgttga tcacgataat 1440
ttccaagttt taaacaagga tatattgcag tttaaatttc ctaaaaacca atcctataaa 1500
atatatggta atatacctta taacataagt acggatataa tacgcaaaat tgtttttgat 1560
agtatagcta atgagattta tttaatcgtg gaatacgggt ttgctaaaag attattaaat 1620
acaaaacgct cattggcatt acttttaatg gcagaagttg atatttctat attaagtatg 1680
gttccaagag aatattttca tcctaaacct aaagtgaata gctcacttat cagattaagt 1740
agaaaaaaat caagaatatc acacaaagat aaacaaaagt ataattattt cgttatgaaa 1800
tgggttaaca aagaatacaa gaaaatattt acaaaaaatc aatttaacaa ttccttaaaa 1860
catgcaggaa ttgacgattt aaacaatatt agctttgaac aattcttatc tcttttcaat 1920
agctataaat tatttaataa gtaagttaag ggatgcagtt catcgatgaa ggcaactaca 1980
gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat 2040
acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat 2100
ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc 2160
tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc 2220
taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa 2280
cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg 2340
actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg 2400
gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt 2460
taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc 2520
aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag 2580
acaaacaatc aacgtttgcg ccaagcttgc atgcgagagt agggaactgc caggcatcaa 2640
ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg 2700
aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 2760
cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 2820
gccatcctga cggatggcct ttttgcgttt ctacaaactc tttttgttta tttttctaaa 2880
tacattcaaa tatgtatccg ctcatgctcc ggatctgcat cgcaggatgc tgctggctac 2940
cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc 3000
tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg 3060
catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta 3120
cccccatgaa cagaaattcc cccttacacg gaggcatcaa gtgaccaaac aggaaaaaac 3180
cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa 3240
cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga 3300
gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 3360
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 3420
gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 3480
tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac 3540
catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct 3600
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3660
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3720
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3780
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3840
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3900
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3960
gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4020
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4080
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4140
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4200
aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 4260
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4320
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4380
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4440
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4500
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4560
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4620
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4680
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4740
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4800
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc 4860
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4920
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4980
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 5040
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 5100
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 5160
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 5220
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 5280
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 5340
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 5400
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 5460
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5520
gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5580
atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 5640
cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 5700
cagggcgcgt cagcgggtgt tcatgtgcgt aactaacttg ccatcttcaa acaggagggc 5760
tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa 5820
aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc 5880
aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc 5940
ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca 6000
agttcctgaa ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt 6060
ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca 6120
catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt 6180
ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa 6240
agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc 6300
aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg 6360
taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag 6420
ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac 6480
gtatcaaaat gtacagcatg ccacgcgtc 6509
<210>178
<211>571
<212>PRT
<213>枯草芽孢杆菌
<400>178
Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg
1 5 10 15
Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His
20 25 30
Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu
35 40 45
Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala
50 55 60
Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val
65 70 75 80
Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu
85 90 95
Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn
100 105 110
Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn
115 120 125
Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp
130 135 140
Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser
145 150 155 160
Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val
165 170 175
Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys
180 185 190
Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile
195 200 205
Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg
210 215 220
Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu
225 230 235 240
Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu
245 250 255
Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly
260 265 270
Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp
275 280 285
Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr
290 295 300
Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln
305 310 315 320
Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile
325 330 335
Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile
340 345 350
Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala
355 360 365
Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu
370 375 380
Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser
385 390 395 400
His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr
405 410 415
Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp
420 425 430
Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val
435 440 445
Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala
450 455 460
Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr
465 470 475 480
Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser
485 490 495
Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe
500 505 510
Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val
515 520 525
Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro
530 535 540
Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys
545 550 555 560
Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu
565 570
<210>179
<211>1665
<212>DNA
<213>乳酸乳球菌
<400>179
atgtctgaga aacaatttgg ggcgaacttg gttgtcgata gtttgattaa ccataaagtg 60
aagtatgtat ttgggattcc aggagcaaaa attgaccggg tttttgattt attagaaaat 120
gaagaaggcc ctcaaatggt cgtgactcgt catgagcaag gagctgcttt catggctcaa 180
gctgtcggtc gtttaactgg cgaacctggt gtagtagttg ttacgagtgg gcctggtgta 240
tcaaaccttg cgactccgct tttgaccgcg acatcagaag gtgatgctat tttggctatc 300
ggtggacaag ttaaacgaag tgaccgtctt aaacgtgcgc accaatcaat ggataatgct 360
ggaatgatgc aatcagcaac aaaatattca gcagaagttc ttgaccctaa tacactttct 420
gaatcaattg ccaacgctta tcgtattgca aaatcaggac atccaggtgc aactttctta 480
tcaatccccc aagatgtaac ggatgccgaa gtatcaatca aagccattca accactttca 540
gaccctaaaa tggggaatgc ctctattgat gacattaatt atttagcaca agcaattaaa 600
aatgctgtat tgccagtaat tttggttgga gctggtgctt cagatgctaa agtcgcttca 660
tccttgcgta atctattgac tcatgttaat attcctgtcg ttgaaacatt ccaaggtgca 720
ggggttattt cacatgattt agaacatact ttttatggac gtatcggtct tttccgcaat 780
caaccaggcg atatgcttct gaaacgttct gaccttgtta ttgctgttgg ttatgaccca 840
attgaatatg aagctcgtaa ctggaatgca gaaattgata gtcgaattat cgttattgat 900
aatgccattg ctgaaattga tacttactac caaccagagc gtgaattaat tggtgatatc 960
gcagcaacat tggataatct tttaccagct gttcgtggct acaaaattcc aaaaggaaca 1020
aaagattatc tcgatggcct tcatgaagtt gctgagcaac acgaatttga tactgaaaat 1080
actgaagaag gtagaatgca ccctcttgat ttggtcagca ctttccaaga aatcgtcaag 1140
gatgatgaaa cagtaaccgt tgacgtaggt tcactctaca tttggatggc acgtcatttc 1200
aaatcatacg aaccacgtca tctcctcttc tcaaacggaa tgcaaacact cggagttgca 1260
cttccttggg caattacagc cgcattgttg cgcccaggta aaaaagttta ttcacactct 1320
ggtgatggag gcttcctttt cacagggcaa gaattggaaa cagctgtacg tttgaatctt 1380
ccaatcgttc aaattatctg gaatgacggc cattatgata tggttaaatt ccaagaagaa 1440
atgaaatatg gtcgttcagc agccgttgat tttggctatg ttgattacgt aaaatatgct 1500
gaagcaatga gagcaaaagg ttaccgtgca cacagcaaag aagaacttgc tgaaattctc 1560
aaatcaatcc cagatactac tggaccggtg gtaattgacg ttcctttgga ctattctgat 1620
aacattaaat tagcagaaaa attattgcct gaagagtttt attga 1665
<210>180
<211>554
<212>PRT
<213>乳酸乳球菌
<400>180
Met Ser Glu Lys Gln Phe Gly Ala Asn Leu Val Val Asp Ser Leu Ile
1 5 10 15
Asn His Lys Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp
20 25 30
Arg Val Phe Asp Leu Leu Glu Asn Glu Glu Gly Pro Gln Met Val Val
35 40 45
Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly Arg
50 55 60
Leu Thr Gly Glu Pro Gly Val Val Val Val Thr Ser Gly Pro Gly Val
65 70 75 80
Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Gly Asp Ala
85 90 95
Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys Arg
100 105 110
Ala His Gln Ser Met Asp Asn Ala Gly Met Met Gln Ser Ala Thr Lys
115 120 125
Tyr Ser Ala Glu Val Leu Asp Pro Asn Thr Leu Ser Glu Ser Ile Ala
130 135 140
Asn Ala Tyr Arg Ile Ala Lys Ser Gly His Pro Gly Ala Thr Phe Leu
145 150 155 160
Ser Ile Pro Gln Asp Val Thr Asp Ala Glu Val Ser Ile Lys Ala Ile
165 170 175
Gln Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp Ile
180 185 190
Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile Leu
195 200 205
Val Gly Ala Gly Ala Ser Asp Ala Lys Val Ala Ser Ser Leu Arg Asn
210 215 220
Leu Leu Thr His Val Asn Ile Pro Val Val Glu Thr Phe Gln Gly Ala
225 230 235 240
Gly Val Ile Ser His Asp Leu Glu His Thr Phe Tyr Gly Arg Ile Gly
245 250 255
Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp Leu
260 265 270
Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn Trp
275 280 285
Asn Ala Glu Ile Asp Ser Arg Ile Ile Val Ile Asp Asn Ala Ile Ala
290 295 300
Glu Ile Asp Thr Tyr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp Ile
305 310 315 320
Ala Ala Thr Leu Asp Asn Leu Leu Pro Ala Val Arg Gly Tyr Lys Ile
325 330 335
Pro Lys Gly Thr Lys Asp Tyr Leu Asp Gly Leu His Glu Val Ala Glu
340 345 350
Gln His Glu Phe Asp Thr Glu Asn Thr Glu Glu Gly Arg Met His Pro
355 360 365
Leu Asp Leu Val Ser Thr Phe Gln Glu Ile Val Lys Asp Asp Glu Thr
370 375 380
Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg His Phe
385 390 395 400
Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr
405 410 415
Leu Gly Val Ala Leu Pro Trp Ala Ile Thr Ala Ala Leu Leu Arg Pro
420 425 430
Gly Lys Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe Thr
435 440 445
Gly Gln Glu Leu Glu Thr Ala Val Arg Leu Asn Leu Pro Ile Val Gln
450 455 460
Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu Glu
465 470 475 480
Met Lys Tyr Gly Arg Ser Ala Ala Val Asp Phe Gly Tyr Val Asp Tyr
485 490 495
Val Lys Tyr Ala Glu Ala Met Arg Ala Lys Gly Tyr Arg Ala His Ser
500 505 510
Lys Glu Glu Leu Ala Glu Ile Leu Lys Ser Ile Pro Asp Thr Thr Gly
515 520 525
Pro Val Val Ile Asp Val Pro Leu Asp Tyr Ser Asp Asn Ile Lys Leu
530 535 540
Ala Glu Lys Leu Leu Pro Glu Glu Phe Tyr
545 550
<210>181
<211>395
<212>PRT
<213>酿酒酵母
<400>181
Met Leu Arg Thr Gln Ala Ala Arg Leu Ile Cys Asn Ser Arg Val Ile
1 5 10 15
Thr Ala Lys Arg Thr Phe Ala Leu Ala Thr Arg Ala Ala Ala Tyr Ser
20 25 30
Arg Pro Ala Ala Arg Phe Val Lys Pro Met Ile Thr Thr Arg Gly Leu
35 40 45
Lys Gln Ile Asn Phe Gly Gly Thr Val Glu Thr Val Tyr Glu Arg Ala
50 55 60
Asp Trp Pro Arg Glu Lys Leu Leu Asp Tyr Phe Lys Asn Asp Thr Phe
65 70 75 80
Ala Leu Ile Gly Tyr Gly Ser Gln Gly Tyr Gly Gln Gly Leu Asn Leu
85 90 95
Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg Lys Asp Gly Ala
100 105 110
Ser Trp Lys Ala Ala Ile Glu Asp Gly Trp Val Pro Gly Lys Asn Leu
115 120 125
Phe Thr Val Glu Asp Ala Ile Lys Arg Gly Ser Tyr Val Met Asn Leu
130 135 140
Leu Ser Asp Ala Ala Gln Ser Glu Thr Trp Pro Ala Ile Lys Pro Leu
145 150 155 160
Leu Thr Lys Gly Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val
165 170 175
Phe Lys Asp Leu Thr His Val Glu Pro Pro Lys Asp Leu Asp Val Ile
180 185 190
Leu Val Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe Lys
195 200 205
Glu Gly Arg Gly Ile Asn Ser Ser Tyr Ala Val Trp Asn Asp Val Thr
210 215 220
Gly Lys Ala His Glu Lys Ala Gln Ala Leu Ala Val Ala Ile Gly Ser
225 230 235 240
Gly Tyr Val Tyr Gln Thr Thr Phe Glu Arg Glu Val Asn Ser Asp Leu
245 250 255
Tyr Gly Glu Arg Gly Cys Leu Met Gly Gly Ile His Gly Met Phe Leu
260 265 270
Ala Gln Tyr Asp Val Leu Arg Glu Asn Gly His Ser Pro Ser Glu Ala
275 280 285
Phe Asn Glu Thr Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile
290 295 300
Gly Lys Tyr Gly Met Asp Tyr Met Tyr Asp Ala Cys Ser Thr Thr Ala
305 310 315 320
Arg Arg Gly Ala Leu Asp Trp Tyr Pro Ile Phe Lys Asn Ala Leu Lys
325 330 335
Pro Val Phe Gln Asp Leu Tyr Glu Ser Thr Lys Asn Gly Thr Glu Thr
340 345 350
Lys Arg Ser Leu Glu Phe Asn Ser Gln Pro Asp Tyr Arg Glu Lys Leu
355 360 365
Glu Lys Glu Leu Asp Thr Ile Arg Asn Met Glu Ile Trp Lys Val Gly
370 375 380
Lys Glu Val Arg Lys Leu Arg Pro Glu Asn Gln
385 390 395
<210>182
<211>993
<212>DNA
<213>海藻甲烷球菌
<400>182
atgaaggtat tctatgactc agattttaaa ttagatgctt taaaagaaaa aacaattgca 60
gtaatcggtt atggaagtca aggtagggca cagtccttaa acatgaaaga cagcggatta 120
aacgttgttg ttggtttaag aaaaaacggt gcttcatgga acaacgctaa agcagacggt 180
cacaatgtaa tgaccattga agaagctgct gaaaaagcgg acatcatcca catcttaata 240
cctgatgaat tacaggcaga agtttatgaa agccagataa aaccatacct aaaagaagga 300
aaaacactaa gcttttcaca tggttttaac atccactatg gattcattgt tccaccaaaa 360
ggagttaacg tggttttagt tgctccaaaa tcacctggaa aaatggttag aagaacatac 420
gaagaaggtt tcggtgttcc aggtttaatc tgtattgaaa ttgatgcaac aaacaacgca 480
tttgatattg tttcagcaat ggcaaaagga atcggtttat caagagctgg agttatccag 540
acaactttca aagaagaaac agaaactgac cttttcggtg aacaagctgt tttatgcggt 600
ggagttaccg aattaatcaa ggcaggattt gaaacactcg ttgaagcagg atacgcacca 660
gaaatggcat actttgaaac ctgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720
aaaggattca aaaacatgtg gaacgatgta agtaacactg cagaatacgg cggacttaca 780
agaagaagca gaatcgttac agctgattca aaagctgcaa tgaaagaaat cttaagagaa 840
atccaagatg gaagattcac aaaagaattc cttctcgaaa aacaggtaag ctatgctcat 900
ttaaaatcaa tgagaagact cgaaggagac ttacaaatcg aagaagtcgg cgcaaaatta 960
agaaaaatgt gcggtcttga aaaagaagaa taa 993
<210>183
<211>330
<212>PRT
<213>海藻甲烷球菌
<400>183
Met Lys Val Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu
1 5 10 15
Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser
20 25 30
Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys
35 40 45
Asn Gly Ala Ser Trp Asn Asn Ala Lys Ala Asp Gly His Asn Val Met
50 55 60
Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile His Ile Leu Ile
65 70 75 80
Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu Ser Gln Ile Lys Pro Tyr
85 90 95
Leu Lys Glu Gly Lys Thr Leu Ser Phe Ser His Gly Phe Asn Ile His
100 105 110
Tyr Gly Phe Ile Val Pro Pro Lys Gly Val Asn Val Val Leu Val Ala
115 120 125
Pro Lys Ser Pro Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe
130 135 140
Gly Val Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala
145 150 155 160
Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala
165 170 175
Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe
180 185 190
Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu Ile Lys Ala
195 200 205
Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu Met Ala Tyr
210 215 220
Phe Glu Thr Cys His Glu Leu Lys Leu Ile Val Asp Leu Ile Tyr Gln
225 230 235 240
Lys Gly Phe Lys Asn Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr
245 250 255
Gly Gly Leu Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala
260 265 270
Ala Met Lys Glu Ile Leu Arg Glu Ile Gln Asp Gly Arg Phe Thr Lys
275 280 285
Glu Phe Leu Leu Glu Lys Gln Val Ser Tyr Ala His Leu Lys Ser Met
290 295 300
Arg Arg Leu Glu Gly Asp Leu Gln Ile Glu Glu Val Gly Ala Lys Leu
305 310 315 320
Arg Lys Met Cys Gly Leu Glu Lys Glu Glu
325 330
<210>184
<211>1476
<212>DNA
<213>枯草芽孢杆菌
<400>184
atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60
cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120
gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180
ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240
aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300
ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360
ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420
gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480
gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540
aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600
caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660
gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720
gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780
atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840
gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900
cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960
gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020
accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080
atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140
atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200
atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260
aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320
ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380
gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440
atgacagata tgaaacgtat tgctgttgcg ggttaa 1476
<210>185
<211>342
<212>PRT
<213>枯草芽孢杆菌
<400>185
Met Val Lys Val Tyr Tyr Asn Gly Asp Ile Lys Glu Asn Val Leu Ala
1 5 10 15
Gly Lys Thr Val Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala His
20 25 30
Ala Leu Asn Leu Lys Glu Ser Gly Val Asp Val Ile Val Gly Val Arg
35 40 45
Gln Gly Lys Ser Phe Thr Gln Ala Gln Glu Asp Gly His Lys Val Phe
50 55 60
Ser Val Lys Glu Ala Ala Ala Gln Ala Glu Ile Ile Met Val Leu Leu
65 70 75 80
Pro Asp Glu Gln Gln Gln Lys Val Tyr Glu Ala Glu Ile Lys Asp Glu
85 90 95
Leu Thr Ala Gly Lys Ser Leu Val Phe Ala His Gly Phe Asn Val His
100 105 110
Phe His Gln Ile Val Pro Pro Ala Asp Val Asp Val Phe Leu Val Ala
115 120 125
Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Glu Gln Gly Ala
130 135 140
Gly Val Pro Ala Leu Phe Ala Ile Tyr Gln Asp Val Thr Gly Glu Ala
145 150 155 160
Arg Asp Lys Ala Leu Ala Tyr Ala Lys Gly Ile Gly Gly Ala Arg Ala
165 170 175
Gly Val Leu Glu Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe
180 185 190
Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Ser Ala Leu Val Lys Ala
195 200 205
Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Gln Pro Glu Leu Ala Tyr
210 215 220
Phe Glu Cys Leu His Glu Leu Lys Leu Ile Val Asp Leu Met Tyr Glu
225 230 235 240
Glu Gly Leu Ala Gly Met Arg Tyr Ser Ile Ser Asp Thr Ala Gln Trp
245 250 255
Gly Asp Phe Val Ser Gly Pro Arg Val Val Asp Ala Lys Val Lys Glu
260 265 270
Ser Met Lys Glu Val Leu Lys Asp Ile Gln Asn Gly Thr Phe Ala Lys
275 280 285
Glu Trp Ile Val Glu Asn Gln Val Asn Arg Pro Arg Phe Asn Ala Ile
290 295 300
Asn Ala Ser Glu Asn Glu His Gln Ile Glu Val Val Gly Arg Lys Leu
305 310 315 320
Arg Glu Met Met Pro Phe Val Lys Gln Gly Lys Lys Lys Glu Ala Val
325 330 335
Val Ser Val Ala Gln Asn
340
<210>186
<211>585
<212>PRT
<213>酿酒酵母
<400>186
Met Gly Leu Leu Thr Lys Val Ala Thr Ser Arg Gln Phe Ser Thr Thr
1 5 10 15
Arg Cys Val Ala Lys Lys Leu Asn Lys Tyr Ser Tyr Ile Ile Thr Glu
20 25 30
Pro Lys Gly Gln Gly Ala Ser Gln Ala Met Leu Tyr Ala Thr Gly Phe
35 40 45
Lys Lys Glu Asp Phe Lys Lys Pro Gln Val Gly Val Gly Ser Cys Trp
50 55 60
Trp Ser Gly Asn Pro Cys Asn Met His Leu Leu Asp Leu Asn Asn Arg
65 70 75 80
Cys Ser Gln Ser Ile Glu Lys Ala Gly Leu Lys Ala Met Gln Phe Asn
85 90 95
Thr Ile Gly Val Ser Asp Gly Ile Ser Met Gly Thr Lys Gly Met Arg
100 105 110
Tyr Ser Leu Gln Ser Arg Glu Ile Ile Ala Asp Ser Phe Glu Thr Ile
115 120 125
Met Met Ala Gln His Tyr Asp Ala Asn Ile Ala Ile Pro Ser Cys Asp
130 135 140
Lys Asn Met Pro Gly Val Met Met Ala Met Gly Arg His Asn Arg Pro
145 150 155 160
Ser Ile Met Val Tyr Gly Gly Thr Ile Leu Pro Gly His Pro Thr Cys
165 170 175
Gly Ser Ser Lys Ile Ser Lys Asn Ile Asp Ile Val Ser Ala Phe Gln
180 185 190
Ser Tyr Gly Glu Tyr Ile Ser Lys Gln Phe Thr Glu Glu Glu Arg Glu
195 200 205
Asp Val Val Glu His Ala Cys Pro Gly Pro Gly Ser Cys Gly Gly Met
210 215 220
Tyr Thr Ala Asn Thr Met Ala Ser Ala Ala Glu Val Leu Gly Leu Thr
225 230 235 240
Ile Pro Asn Ser Ser Ser Phe Pro Ala Val Ser Lys Glu Lys Leu Ala
245 250 255
Glu Cys Asp Asn Ile Gly Glu Tyr Ile Lys Lys Thr Met Glu Leu Gly
260 265 270
Ile Leu Pro Arg Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn Ala Ile
275 280 285
Thr Tyr Val Val Ala Thr Gly Gly Ser Thr Asn Ala Val Leu His Leu
290 295 300
Val Ala Val Ala His Ser Ala Gly Val Lys Leu Ser Pro Asp Asp Phe
305 310 315 320
Gln Arg Ile Ser Asp Thr Thr Pro Leu Ile Gly Asp Phe Lys Pro Ser
325 330 335
Gly Lys Tyr Val Met Ala Asp Leu Ile Asn Val Gly Gly Thr Gln Ser
340 345 350
Val Ile Lys Tyr Leu Tyr Glu Asn Asn Met Leu His Gly Asn Thr Met
355 360 365
Thr Val Thr Gly Asp Thr Leu Ala Glu Arg Ala Lys Lys Ala Pro Ser
370 375 380
Leu Pro Glu Gly Gln Glu Ile Ile Lys Pro Leu Ser His Pro Ile Lys
385 390 395 400
Ala Asn Gly His Leu Gln Ile Leu Tyr Gly Ser Leu Ala Pro Gly Gly
405 410 415
Ala Val Gly Lys Ile Thr Gly Lys Glu Gly Thr Tyr Phe Lys Gly Arg
420 425 430
Ala Arg Val Phe Glu Glu Glu Gly Ala Phe Ile Glu Ala Leu Glu Arg
435 440 445
Gly Glu Ile Lys Lys Gly Glu Lys Thr Val Val Val Ile Arg Tyr Glu
450 455 460
Gly Pro Arg Gly Ala Pro Gly Met Pro Glu Met Leu Lys Pro Ser Ser
465 470 475 480
Ala Leu Met Gly Tyr Gly Leu Gly Lys Asp Val Ala Leu Leu Thr Asp
485 490 495
Gly Arg Phe Ser Gly Gly Ser His Gly Phe Leu Ile Gly His Ile Val
500 505 510
Pro Glu Ala Ala Glu Gly Gly Pro Ile Gly Leu Val Arg Asp Gly Asp
515 520 525
Glu Ile Ile Ile Asp Ala Asp Asn Asn Lys Ile Asp Leu Leu Val Ser
530 535 540
Asp Lys Glu Met Ala Gln Arg Lys Gln Ser Trp Val Ala Pro Pro Pro
545 550 555 560
Arg Tyr Thr Arg Gly Thr Leu Ser Lys Tyr Ala Lys Leu Val Ser Asn
565 570 575
Ala Ser Asn Gly Cys Val Leu Asp Ala
580 585
<210>187
<211>1653
<212>DNA
<213>海藻甲烷球菌
<400>187
atgataagtg ataacgtcaa aaagggagtt ataagaactc caaaccgagc tcttttaaag 60
gcttgcggat atacagacga agacatggaa aaaccattta ttggaattgt aaacagcttt 120
acagaagttg ttcccggcca cattcactta agaacattat cagaagcggc taaacatggt 180
gtttatgcaa acggtggaac accatttgaa tttaatacca ttggaatttg cgacggtatt 240
gcaatgggcc acgaaggtat gaaatactct ttaccttcaa gagaaattat tgcagacgct 300
gttgaatcaa tggcaagagc acatggattt gatggtcttg ttttaattcc tacgtgtgat 360
aaaatcgttc ctggaatgat aatgggtgct ttaagactaa acattccatt tattgtagtt 420
actggaggac caatgcttcc cggagaattc caaggtaaaa aatacgaact tatcagcctt 480
tttgaaggtg tcggagaata ccaagttgga aaaattactg aagaagagtt aaagtgcatt 540
gaagactgtg catgttcagg tgctggaagt tgtgcagggc tttacactgc aaacagtatg 600
gcctgcctta cagaagcttt gggactctct cttccaatgt gtgcaacaac gcatgcagtt 660
gatgcccaaa aagttaggct tgctaaaaaa agtggctcaa aaattgttga tatggtaaaa 720
gaagacctaa aaccaacaga catattaaca aaagaagctt ttgaaaatgc tattttagtt 780
gaccttgcac ttggtggatc aacaaacaca acattacaca ttcctgcaat tgcaaatgaa 840
attgaaaata aattcataac tctcgatgac tttgacaggt taagcgatga agttccacac 900
attgcatcaa tcaaaccagg tggagaacac tacatgattg atttacacaa tgctggaggt 960
attcctgcgg tattgaacgt tttaaaagaa aaaattagag atacaaaaac agttgatgga 1020
agaagcattt tggaaatcgc agaatctgtt aaatacataa attacgacgt tataagaaaa 1080
gtggaagctc cggttcacga aactgctggt ttaagggttt taaagggaaa tcttgctcca 1140
aacggttgcg ttgtaaaaat cggtgcagta catccgaaaa tgtacaaaca cgatggacct 1200
gcaaaagttt acaattccga agatgaagca atttctgcga tacttggcgg aaaaattgta 1260
gaaggggacg ttatagtaat cagatacgaa ggaccatcag gaggccctgg aatgagagaa 1320
atgctctccc caacttcagc aatctgtgga atgggtcttg atgacagcgt tgcattgatt 1380
actgatggaa gattcagtgg tggaagtagg ggcccatgta tcggacacgt ttctccagaa 1440
gctgcagctg gcggagtaat tgctgcaatt gaaaacgggg atatcatcaa aatcgacatg 1500
attgaaaaag aaataaatgt tgatttagat gaatcagtca ttaaagaaag actctcaaaa 1560
ctgggagaat ttgagcctaa aatcaaaaaa ggctatttat caagatactc aaaacttgtc 1620
tcatctgctg acgaaggggc agttttaaaa taa 1653
<210>188
<211>550
<212>PRT
<213>海藻甲烷球菌
<400>188
Met Ile Ser Asp Asn Val Lys Lys Gly Val Ile Arg Thr Pro Asn Arg
1 5 10 15
Ala Leu Leu Lys Ala Cys Gly Tyr Thr Asp Glu Asp Met Glu Lys Pro
20 25 30
Phe Ile Gly Ile Val Asn Ser Phe Thr Glu Val Val Pro Gly His Ile
35 40 45
His Leu Arg Thr Leu Ser Glu Ala Ala Lys His Gly Val Tyr Ala Asn
50 55 60
Gly Gly Thr Pro Phe Glu Phe Asn Thr Ile Gly Ile Cys Asp Gly Ile
65 70 75 80
Ala Met Gly His Glu Gly Met Lys Tyr Ser Leu Pro Ser Arg Glu Ile
85 90 95
Ile Ala Asp Ala Val Glu Ser Met Ala Arg Ala His Gly Phe Asp Gly
100 105 110
Leu Val Leu Ile Pro Thr Cys Asp Lys Ile Val Pro Gly Met Ile Met
115 120 125
Gly Ala Leu Arg Leu Asn Ile Pro Phe Ile Val Val Thr Gly Gly Pro
130 135 140
Met Leu Pro Gly Glu Phe Gln Gly Lys Lys Tyr Glu Leu Ile Ser Leu
145 150 155 160
Phe Glu Gly Val Gly Glu Tyr Gln Val Gly Lys Ile Thr Glu Glu Glu
165 170 175
Leu Lys Cys Ile Glu Asp Cys Ala Cys Ser Gly Ala Gly Ser Cys Ala
180 185 190
Gly Leu Tyr Thr Ala Asn Ser Met Ala Cys Leu Thr Glu Ala Leu Gly
195 200 205
Leu Ser Leu Pro Met Cys Ala Thr Thr His Ala Val Asp Ala Gln Lys
210 215 220
Val Arg Leu Ala Lys Lys Ser Gly Ser Lys Ile Val Asp Met Val Lys
225 230 235 240
Glu Asp Leu Lys Pro Thr Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn
245 250 255
Ala Ile Leu Val Asp Leu Ala Leu Gly Gly Ser Thr Asn Thr Thr Leu
260 265 270
His Ile Pro Ala Ile Ala Asn Glu Ile Glu Asn Lys Phe Ile Thr Leu
275 280 285
Asp Asp Phe Asp Arg Leu Ser Asp Glu Val Pro His Ile Ala Ser Ile
290 295 300
Lys Pro Gly Gly Glu His Tyr Met Ile Asp Leu His Asn Ala Gly Gly
305 310 315 320
Ile Pro Ala Val Leu Asn Val Leu Lys Glu Lys Ile Arg Asp Thr Lys
325 330 335
Thr Val Asp Gly Arg Ser Ile Leu Glu Ile Ala Glu Ser Val Lys Tyr
340 345 350
Ile Asn Tyr Asp Val Ile Arg Lys Val Glu Ala Pro Val His Glu Thr
355 360 365
Ala Gly Leu Arg Val Leu Lys Gly Asn Leu Ala Pro Asn Gly Cys Val
370 375 380
Val Lys Ile Gly Ala Val His Pro Lys Met Tyr Lys His Asp Gly Pro
385 390 395 400
Ala Lys Val Tyr Asn Ser Glu Asp Glu Ala Ile Ser Ala Ile Leu Gly
405 410 415
Gly Lys Ile Val Glu Gly Asp Val Ile Val Ile Arg Tyr Glu Gly Pro
420 425 430
Ser Gly Gly Pro Gly Met Arg Glu Met Leu Ser Pro Thr Ser Ala Ile
435 440 445
Cys Gly Met Gly Leu Asp Asp Ser Val Ala Leu Ile Thr Asp Gly Arg
450 455 460
Phe Ser Gly Gly Ser Arg Gly Pro Cys Ile Gly His Val Ser Pro Glu
465 470 475 480
Ala Ala Ala Gly Gly Val Ile Ala Ala Ile Glu Asn Gly Asp Ile Ile
485 490 495
Lys Ile Asp Met Ile Glu Lys Glu Ile Asn Val Asp Leu Asp Glu Ser
500 505 510
Val Ile Lys Glu Arg Leu Ser Lys Leu Gly Glu Phe Glu Pro Lys Ile
515 520 525
Lys Lys Gly Tyr Leu Ser Arg Tyr Ser Lys Leu Val Ser Ser Ala Asp
530 535 540
Glu Gly Ala Val Leu Lys
545 550
<210>189
<211>1677
<212>DNA
<213>枯草芽孢杆菌
<400>189
atggcagaat tacgcagtaa tatgatcaca caaggaatcg atagagctcc gcaccgcagt 60
ttgcttcgtg cagcaggggt aaaagaagag gatttcggca agccgtttat tgcggtgtgt 120
aattcataca ttgatatcgt tcccggtcat gttcacttgc aggagtttgg gaaaatcgta 180
aaagaagcaa tcagagaagc agggggcgtt ccgtttgaat ttaataccat tggggtagat 240
gatggcatcg caatggggca tatcggtatg agatattcgc tgccaagccg tgaaattatc 300
gcagactctg tggaaacggt tgtatccgca cactggtttg acggaatggt ctgtattccg 360
aactgcgaca aaatcacacc gggaatgctt atggcggcaa tgcgcatcaa cattccgacg 420
atttttgtca gcggcggacc gatggcggca ggaagaacaa gttacgggcg aaaaatctcc 480
ctttcctcag tattcgaagg ggtaggcgcc taccaagcag ggaaaatcaa cgaaaacgag 540
cttcaagaac tagagcagtt cggatgccca acgtgcgggt cttgctcagg catgtttacg 600
gcgaactcaa tgaactgtct gtcagaagca cttggtcttg ctttgccggg taatggaacc 660
attctggcaa catctccgga acgcaaagag tttgtgagaa aatcggctgc gcaattaatg 720
gaaacgattc gcaaagatat caaaccgcgt gatattgtta cagtaaaagc gattgataac 780
gcgtttgcac tcgatatggc gctcggaggt tctacaaata ccgttcttca tacccttgcc 840
cttgcaaacg aagccggcgt tgaatactct ttagaacgca ttaacgaagt cgctgagcgc 900
gtgccgcact tggctaagct ggcgcctgca tcggatgtgt ttattgaaga tcttcacgaa 960
gcgggcggcg tttcagcggc tctgaatgag ctttcgaaga aagaaggagc gcttcattta 1020
gatgcgctga ctgttacagg aaaaactctt ggagaaacca ttgccggaca tgaagtaaag 1080
gattatgacg tcattcaccc gctggatcaa ccattcactg aaaagggagg ccttgctgtt 1140
ttattcggta atctagctcc ggacggcgct atcattaaaa caggcggcgt acagaatggg 1200
attacaagac acgaagggcc ggctgtcgta ttcgattctc aggacgaggc gcttgacggc 1260
attatcaacc gaaaagtaaa agaaggcgac gttgtcatca tcagatacga agggccaaaa 1320
ggcggacctg gcatgccgga aatgctggcg ccaacatccc aaatcgttgg aatgggactc 1380
gggccaaaag tggcattgat tacggacgga cgtttttccg gagcctcccg tggcctctca 1440
atcggccacg tatcacctga ggccgctgag ggcgggccgc ttgcctttgt tgaaaacgga 1500
gaccatatta tcgttgatat tgaaaaacgc atcttggatg tacaagtgcc agaagaagag 1560
tgggaaaaac gaaaagcgaa ctggaaaggt tttgaaccga aagtgaaaac cggctacctg 1620
gcacgttatt ctaaacttgt gacaagtgcc aacaccggcg gtattatgaa aatctag 1677
<210>190
<211>558
<212>PRT
<213>枯草芽孢杆菌
<400>190
Met Ala Glu Leu Arg Ser Asn Met Ile Thr Gln Gly Ile Asp Arg Ala
1 5 10 15
Pro His Arg Ser Leu Leu Arg Ala Ala Gly Val Lys Glu Glu Asp Phe
20 25 30
Gly Lys Pro Phe Ile Ala Val Cys Asn Ser Tyr Ile Asp Ile Val Pro
35 40 45
Gly His Val His Leu Gln Glu Phe Gly Lys Ile Val Lys Glu Ala Ile
50 55 60
Arg Glu Ala Gly Gly Val Pro Phe Glu Phe Asn Thr Ile Gly Val Asp
65 70 75 80
Asp Gly Ile Ala Met Gly His Ile Gly Met Arg Tyr Ser Leu Pro Ser
85 90 95
Arg Glu Ile Ile Ala Asp Ser Val Glu Thr Val Val Ser Ala His Trp
100 105 110
Phe Asp Gly Met Val Cys Ile Pro Asn Cys Asp Lys Ile Thr Pro Gly
115 120 125
Met Leu Met Ala Ala Met Arg Ile Asn Ile Pro Thr Ile Phe Val Ser
130 135 140
Gly Gly Pro Met Ala Ala Gly Arg Thr Ser Tyr Gly Arg Lys Ile Ser
145 150 155 160
Leu Ser Ser Val Phe Glu Gly Val Gly Ala Tyr Gln Ala Gly Lys Ile
165 170 175
Asn Glu Asn Glu Leu Gln Glu Leu Glu Gln Phe Gly Cys Pro Thr Cys
180 185 190
Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Ser
195 200 205
Glu Ala Leu Gly Leu Ala Leu Pro Gly Asn Gly Thr Ile Leu Ala Thr
210 215 220
Ser Pro Glu Arg Lys Glu Phe Val Arg Lys Ser Ala Ala Gln Leu Met
225 230 235 240
Glu Thr Ile Arg Lys Asp Ile Lys Pro Arg Asp Ile Val Thr Val Lys
245 250 255
Ala Ile Asp Asn Ala Phe Ala Leu Asp Met Ala Leu Gly Gly Ser Thr
260 265 270
Asn Thr Val Leu His Thr Leu Ala Leu Ala Asn Glu Ala Gly Val Glu
275 280 285
Tyr Ser Leu Glu Arg Ile Asn Glu Val Ala Glu Arg Val Pro His Leu
290 295 300
Ala Lys Leu Ala Pro Ala Ser Asp ValPhe Ile Glu Asp Leu His Glu
305 310 315 320
Ala Gly Gly Val Ser Ala Ala Leu Asn Glu Leu Ser Lys Lys Glu Gly
325 330 335
Ala Leu His Leu Asp Ala Leu Thr Val Thr Gly Lys Thr Leu Gly Glu
340 345 350
Thr Ile Ala Gly His Glu Val Lys Asp Tyr Asp Val Ile His Pro Leu
355 360 365
Asp Gln Pro Phe Thr Glu Lys Gly Gly Leu Ala Val Leu Phe Gly Asn
370 375 380
Leu Ala Pro Asp Gly Ala Ile Ile Lys Thr Gly Gly Val Gln Asn Gly
385 390 395 400
Ile Thr Arg His Glu Gly Pro Ala Val Val Phe Asp Ser Gln Asp Glu
405 410 415
Ala Leu Asp Gly Ile Ile Asn Arg Lys Val Lys Glu Gly Asp Val Val
420 425 430
Ile Ile Arg Tyr Glu Gly Pro Lys Gly Gly Pro Gly Met Pro Glu Met
435 440 445
Leu Ala Pro Thr Ser Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val
450 455 460
Ala Leu Ile Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser
465 470 475 480
Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala Phe
485 490 495
Val Glu Asn Gly Asp His Ile Ile Val Asp Ile Glu Lys Arg Ile Leu
500 505 510
Asp Val Gln Val Pro Glu Glu Glu Trp Glu Lys Arg Lys Ala Asn Trp
515 520 525
Lys Gly Phe Glu Pro Lys Val Lys Thr Gly Tyr Leu Ala Arg Tyr Ser
530 535 540
Lys Leu Val Thr Ser Ala Asn Thr Gly Gly Ile Met Lys Ile
545 550 555
<210>191
<211>1647
<212>DNA
<213>乳酸乳球菌
<400>191
atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60
tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccacaaggat 120
atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga tggctatgct 180
cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240
aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300
acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360
aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420
aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc 480
tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540
aaaaaggaaa actcaacttc aaatacaagt gaccaagaaa ttttgaacaa aattcaagaa 600
agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660
ttagaaaaaa cagtcactca atttatttca aagacaaaac tacctattac gacattaaac 720
tttggtaaaa gttcagttga tgaagccctc ccttcatttt taggaatcta taatggtaca 780
ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatctt gatgcttgga 840
gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga aaataaaatg 900
atttcactga atatagatga aggaaaaata tttaacgaaa gaatccaaaa ttttgatttt 960
gaatccctca tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020
gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080
caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140
ttctttggcg cttcatcaat tttcttaaaa tcaaagagtc attttattgg tcaaccctta 1200
tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttacagtgca agaattagga 1320
ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380
gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440
tcaaaattac cagaatcgtt tggagcaaca gaagatcgag tagtctcaaa aatcgttaga 1500
actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560
tggattgagt taattttggc aaaagaaggt gcaccaaaag tactgaaaaa aatgggcaaa 1620
ctatttgctg aacaaaataa atcataa 1647
<210>192
<211>1644
<212>DNA
<213>乳酸乳球菌
<400>192
atgtatacag taggagatta cctgttagac cgattacacg agttgggaat tgaagaaatt 60
tttggagttc ctggtgacta taacttacaa tttttagatc aaattatttc acgcgaagat 120
atgaaatgga ttggaaatgc taatgaatta aatgcttctt atatggctga tggttatgct 180
cgtactaaaa aagctgccgc atttctcacc acatttggag tcggcgaatt gagtgcgatc 240
aatggactgg caggaagtta tgccgaaaat ttaccagtag tagaaattgt tggttcacca 300
acttcaaaag tacaaaatga cggaaaattt gtccatcata cactagcaga tggtgatttt 360
aaacacttta tgaagatgca tgaacctgtt acagcagcgc ggactttact gacagcagaa 420
aatgccacat atgaaattga ccgagtactt tctcaattac taaaagaaag aaaaccagtc 480
tatattaact taccagtcga tgttgctgca gcaaaagcag agaagcctgc attatcttta 540
gaaaaagaaa gctctacaac aaatacaact gaacaagtga ttttgagtaa gattgaagaa 600
agtttgaaaa atgcccaaaa accagtagtg attgcaggac acgaagtaat tagttttggt 660
ttagaaaaaa cggtaactca gtttgtttca gaaacaaaac taccgattac gacactaaat 720
tttggtaaaa gtgctgttga tgaatctttg ccctcatttt taggaatata taacgggaaa 780
ctttcagaaa tcagtcttaa aaattttgtg gagtccgcag actttatcct aatgcttgga 840
gtgaagctta cggactcctc aacaggtgca ttcacacatc atttagatga aaataaaatg 900
atttcactaa acatagatga aggaataatt ttcaataaag tggtagaaga ttttgatttt 960
agagcagtgg tttcttcttt atcagaatta aaaggaatag aatatgaagg acaatatatt 1020
gataagcaat atgaagaatt tattccatca agtgctccct tatcacaaga ccgtctatgg 1080
caggcagttg aaagtttgac tcaaagcaat gaaacaatcg ttgctgaaca aggaacctca 1140
ttttttggag cttcaacaat tttcttaaaa tcaaatagtc gttttattgg acaaccttta 1200
tggggttcta ttggatatac ttttccagcg gctttaggaa gccaaattgc ggataaagag 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttaccgtaca agaattagga 1320
ctatcaatca gagaaaaact caatccaatt tgttttatca taaataatga tggttataca 1380
gttgaaagag aaatccacgg acctactcaa agttataacg acattccaat gtggaattac 1440
tcgaaattac cagaaacatt tggagcaaca gaagatcgtg tagtatcaaa aattgttaga 1500
acagagaatg aatttgtgtc tgtcatgaaa gaagcccaag cagatgtcaa tagaatgtat 1560
tggatagaac tagttttgga aaaagaagat gcgccaaaat tactgaaaaa aatgggtaaa 1620
ttatttgctg agcaaaataa atag 1644
<210>193
<211>547
<212>PRT
<213>乳酸乳球菌
<400>193
Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly
1 5 10 15
Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu
20 25 30
Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn
35 40 45
Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys
50 55 60
Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile
65 70 75 80
Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile
85 90 95
Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His
100 105 110
His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu
115 120 125
Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr
130 135 140
Glu Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val
145 150 155 160
Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro
165 170 175
Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln
180 185 190
Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro
195 200 205
Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr
210 215 220
Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn
225 230 235 240
Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile
245 250 255
Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser
260 265 270
Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr
275 280 285
Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn
290 295 300
Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe
305 310 315 320
Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu
325 330 335
Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala
340 345 350
Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln
355 360 365
Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380
Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu
385 390 395 400
Trp Gly Ser Ile Gly TyrThr Phe Pro Ala Ala Leu Gly Ser Gln Ile
405 410 415
Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430
Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn
435 440 445
Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu
450 455 460
Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr
465 470 475 480
Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser
485 490 495
Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala
500 505 510
Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys
515 520 525
Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu
530 535 540
Gln Asn Lys
545
<210>194
<211>1653
<212>DNA
<213>鼠伤寒沙门氏菌
<400>194
ttatcccccg ttgcgggctt ccagcgcccg ggtcacggta cgcagtaatt ccggcagatc 60
ggcttttggc aacatcactt caataaatga cagacgttgt gggcgcgcca accgttcgag 120
gacctctgcc agttggatag cctgcgtcac ccgccagcac tccgcctgtt gcgccgcgtt 180
tagcgccggt ggtatctgcg tccagttcca gctcgcgatg tcgttatacc gctgggccgc 240
gccgtgaatg gcgcgctcta cggtatagcc gtcattgttg agcagcagga tgaccggcgc 300
ctgcccgtcg cgtaacatcg agcccatctc ctgaatcgtg agctgcgccg cgccatcgcc 360
gataatcaga atcacccgcc gatcgggaca ggcggtttgc gcgccaaacg cggcgggcaa 420
ggaatagccg atagaccccc acagcggctg taacacaact tccgcgccgt caggaagcga 480
cagcgcggca gcgccaaaag ctgctgtccc ctggtcgaca aggataatat ctccgggttt 540
gagatactgc tgtaaggttt gccagaagct ttcctgggtc agttctcctt tatcaatccg 600
cactggctgt ccggcggaac gcgtcggcgg cggcgcaaaa gcgcattcca ggcacagttc 660
gcgcagcgta gacaccgcct gcgccatcgg gaggttgaac caggtttcgc cgatgcgcga 720
cgcgtaaggc tgaatctcca gcgtgcgttc cgccggtaat tgttgggtaa atccggccgt 780
aagggtatcg acaaaacggg tgccgacgca gataacccta tcggcgtcct ctatggcctg 840
acgcacttct ttgctgctgg cgccagcgct ataggtgcca acgaagttcg ggtgctgttc 900
atcaaaaagc cccttcccca tcagtagtgt cgcatgagcg atgggcgttt ccgccatcca 960
gcgctgcaac agtggtcgta aaccaaaacg cccggcaaga aagtcggcca atagcgcaat 1020
gcgccgactg ttcatcaggc actgacgggc gtgataacga aaggccgtct ccacgccgct 1080
ttgcgcttca tgcacgggca acgccagcgc ctgcgtaggt gggatggccg tttttttcgc 1140
cacatcggcg ggcaacatga tgtatcctgg cctgcgtgcg gcaagcattt cacccaacac 1200
gcggtcaatc tcgaaacagg cgttctgttc atctaatatt gcgctggcag cggatatcgc 1260
ctgactcatg cgataaaaat gacgaaaatc gccgtcaccg agggtatggt gcatcaattc 1320
gccacgctgc tgcgcagcgc tacagggcgc gccgacgata tgcaagaccg ggacatattc 1380
cgcgtaactg cccgcgatac cgttaatagc gctaagttct cccacgccaa aggtggtgag 1440
tagcgctcca gcgcccgaca tgcgcgcata gccgtccgcg gcataagcgg cgttcagctc 1500
attggcgcat cccacccaac gcagggtcgg gtggtcaatc acatggtcaa gaaactgcaa 1560
gttataatcg cccggtacgc caaaaagatg gccaatgccg catcctgcca gtctgtccag 1620
caaatagtcg gccacggtat aggggttttg cat 1653
<210>195
<211>550
<212>PRT
<213>鼠伤寒沙门氏菌
<400>195
Met Gln Asn Pro Tyr Thr Val Ala Asp Tyr Leu Leu Asp Arg Leu Ala
1 5 10 15
Gly Cys Gly Ile Gly His Leu Phe Gly Val Pro Gly Asp Tyr Asn Leu
20 25 30
Gln Phe Leu Asp His Val Ile Asp His Pro Thr Leu Arg Trp Val Gly
35 40 45
Cys Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg
50 55 60
Met Ser Gly Ala Gly Ala Leu Leu Thr Thr Phe Gly Val Gly Glu Leu
65 70 75 80
Ser Ala Ile Asn Gly Ile Ala Gly Ser Tyr Ala Glu Tyr Val Pro Val
85 90 95
Leu His Ile Val Gly Ala Pro Cys Ser Ala Ala Gln Gln Arg Gly Glu
100 105 110
Leu Met His His Thr Leu Gly Asp Gly Asp Phe Arg His Phe Tyr Arg
115 120 125
Met Ser Gln Ala Ile Ser Ala Ala Ser Ala Ile Leu Asp Glu Gln Asn
130 135 140
Ala Cys Phe Glu Ile Asp Arg Val Leu Gly Glu Met Leu Ala Ala Arg
145 150 155 160
Arg Pro Gly Tyr Ile Met Leu Pro Ala Asp Val Ala Lys Lys Thr Ala
165 170 175
Ile Pro Pro Thr Gln Ala Leu Ala Leu Pro Val His Glu Ala Gln Ser
180 185 190
Gly Val Glu Thr Ala Phe Arg Tyr His Ala Arg Gln Cys Leu Met Asn
195 200 205
Ser Arg Arg Ile Ala Leu Leu Ala Asp Phe Leu Ala Gly Arg Phe Gly
210 215 220
Leu Arg Pro Leu Leu Gln Arg Trp Met Ala Glu Thr Pro Ile Ala His
225 230 235 240
Ala Thr Leu Leu Met Gly Lys Gly Leu Phe Asp Glu Gln His Pro Asn
245 250 255
Phe Val Gly Thr Tyr Ser Ala Gly Ala Ser Ser Lys Glu Val Arg Gln
260 265 270
Ala Ile Glu Asp Ala Asp Arg Val Ile Cys Val Gly Thr Arg Phe Val
275 280 285
Asp Thr Leu Thr Ala Gly Phe Thr Gln Gln Leu Pro Ala Glu Arg Thr
290 295 300
Leu Glu Ile Gln Pro Tyr Ala Ser Arg Ile Gly Glu Thr Trp Phe Asn
305 310 315 320
Leu Pro Met Ala Gln Ala Val Ser Thr Leu Arg Glu Leu Cys Leu Glu
325 330 335
Cys Ala Phe Ala Pro Pro Pro Thr Arg Ser Ala Gly Gln Pro Val Arg
340 345 350
Ile Asp Lys Gly Glu Leu Thr Gln Glu Ser Phe Trp Gln Thr Leu Gln
355 360 365
Gln Tyr Leu Lys Pro Gly Asp Ile Ile Leu Val Asp Gln Gly Thr Ala
370 375 380
Ala Phe Gly Ala Ala Ala Leu Ser Leu Pro Asp Gly Ala Glu Val Val
385 390 395 400
Leu Gln Pro Leu Trp Gly Ser Ile Gly Tyr Ser Leu Pro Ala Ala Phe
405 410 415
Gly Ala Gln Thr Ala Cys Pro Asp Arg Arg Val Ile Leu Ile Ile Gly
420 425 430
Asp Gly Ala Ala Gln Leu Thr Ile Gln Glu Met Gly Ser Met Leu Arg
435 440 445
Asp Gly Gln Ala Pro Val Ile Leu Leu Leu Asn Asn Asp Gly Tyr Thr
450 455 460
Val Glu Arg Ala Ile His Gly Ala Ala Gln Arg Tyr Asn Asp Ile Ala
465 470 475 480
Ser Trp Asn Trp Thr Gln Ile Pro Pro Ala Leu Asn Ala Ala Gln Gln
485 490 495
Ala Glu Cys Trp Arg Val Thr Gln Ala Ile Gln Leu Ala Glu Val Leu
500 505 510
Glu Arg Leu Ala Arg Pro Gln Arg Leu Ser Phe Ile Glu Val Met Leu
515 520 525
Pro Lys Ala Asp Leu Pro Glu Leu Leu Arg Thr Val Thr Arg Ala Leu
530 535 540
Glu Ala Arg Asn Gly Gly
545 550
<210>196
<211>1665
<212>DNA
<213>丙醇丁酮梭菌
<400>196
ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60
cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120
tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180
ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240
agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300
ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360
ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420
agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480
cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540
aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600
gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660
tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720
gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780
tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840
attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900
aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960
cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020
gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080
ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140
ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200
gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260
ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320
caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380
ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440
aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500
agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560
aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620
cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 1665
<210>197
<211>554
<212>PRT
<213>丙醇丁酮梭菌
<400>197
Met Lys Ser Glu Tyr Thr Ile Gly Arg Tyr Leu Leu Asp Arg Leu Ser
1 5 10 15
Glu Leu Gly Ile Arg His Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu
20 25 30
Ser Phe Leu Asp Tyr Ile Met Glu Tyr Lys Gly Ile Asp Trp Val Gly
35 40 45
Asn Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp Gly Tyr Ala Arg
50 55 60
Ile Asn Gly Ile Gly Ala Ile Leu Thr Thr Phe Gly Val Gly Glu Leu
65 70 75 80
Ser Ala Ile Asn Ala Ile Ala Gly Ala Tyr Ala Glu Gln Val Pro Val
85 90 95
Val Lys Ile Thr Gly Ile Pro Thr Ala Lys Val Arg Asp Asn Gly Leu
100 105 110
Tyr Val His His Thr Leu Gly Asp Gly Arg Phe Asp His Phe Phe Glu
115 120 125
Met Phe Arg Glu Val Thr Val Ala Glu Ala Leu Leu Ser Glu Glu Asn
130 135 140
Ala Ala Gln Glu Ile Asp Arg Val Leu Ile Ser Cys Trp Arg Gln Lys
145 150 155 160
Arg Pro Val Leu Ile Asn Leu Pro Ile Asp Val Tyr Asp Lys Pro Ile
165 170 175
Asn Lys Pro Leu Lys Pro Leu Leu Asp Tyr Thr Ile Ser Ser Asn Lys
180 185 190
Glu Ala Ala Cys Glu Phe Val Thr Glu Ile Val Pro Ile Ile Asn Arg
195 200 205
Ala Lys Lys Pro Val Ile Leu Ala Asp Tyr Gly Val Tyr Arg Tyr Gln
210 215 220
Val Gln His Val Leu Lys Asn Leu Ala Glu Lys Thr Gly Phe Pro Val
225 230 235 240
Ala Thr Leu Ser Met Gly Lys Gly Val Phe Asn Glu Ala His Pro Gln
245 250 255
Phe Ile Gly Val Tyr Asn Gly Asp Val Ser Ser Pro Tyr Leu Arg Gln
260 265 270
Arg Val Asp Glu Ala Asp Cys Ile Ile Ser Val Gly Val Lys Leu Thr
275 280 285
Asp Ser Thr Thr Gly Gly Phe Ser His Gly Phe Ser Lys Arg Asn Val
290 295 300
Ile His Ile Asp Pro Phe Ser Ile Lys Ala Lys Gly Lys Lys Tyr Ala
305 310 315 320
Pro Ile Thr Met Lys Asp Ala Leu Thr Glu Leu Thr Ser Lys Ile Glu
325 330 335
His Arg Asn Phe Glu Asp Leu Asp Ile Lys Pro Tyr Lys Ser Asp Asn
340 345 350
Gln Lys Tyr Phe Ala Lys Glu Lys Pro Ile Thr Gln Lys Arg Phe Phe
355 360 365
Glu Arg Ile Ala His Phe Ile Lys Glu Lys Asp Val Leu Leu Ala Glu
370 375 380
Gln Gly Thr Cys Phe Phe Gly Ala Ser Thr Ile Gln Leu Pro Lys Asp
385 390 395 400
Ala Thr Phe Ile Gly Gln Pro Leu Trp Gly Ser Ile Gly Tyr Thr Leu
405 410 415
Pro Ala Leu Leu Gly Ser Gln Leu Ala Asp Gln Lys Arg Arg Asn Ile
420 425 430
Leu Leu Ile Gly Asp Gly Ala Phe Gln Met Thr Ala Gln Glu Ile Ser
435 440 445
Thr Met Leu Arg Leu Gln Ile Lys Pro Ile Ile Phe Leu Ile Asn Asn
450 455 460
Asp Gly Tyr Thr Ile Glu Arg Ala Ile His Gly Arg Glu Gln Val Tyr
465 470 475 480
Asn Asn Ile Gln Met Trp Arg Tyr His Asn Val Pro Lys Val Leu Gly
485 490 495
Pro Lys Glu Cys Ser Leu Thr Phe Lys Val Gln Ser Glu Thr Glu Leu
500 505 510
Glu Lys Ala Leu Leu Val Ala Asp Lys Asp Cys Glu His Leu Ile Phe
515 520 525
Ile Glu Val Val Met Asp Arg Tyr Asp Lys Pro Glu Pro Leu Glu Arg
530 535 540
Leu Ser Lys Arg Phe Ala Asn Gln Asn Asn
545 550
<210>198
<211>939
<212>DNA
<213>酿酒酵母
<400>198
atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60
ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120
gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180
gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240
aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300
ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360
agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420
gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480
ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540
gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600
ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660
ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720
aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780
gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840
cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900
gttgatatga agtggggatc cttcccaatt ttccaatga 939
<210>199
<211>312
<212>PRT
<213>酿酒酵母
<400>199
Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr
1 5 10 15
Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp
20 25 30
Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg
35 40 45
His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg
50 55 60
Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr
65 70 75 80
Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys
85 90 95
Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His
100 105 110
Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu
115 120 125
Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu
130 135 140
Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr
145 150 155 160
Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile
165 170 175
Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn
180 185 190
Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe
195 200 205
Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser
210 215 220
Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys
225 230 235 240
Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln
245 250 255
Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val
260 265 270
Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile
275 280 285
Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys
290 295 300
Trp Gly Ser Phe Pro Ile Phe Gln
305 310
<210>200
<211>1083
<212>DNA
<213>酿酒酵母
<400>200
ctagtctgaa aattctttgt cgtagccgac taaggtaaat ctatatctaa cgtcaccctt 60
ttccatcctt tcgaaggctt catggacgcc ggcttcacca acaggtaatg tttccaccca 120
aattttgata tctttttcag agactaattt caagagttgg ttcaattctt tgatggaacc 180
taaagcactg taagaaatgg agacagcctt taagccatat ggctttagcg ataacatttc 240
gtgttgttct ggtatagaga ttgagacaat tctaccacca accttcatag cctttggcat 300
aatgttgaag tcaatgtcgg taagggagga agcacagact acaatcaggt cgaaggtgtc 360
aaagtacttt tcaccccaat caccttcttc taatgtagca atgtagtgat cggcgcccat 420
cttcattgca tcttctcttt ttctcgaaga acgagaaata acatacgtct ctgcccccat 480
ggctttggaa atcaatgtac ccatactgcc gataccacca agaccaacta taccaacttt 540
tttacctgga ccgcaaccgt tacgaaccaa tggagagtac acagtcaaac caccacataa 600
tagtggagca gccaaatgtg atggaatatt ctctgggata ggcaccacaa aatgttcatg 660
aactctgacg tagtttgcat agccaccctg cgacacatag ccgtcttcat aaggctgact 720
gtatgtggta acaaacttgg tgcagtatgg ttcattatca ttcttacaac ggtcacattc 780
caagcatgaa aagacttgag cacctacacc aacacgttga ccgactttca acccactgtt 840
tgacttgggc cctagcttga caactttacc aacgatttca tgaccaacga ctagcggcat 900
cttcatattg ccccaatgac cagctgcaca atgaatatca ctaccgcaga caccacatgc 960
ttcgatctta atgtcaatgt catgatcgta aaatggtttt gggtcatact ttgtcttctt 1020
tgggtttttc caatcttcgt gtgattgaat agcgatacct tcaaatttct caggataaga 1080
cat 1083
<210>201
<211>360
<212>PRT
<213>酿酒酵母
<400>201
Met Ser Tyr Pro Glu Lys Phe Glu Gly Ile Ala Ile Gln Ser His Glu
1 5 10 15
Asp Trp Lys Asn Pro Lys Lys Thr Lys Tyr Asp Pro Lys Pro Phe Tyr
20 25 30
Asp His Asp Ile Asp Ile Lys Ile Glu Ala Cys Gly Val Cys Gly Ser
35 40 45
Asp Ile His Cys Ala Ala Gly His Trp Gly Asn Met Lys Met Pro Leu
50 55 60
Val Val Gly His Glu Ile Val Gly Lys Val Val Lys Leu Gly Pro Lys
65 70 75 80
Ser Asn Ser Gly Leu Lys Val Gly Gln Arg Val Gly Val Gly Ala Gln
85 90 95
Val Phe Ser Cys Leu Glu Cys Asp Arg Cys Lys Asn Asp Asn Glu Pro
100 105 110
Tyr Cys Thr Lys Phe Val Thr Thr Tyr Ser Gln Pro Tyr Glu Asp Gly
115 120 125
Tyr Val Ser Gln Gly Gly Tyr Ala Asn Tyr Val Arg Val His Glu His
130 135 140
Phe Val Val Pro Ile Pro Glu Asn Ile Pro Ser His Leu Ala Ala Pro
145 150 155 160
Leu Leu Cys Gly Gly Leu Thr Val Tyr Ser Pro Leu Val Arg Asn Gly
165 170 175
Cys Gly Pro Gly Lys Lys Val Gly Ile Val Gly Leu Gly Gly Ile Gly
180 185 190
Ser Met Gly Thr Leu Ile Ser Lys Ala Met Gly Ala Glu Thr Tyr Val
195 200 205
Ile Ser Arg Ser Ser Arg Lys Arg Glu Asp Ala Met Lys Met Gly Ala
210 215 220
Asp His Tyr Ile Ala Thr Leu Glu Glu Gly Asp Trp Gly Glu Lys Tyr
225 230 235 240
Phe Asp Thr Phe Asp Leu Ile Val Val Cys Ala Ser Ser Leu Thr Asp
245 250 255
Ile Asp Phe Asn Ile Met Pro Lys Ala Met Lys Val Gly Gly Arg Ile
260 265 270
Val Ser Ile Ser Ile Pro Glu Gln His Glu Met Leu Ser Leu Lys Pro
275 280 285
Tyr Gly Leu Lys Ala Val Ser Ile Ser Tyr Ser Ala Leu Gly Ser Ile
290 295 300
Lys Glu Leu Asn Gln Leu Leu Lys Leu Val Ser Glu Lys Asp Ile Lys
305 310 315 320
Ile Trp Val Glu Thr Leu Pro Val Gly Glu Ala Gly Val His Glu Ala
325 330 335
Phe Glu Arg Met Glu Lys Gly Asp Val Arg Tyr Arg Phe Thr Leu Val
340 345 350
Gly Tyr Asp Lys Glu Phe Ser Asp
355 360
<210>202
<211>1170
<212>DNA
<213>丙醇丁酮梭菌
<400>202
ttaataagat tttttaaata tctcaagaac atcctctgca tttattggtc ttaaacttcc 60
tattgttcct ccagaatttc taacagcttg ctttgccatt agttctagtt tatcttttcc 120
tattccaact tctctaagct ttgaaggaat acccaatgaa ttaaagtatt ctctcgtatt 180
tttaatagcc tctcgtgcta tttcatagtt atctttgttc ttgtctattc cccaaacatt 240
tattccataa gaaacaaatt tatgaagtgt atcgtcattt agaatatatt ccatccaatt 300
aggtgttaaa attgcaagtc ctacaccatg tgttatatca taatatgcac ttaactcgtg 360
ttccatagga tgacaactcc attttctatc cttaccaagt gataatagac catttatagc 420
taaacttgaa gcccacatca aattagctct agcctcgtaa tcatcagtct tctccattgc 480
tatttttcca tactttatac atgttcttaa gattgcttct gctataccgt cctgcacata 540
agcaccttca acaccactaa agtaagattc aaaggtgtga ctcataatgt cagctgttcc 600
cgctgctgtt tgatttttag gtactgtaaa agtatatgta ggatctaaca ctgaaaattt 660
aggtctcata tcatcatgtc ctactccaag cttttcatta gtctccatat ttgaaattac 720
tgcaatttga tccatttcag accctgttgc tgaaagagta agtatacttg caattggaag 780
aactttagtt attttagatg gatctttaac catgtcccat gtatcgccat cataataaac 840
tccagctgca attaccttag aacagtctat tgcacttcct ccccctattg ctaatactaa 900
atccacatta ttttctctac atatttctat gccttttttt actgttgtta tcctaggatt 960
tggctctact cctgaaagtt catagaaagc tatattgttt tcttttaata tagctgttgc 1020
tctatcatat ataccgttcc tttttatact tcctccgcca taaactataa gcactcttga 1080
gccatatttc ttaatttctt ctccaattac gtctattttt ccttttccaa aaaaaacttt 1140
agttggtatt gaataatcaa aacttagcat 1170
<210>203
<211>389
<212>PRT
<213>丙醇丁酮梭菌
<400>203
Met Leu Ser Phe Asp Tyr Ser Ile Pro Thr Lys Val Phe Phe Gly Lys
1 5 10 15
Gly Lys Ile Asp Val Ile Gly Glu Glu Ile Lys Lys Tyr Gly Ser Arg
20 25 30
Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr
35 40 45
Asp Arg Ala Thr Ala Ile Leu Lys Glu Asn Asn Ile Ala Phe Tyr Glu
50 55 60
Leu Ser Gly Val Glu Pro Asn Pro Arg Ile Thr Thr Val Lys Lys Gly
65 70 75 80
Ile Glu Ile Cys Arg Glu Asn Asn Val Asp Leu Val Leu Ala Ile Gly
85 90 95
Gly Gly Ser Ala Ile Asp Cys Ser Lys Val Ile Ala Ala Gly Val Tyr
100 105 110
Tyr Asp Gly Asp Thr Trp Asp Met Val Lys Asp Pro Ser Lys Ile Thr
115 120 125
Lys Val Leu Pro Ile Ala Ser Ile Leu Thr Leu Ser Ala Thr Gly Ser
130 135 140
Glu Met Asp Gln Ile Ala Val Ile Ser Asn Met Glu Thr Asn Glu Lys
145 150 155 160
Leu Gly Val Gly His Asp Asp Met Arg Pro Lys Phe Ser Val Leu Asp
165 170 175
Pro Thr Tyr Thr Phe Thr Val Pro Lys Asn Gln Thr Ala Ala Gly Thr
180 185 190
Ala Asp Ile Met Ser His Thr Phe Glu Ser Tyr Phe Ser Gly Val Glu
195 200 205
Gly Ala Tyr Val Gln Asp Gly Ile Ala Glu Ala Ile Leu Arg Thr Cys
210 215 220
Ile Lys Tyr Gly Lys Ile Ala Met Glu Lys Thr Asp Asp Tyr Glu Ala
225 230 235 240
Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu
245 250 255
Ser Leu Gly Lys Asp Arg Lys Trp Ser Cys His Pro Met Glu His Glu
260 265 270
Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu
275 280 285
Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asp Asp Thr Leu His Lys
290 295 300
Phe Val Ser Tyr Gly Ile Asn Val Trp Gly Ile Asp Lys Asn Lys Asp
305 310 315 320
Asn Tyr Glu Ile Ala Arg Glu Ala Ile Lys Asn Thr Arg Glu Tyr Phe
325 330 335
Asn Ser Leu Gly Ile Pro Ser Lys Leu Arg Glu Val Gly Ile Gly Lys
340 345 350
Asp Lys Leu Glu Leu Met Ala Lys Gln Ala Val Arg Asn Ser Gly Gly
355 360 365
Thr Ile Gly Ser Leu Arg Pro Ile Asn Ala Glu Asp Val Leu Glu Ile
370 375 380
Phe Lys Lys Ser Tyr
385
<210>204
<211>390
<212>PRT
<213>丙醇丁酮梭菌
<400>204
Met Val Asp Phe Glu Tyr Ser Ile Pro Thr Arg Ile Phe Phe Gly Lys
1 5 10 15
Asp Lys Ile Asn Val Leu Gly Arg Glu Leu Lys Lys Tyr Gly Ser Lys
20 25 30
Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr
35 40 45
Asp Lys Ala Val Ser Ile Leu Glu Lys Asn Ser Ile Lys Phe Tyr Glu
50 55 60
Leu Ala Gly Val Glu Pro Asn Pro Arg Val Thr Thr Val Glu Lys Gly
65 70 75 80
Val Lys Ile Cys Arg Glu Asn Gly Val Glu Val Val Leu Ala Ile Gly
85 90 95
Gly Gly Ser Ala Ile Asp Cys Ala Lys Val Ile Ala Ala Ala Cys Glu
100 105 110
Tyr Asp Gly Asn Pro Trp Asp Ile Val Leu Asp Gly Ser Lys Ile Lys
115 120 125
Arg Val Leu Pro Ile Ala Ser Ile Leu Thr Ile Ala Ala Thr Gly Ser
130 135 140
Glu Met Asp Thr Trp Ala Val Ile Asn Asn Met Asp Thr Asn Glu Lys
145 150 155 160
Leu Ile Ala Ala His Pro Asp Met Ala Pro Lys Phe Ser Ile Leu Asp
165 170 175
Pro Thr Tyr Thr Tyr Thr Val Pro Thr Asn Gln Thr Ala Ala Gly Thr
180 185 190
Ala Asp Ile Met Ser His Ile Phe Glu Val Tyr Phe Ser Asn Thr Lys
195 200 205
Thr Ala Tyr Leu Gln Asp Arg Met Ala Glu Ala Leu Leu Arg Thr Cys
210 215 220
Ile Lys Tyr Gly Gly Ile Ala Leu Glu Lys Pro Asp Asp Tyr Glu Ala
225 230 235 240
Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu
245 250 255
Thr Tyr Gly Lys Asp Thr Asn Trp Ser Val His Leu Met Glu His Glu
260 265 270
Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu
275 280 285
Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asn Asp Thr Val Tyr Lys
290 295 300
Phe Val Glu Tyr Gly Val Asn Val Trp Gly Ile Asp Lys Glu Lys Asn
305 310 315 320
His Tyr Asp Ile Ala His Gln Ala Ile Gln Lys Thr Arg Asp Tyr Phe
325 330 335
Val Asn Val Leu Gly Leu Pro Ser Arg Leu Arg Asp Val Gly Ile Glu
340 345 350
Glu Glu Lys Leu Asp Ile Met Ala Lys Glu Ser Val Lys Leu Thr Gly
355 360 365
Gly Thr Ile Gly Asn Leu Arg Pro Val Asn Ala Ser Glu Val Leu Gln
370 375 380
Ile Phe Lys Lys Ser Val
385 390
<210>205
<211>993
<212>DNA
<213>枯草芽孢杆菌
<400>205
atgagtacaa accgacatca agcactaggg ctgactgatc aggaagccgt tgatatgtat 60
agaaccatgc tgttagcaag aaaaatcgat gaaagaatgt ggctgttaaa ccgttctggc 120
aaaattccat ttgtaatctc ttgtcaagga caggaagcag cacaggtagg agcggctttc 180
gcacttgacc gtgaaatgga ttatgtattg ccgtactaca gagacatggg tgtcgtgctc 240
gcgtttggca tgacagcaaa ggacttaatg atgtccgggt ttgcaaaagc agcagatccg 300
aactcaggag gccgccagat gccgggacat ttcggacaaa agaaaaaccg cattgtgacg 360
ggatcatctc cggttacaac gcaagtgccg cacgcagtcg gtattgcgct tgcgggacgt 420
atggagaaaa aggatatcgc agcctttgtt acattcgggg aagggtcttc aaaccaaggc 480
gatttccatg aaggggcaaa ctttgccgct gtccataagc tgccggttat tttcatgtgt 540
gaaaacaaca aatacgcaat ctcagtgcct tacgataagc aagtcgcatg tgagaacatt 600
tccgaccgtg ccataggcta tgggatgcct ggcgtaactg tgaatggaaa tgatccgctg 660
gaagtttatc aagcggttaa agaagcacgc gaaagggcac gcagaggaga aggcccgaca 720
ttaattgaaa cgatttctta ccgccttaca ccacattcca gtgatgacga tgacagcagc 780
tacagaggcc gtgaagaagt agaggaagcg aaaaaaagtg atcccctgct tacttatcaa 840
gcttacttaa aggaaacagg cctgctgtcc gatgagatag aacaaaccat gctggatgaa 900
attatggcaa tcgtaaatga agcgacggat gaagcggaga acgccccata tgcagctcct 960
gagtcagcgc ttgattatgt ttatgcgaag tag 993
<210>206
<211>330
<212>PRT
<213>枯草芽孢杆菌
<400>206
Met Ser Thr Asn Arg His Gln Ala Leu Gly Leu Thr Asp Gln Glu Ala
1 5 10 15
Val Asp Met Tyr Arg Thr Met Leu Leu Ala Arg Lys Ile Asp Glu Arg
20 25 30
Met Trp Leu Leu Asn Arg Ser Gly Lys Ile Pro Phe Val Ile Ser Cys
35 40 45
Gln Gly Gln Glu Ala Ala Gln Val Gly Ala Ala Phe Ala Leu Asp Arg
50 55 60
Glu Met Asp Tyr Val Leu Pro Tyr Tyr Arg Asp Met Gly Val Val Leu
65 70 75 80
Ala Phe Gly Met Thr Ala Lys Asp Leu Met Met Ser Gly Phe Ala Lys
85 90 95
Ala Ala Asp Pro Asn Ser Gly Gly Arg Gln Met Pro Gly His Phe Gly
100 105 110
Gln Lys Lys Asn Arg Ile Val Thr Gly Ser Ser Pro Val Thr Thr Gln
115 120 125
Val Pro His Ala Val Gly Ile Ala Leu Ala Gly Arg Met Glu Lys Lys
130 135 140
Asp Ile Ala Ala Phe Val Thr Phe Gly Glu Gly Ser Ser Asn Gln Gly
145 150 155 160
Asp Phe His Glu Gly Ala Asn Phe Ala Ala Val His Lys Leu Pro Val
165 170 175
Ile Phe Met Cys Glu Asn Asn Lys Tyr Ala Ile Ser Val Pro Tyr Asp
180 185 190
Lys Gln Val Ala Cys Glu Asn Ile Ser Asp Arg Ala Ile Gly Tyr Gly
195 200 205
Met Pro Gly Val Thr Val Asn Gly Asn Asp Pro Leu Glu Val Tyr Gln
210 215 220
Ala Val Lys Glu Ala Arg Glu Arg Ala Arg Arg Gly Glu Gly Pro Thr
225 230 235 240
Leu Ile Glu Thr Ile Ser Tyr Arg Leu Thr Pro His Ser Ser Asp Asp
245 250 255
Asp Asp Ser Ser Tyr Arg Gly Arg Glu Glu Val Glu Glu Ala Lys Lys
260 265 270
Ser Asp Pro Leu Leu Thr Tyr Gln Ala Tyr Leu Lys Glu Thr Gly Leu
275 280 285
Leu Ser Asp Glu Ile Glu Gln Thr Met Leu Asp Glu Ile Met Ala Ile
290 295 300
Val Asn Glu Ala Thr Asp Glu Ala Glu Asn Ala Pro Tyr Ala Ala Pro
305 310 315 320
Glu Ser Ala Leu Asp Tyr Val Tyr Ala Lys
325 330
<210>207
<211>984
<212>DNA
<213>枯草芽孢杆菌
<400>207
atgtcagtaa tgtcatatat tgatgcaatc aatttggcga tgaaagaaga aatggaacga 60
gattctcgcg ttttcgtcct tggggaagat gtaggaagaa aaggcggtgt gtttaaagcg 120
acagcgggac tctatgaaca atttggggaa gagcgcgtta tggatacgcc gcttgctgaa 180
tctgcaatcg caggagtcgg tatcggagcg gcaatgtacg gaatgagacc gattgctgaa 240
atgcagtttg ctgatttcat tatgccggca gtcaaccaaa ttatttctga agcggctaaa 300
atccgctacc gcagcaacaa tgactggagc tgtccgattg tcgtcagagc gccatacggc 360
ggaggcgtgc acggagccct gtatcattct caatcagtcg aagcaatttt cgccaaccag 420
cccggactga aaattgtcat gccatcaaca ccatatgacg cgaaagggct cttaaaagcc 480
gcagttcgtg acgaagaccc cgtgctgttt tttgagcaca agcgggcata ccgtctgata 540
aagggcgagg ttccggctga tgattatgtc ctgccaatcg gcaaggcgga cgtaaaaagg 600
gaaggcgacg acatcacagt gatcacatac ggcctgtgtg tccacttcgc cttacaagct 660
gcagaacgtc tcgaaaaaga tggcatttca gcgcatgtgg tggatttaag aacagtttac 720
ccgcttgata aagaagccat catcgaagct gcgtccaaaa ctggaaaggt tcttttggtc 780
acagaagata caaaagaagg cagcatcatg agcgaagtag ccgcaattat atccgagcat 840
tgtctgttcg acttagacgc gccgatcaaa cggcttgcag gtcctgatat tccggctatg 900
ccttatgcgc cgacaatgga aaaatacttt atggtcaacc ctgataaagt ggaagcggcg 960
atgagagaat tagcggagtt ttaa 984
<210>208
<211>327
<212>PRT
<213>枯草芽孢杆菌
<400>208
Met Ser Val Met Ser Tyr Ile Asp Ala Ile Asn Leu Ala Met Lys Glu
1 5 10 15
Glu Met Glu Arg Asp Ser Arg Val Phe Val Leu Gly Glu Asp Val Gly
20 25 30
Arg Lys Gly Gly Val Phe Lys Ala Thr Ala Gly Leu Tyr Glu Gln Phe
35 40 45
Gly Glu Glu Arg Val Met Asp Thr Pro Leu Ala Glu Ser Ala Ile Ala
50 55 60
Gly Val Gly Ile Gly Ala Ala Met Tyr Gly Met Arg Pro Ile Ala Glu
65 70 75 80
Met Gln Phe Ala Asp Phe Ile Met Pro Ala Val Asn Gln Ile Ile Ser
85 90 95
Glu Ala Ala Lys Ile Arg Tyr Arg Ser Asn Asn Asp Trp Ser Cys Pro
100 105 110
Ile Val Val Arg Ala Pro Tyr Gly Gly Gly Val His Gly Ala Leu Tyr
115 120 125
His Ser Gln Ser Val Glu Ala Ile Phe Ala Asn Gln Pro Gly Leu Lys
130 135 140
Ile Val Met Pro Ser Thr Pro Tyr Asp Ala Lys Gly Leu Leu Lys Ala
145 150 155 160
Ala Val Arg Asp Glu Asp Pro Val Leu Phe Phe Glu His Lys Arg Ala
165 170 175
Tyr Arg Leu Ile Lys Gly Glu Val Pro Ala Asp Asp Tyr Val Leu Pro
180 185 190
Ile Gly Lys Ala Asp Val Lys Arg Glu Gly Asp Asp Ile Thr Val Ile
195 200 205
Thr Tyr Gly Leu Cys Val His Phe Ala Leu Gln Ala Ala Glu Arg Leu
210 215 220
Glu Lys Asp Gly Ile Ser Ala His Val Val Asp Leu Arg Thr Val Tyr
225 230 235 240
Pro Leu Asp Lys Glu Ala Ile Ile Glu Ala Ala Ser Lys Thr Gly Lys
245 250 255
Val Leu Leu Val Thr Glu Asp Thr Lys Glu Gly Ser Ile Met Ser Glu
260 265 270
Val Ala Ala Ile Ile Ser Glu His Cys Leu Phe Asp Leu Asp Ala Pro
275 280 285
Ile Lys Arg Leu Ala Gly Pro Asp Ile Pro Ala Met ProTyr Ala Pro
290 295 300
Thr Met Glu Lys Tyr Phe Met Val Asn Pro Asp Lys Val Glu Ala Ala
305 310 315 320
Met Arg Glu Leu Ala Glu Phe
325
<210>209
<211>1275
<212>DNA
<213>枯草芽孢杆菌
<400>209
atggcaattg aacaaatgac gatgccgcag cttggagaaa gcgtaacaga ggggacgatc 60
agcaaatggc ttgtcgcccc cggtgataaa gtgaacaaat acgatccgat cgcggaagtc 120
atgacagata aggtaaatgc agaggttccg tcttctttta ctggtacgat aacagagctt 180
gtgggagaag aaggccaaac cctgcaagtc ggagaaatga tttgcaaaat tgaaacagaa 240
ggcgcgaatc cggctgaaca aaaacaagaa cagccagcag catcagaagc cgctgagaac 300
cctgttgcaa aaagtgctgg agcagccgat cagcccaata aaaagcgcta ctcgccagct 360
gttctccgtt tggccggaga gcacggcatt gacctcgatc aagtgacagg aactggtgcc 420
ggcgggcgca tcacacgaaa agatattcag cgcttaattg aaacaggcgg cgtgcaagaa 480
cagaatcctg aggagctgaa aacagcagct cctgcaccga agtctgcatc aaaacctgag 540
ccaaaagaag agacgtcata tcctgcgtct gcagccggtg ataaagaaat ccctgtcaca 600
ggtgtaagaa aagcaattgc ttccaatatg aagcgaagca aaacagaaat tccgcatgct 660
tggacgatga tggaagtcga cgtcacaaat atggttgcat atcgcaacag tataaaagat 720
tcttttaaga agacagaagg ctttaattta acgttcttcg ccttttttgt aaaagcggtc 780
gctcaggcgt taaaagaatt cccgcaaatg aatagcatgt gggcggggga caaaattatt 840
cagaaaaagg atatcaatat ttcaattgca gttgccacag aggattcttt atttgttccg 900
gtgattaaaa acgctgatga aaaaacaatt aaaggcattg cgaaagacat taccggccta 960
gctaaaaaag taagagacgg aaaactcact gcagatgaca tgcagggagg cacgtttacc 1020
gtcaacaaca caggttcgtt cgggtctgtt cagtcgatgg gcattatcaa ctaccctcag 1080
gctgcgattc ttcaagtaga atccatcgtc aaacgcccgg ttgtcatgga caatggcatg 1140
attgctgtca gagacatggt taatctgtgc ctgtcattag atcacagagt gcttgacggt 1200
ctcgtgtgcg gacgattcct cggacgagtg aaacaaattt tagaatcgat tgacgagaag 1260
acatctgttt actaa 1275
<210>210
<211>424
<212>PRT
<213>枯草芽孢杆菌
<400>210
Met Ala Ile Glu Gln Met Thr Met Pro Gln Leu Gly Glu Ser Val Thr
1 5 10 15
Glu Gly Thr Ile Ser Lys Trp Leu Val Ala Pro Gly Asp Lys Val Asn
20 25 30
Lys Tyr Asp Pro Ile Ala Glu Val Met Thr Asp Lys Val Asn Ala Glu
35 40 45
Val Pro Ser Ser Phe Thr Gly Thr Ile Thr Glu Leu Val Gly Glu Glu
50 55 60
Gly Gln Thr Leu Gln Val Gly Glu Met Ile Cys Lys Ile Glu Thr Glu
65 70 75 80
Gly Ala Asn Pro Ala Glu Gln Lys Gln Glu Gln Pro Ala Ala Ser Glu
85 90 95
Ala Ala Glu Asn Pro Val Ala Lys Ser Ala Gly Ala Ala Asp Gln Pro
100 105 110
Asn Lys Lys Arg Tyr Ser Pro Ala Val Leu Arg Leu Ala Gly Glu His
115 120 125
Gly Ile Asp Leu Asp Gln Val Thr Gly Thr Gly Ala Gly Gly Arg Ile
130 135 140
Thr Arg Lys Asp Ile Gln Arg Leu Ile Glu Thr Gly Gly Val Gln Glu
145 150 155 160
Gln Asn Pro Glu Glu Leu Lys Thr Ala Ala Pro Ala Pro Lys Ser Ala
165 170 175
Ser Lys Pro Glu Pro Lys Glu Glu Thr Ser Tyr Pro Ala Ser Ala Ala
180 185 190
Gly Asp Lys Glu Ile Pro Val Thr Gly Val Arg Lys Ala Ile Ala Ser
195 200 205
Asn Met Lys Arg Ser Lys Thr Glu Ile Pro His Ala Trp Thr Met Met
210 215 220
Glu Val Asp Val Thr Asn Met Val Ala Tyr Arg Asn Ser Ile Lys Asp
225 230 235 240
Ser Phe Lys Lys Thr Glu Gly Phe Asn Leu Thr Phe Phe Ala Phe Phe
245 250 255
Val Lys Ala Val Ala Gln Ala Leu Lys Glu Phe Pro Gln Met Asn Ser
260 265 270
Met Trp Ala Gly Asp Lys Ile Ile Gln Lys Lys Asp Ile Asn Ile Ser
275 280 285
Ile Ala Val Ala Thr Glu Asp Ser Leu Phe Val Pro Val Ile Lys Asn
290 295 300
Ala Asp Glu Lys Thr Ile Lys Gly Ile Ala Lys Asp Ile Thr Gly Leu
305 310 315 320
Ala Lys Lys Val Arg Asp Gly Lys Leu Thr Ala Asp Asp Met Gln Gly
325 330 335
Gly Thr Phe Thr Val Asn Asn Thr Gly Ser Phe Gly Ser Val Gln Ser
340 345 350
Met Gly Ile Ile Asn Tyr Pro Gln Ala Ala Ile Leu Gln Val Glu Ser
355 360 365
Ile Val Lys Arg Pro Val Val Met Asp Asn Gly Met Ile Ala Val Arg
370 375 380
Asp Met Val Asn Leu Cys Leu Ser Leu Asp His Arg Val Leu Asp Gly
385 390 395 400
Leu Val Cys Gly Arg Phe Leu Gly Arg Val Lys Gln Ile Leu Glu Ser
405 410 415
Ile Asp Glu Lys Thr Ser Val Tyr
420
<210>211
<211>1374
<212>DNA
<213>枯草芽孢杆菌
<400>211
atggcaactg agtatgacgt agtcattctg ggcggcggta ccggcggtta tgttgcggcc 60
atcagagccg ctcagctcgg cttaaaaaca gccgttgtgg aaaaggaaaa actcggggga 120
acatgtctgc ataaaggctg tatcccgagt aaagcgctgc ttagaagcgc agaggtatac 180
cggacagctc gtgaagccga tcaattcgga gtggaaacgg ctggcgtgtc cctcaacttt 240
gaaaaagtgc agcagcgtaa gcaagccgtt gttgataagc ttgcagcggg tgtaaatcat 300
ttaatgaaaa aaggaaaaat tgacgtgtac accggatatg gacgtatcct tggaccgtca 360
atcttctctc cgctgccggg aacaatttct gttgagcggg gaaatggcga agaaaatgac 420
atgctgatcc cgaaacaagt gatcattgca acaggatcaa gaccgagaat gcttccgggt 480
cttgaagtgg acggtaagtc tgtactgact tcagatgagg cgctccaaat ggaggagctg 540
ccacagtcaa tcatcattgt cggcggaggg gttatcggta tcgaatgggc gtctatgctt 600
catgattttg gcgttaaggt aacggttatt gaatacgcgg atcgcatatt gccgactgaa 660
gatctagaga tttcaaaaga aatggaaagt cttcttaaga aaaaaggcat ccagttcata 720
acaggggcaa aagtgctgcc tgacacaatg acaaaaacat cagacgatat cagcatacaa 780
gcggaaaaag acggagaaac cgttacctat tctgctgaga aaatgcttgt ttccatcggc 840
agacaggcaa atatcgaagg catcggccta gagaacaccg atattgttac tgaaaatggc 900
atgatttcag tcaatgaaag ctgccaaacg aaggaatctc atatttatgc aatcggagac 960
gtaatcggtg gcctgcagtt agctcacgtt gcttcacatg agggaattat tgctgttgag 1020
cattttgcag gtctcaatcc gcatccgctt gatccgacgc ttgtgccgaa gtgcatttac 1080
tcaagccctg aagctgccag tgtcggctta accgaagacg aagcaaaggc gaacgggcat 1140
aatgtcaaaa tcggcaagtt cccatttatg gcgattggaa aagcgcttgt atacggtgaa 1200
agcgacggtt ttgtcaaaat cgtggctgac cgagatacag atgatattct cggcgttcat 1260
atgattggcc cgcatgtcac cgacatgatt tctgaagcgg gtcttgccaa agtgctggac 1320
gcaacaccgt gggaggtcgg gcaaacgatt tcacccgcat ccaacgcttt ctga 1374
<210>212
<211>457
<212>PRT
<213>枯草芽孢杆菌
<400>212
Met Ala Thr Glu Tyr Asp Val Val Ile Leu Gly Gly Gly Thr Gly Gly
1 5 10 15
Tyr Val Ala Ala Ile Arg Ala Ala Gln Leu Gly Leu Lys Thr Ala Val
20 25 30
Val Glu Lys Glu Lys Leu Gly Gly Thr Cys Leu His Lys Gly Cys Ile
35 40 45
Pro Ser Lys Ala Leu Leu Arg Ser Ala Glu Val Tyr Arg Thr Ala Arg
50 55 60
Glu Ala Asp Gln Phe Gly Val Glu Thr Ala Gly Val Ser Leu Asn Phe
65 70 75 80
Glu Lys Val Gln Gln Arg Lys Gln Ala Val Val Asp Lys Leu Ala Ala
85 90 95
Gly Val Asn His Leu Met Lys Lys Gly Lys Ile Asp Val Tyr Thr Gly
100 105 110
Tyr Gly Arg Ile Leu Gly Pro Ser Ile Phe Ser Pro Leu Pro Gly Thr
115 120 125
Ile Ser Val Glu Arg Gly Asn Gly Glu Glu Asn Asp Met Leu Ile Pro
130 135 140
Lys Gln Val Ile Ile Ala Thr Gly Ser Arg Pro Arg Met Leu Pro Gly
145 150 155c160
Leu Glu Val Asp Gly Lys Ser Val Leu Thr Ser Asp Glu Ala Leu Gln
165 170 175
Met Glu Glu Leu Pro Gln Ser Ile Ile Ile Val Gly Gly Gly Val Ile
180 185 190
Gly Ile Glu Trp Ala Ser Met Leu His Asp Phe Gly Val Lys Val Thr
195 200 205
Val Ile Glu Tyr Ala Asp Arg Ile Leu Pro Thr Glu Asp Leu Glu Ile
210 215 220
Ser Lys Glu Met Glu Ser Leu Leu Lys Lys Lys Gly Ile Gln Phe Ile
225 230 235 240
Thr Gly Ala Lys Val Leu Pro Asp Thr Met Thr Lys Thr Ser Asp Asp
245 250 255
Ile Ser Ile Gln Ala Glu Lys Asp Gly Glu Thr Val Thr Tyr Ser Ala
260 265 270
Glu Lys Met Leu Val Ser Ile Gly Arg Gln Ala Asn Ile Glu Gly Ile
275 280 285
Gly Leu Glu Asn Thr Asp Ile Val Thr Glu Asn Gly Met Ile Ser Val
290 295 300
Asn Glu Ser Cys Gln Thr Lys Glu Ser His Ile Tyr Ala Ile Gly Asp
305 310 315 320
Val Ile Gly Gly Leu Gln Leu Ala His Val Ala Ser His Glu Gly Ile
325 330 335
Ile Ala Val Glu His Phe Ala Gly Leu Asn Pro His Pro Leu Asp Pro
340 345 350
Thr Leu Val Pro Lys Cys Ile Tyr Ser Ser Pro Glu Ala Ala Ser Val
355 360 365
Gly Leu Thr Glu Asp Glu Ala Lys Ala Asn Gly His Asn Val Lys Ile
370 375 380
Gly Lys Phe Pro Phe Met Ala Ile Gly Lys Ala Leu Val Tyr Gly Glu
385 390 395 400
Ser Asp Gly Phe Val Lys Ile Val Ala Asp Arg Asp Thr Asp Asp Ile
405 410 415
Leu Gly Val His Met Ile Gly Pro His Val Thr Asp Met Ile Ser Glu
420 425 430
Ala Gly Leu Ala Lys Val Leu Asp Ala Thr Pro Trp Glu Val Gly Gln
435 440 445
Thr Ile Ser Pro Ala Ser Asn Ala Phe
450 455
<210>213
<211>1233
<212>DNA
<213>恶臭假单胞菌
<400>213
atgaacgagt acgcccccct gcgtttgcat gtgcccgagc ccaccggccg gccaggctgc 60
cagaccgatt tttcctacct gcgcctgaac gatgcaggtc aagcccgtaa accccctgtc 120
gatgtcgacg ctgccgacac cgccgacctg tcctacagcc tggtccgcgt gctcgacgag 180
caaggcgacg cccaaggccc gtgggctgaa gacatcgacc cgcagatcct gcgccaaggc 240
atgcgcgcca tgctcaagac gcggatcttc gacagccgca tggtggttgc ccagcgccag 300
aagaagatgt ccttctacat gcagagcctg ggcgaagaag ccatcggcag cggccaggcg 360
ctggcgctta accgcaccga catgtgcttc cccacctacc gtcagcaaag catcctgatg 420
gcccgcgacg tgtcgctggt ggagatgatc tgccagttgc tgtccaacga acgcgacccc 480
ctcaagggcc gccagctgcc gatcatgtac tcggtacgcg aggccggctt cttcaccatc 540
agcggcaacc tggcgaccca gttcgtgcag gcggtcggct gggccatggc ctcggcgatc 600
aagggcgata ccaagattgc ctcggcctgg atcggcgacg gcgccactgc cgaatcggac 660
ttccacaccg ccctcacctt tgcccacgtt taccgcgccc cggtgatcct caacgtggtc 720
aacaaccagt gggccatctc aaccttccag gccatcgccg gtggcgagtc gaccaccttc 780
gccggccgtg gcgtgggctg cggcatcgct tcgctgcggg tggacggcaa cgacttcgtc 840
gccgtttacg ccgcttcgcg ctgggctgcc gaacgtgccc gccgtggttt gggcccgagc 900
ctgatcgagt gggtcaccta ccgtgccggc ccgcactcga cctcggacga cccgtccaag 960
taccgccctg ccgatgactg gagccacttc ccgctgggtg acccgatcgc ccgcctgaag 1020
cagcacctga tcaagatcgg ccactggtcc gaagaagaac accaggccac cacggccgag 1080
ttcgaagcgg ccgtgattgc tgcgcaaaaa gaagccgagc agtacggcac cctggccaac 1140
ggtcacatcc cgagcgccgc ctcgatgttc gaggacgtgt acaaggagat gcccgaccac 1200
ctgcgccgcc aacgccagga actgggggtt tga 1233
<210>214
<211>410
<212>PRT
<213>恶臭假单胞菌
<400>214
Met Asn Glu Tyr Ala Pro Leu Arg Leu His Val Pro Glu Pro Thr Gly
1 5 10 15
Arg Pro Gly Cys Gln Thr Asp Phe Ser Tyr Leu Arg Leu Asn Asp Ala
20 25 30
Gly Gln Ala Arg Lys Pro Pro Val Asp Val Asp Ala Ala Asp Thr Ala
35 40 45
Asp Leu Ser Tyr Ser Leu Val Arg Val Leu Asp Glu Gln Gly Asp Ala
50 55 60
Gln Gly Pro Trp Ala Glu Asp Ile Asp Pro Gln Ile Leu Arg Gln Gly
65 70 75 80
Met Arg Ala Met Leu Lys Thr Arg Ile Phe Asp Ser Arg Met Val Val
85 90 95
Ala Gln Arg Gln Lys Lys Met Ser Phe Tyr Met Gln Ser Leu Gly Glu
100 105 110
Glu Ala Ile Gly Ser Gly Gln Ala Leu Ala Leu Asn Arg Thr Asp Met
115 120 125
Cys Phe Pro Thr Tyr Arg Gln Gln Ser Ile Leu Met Ala Arg Asp Val
130 135 140
Ser Leu Val Glu Met Ile Cys Gln Leu Leu Ser Asn Glu Arg Asp Pro
145 150 155 160
Leu Lys Gly Arg Gln Leu Pro Ile Met Tyr Ser Val Arg Glu Ala Gly
165 170 175
Phe Phe Thr Ile Ser Gly Asn Leu Ala Thr Gln Phe Val Gln Ala Val
180 185 190
Gly Trp Ala Met Ala Ser Ala Ile Lys Gly Asp Thr Lys Ile Ala Ser
195 200 205
Ala Trp Ile Gly Asp Gly Ala Thr Ala Glu Ser Asp Phe His Thr Ala
210 215 220
Leu Thr Phe Ala His Val Tyr Arg Ala Pro Val Ile Leu Asn Val Val
225 230 235 240
Asn Asn Gln Trp Ala Ile Ser Thr Phe Gln Ala Ile Ala Gly Gly Glu
245 250 255
Ser Thr Thr Phe Ala Gly Arg Gly Val Gly Cys Gly Ile Ala Ser Leu
260 265 270
Arg Val Asp Gly Asn Asp Phe Val Ala Val Tyr Ala Ala Ser Arg Trp
275 280 285
Ala Ala Glu Arg Ala Arg Arg Gly Leu Gly Pro Ser Leu Ile Glu Trp
290 295 300
Val Thr Tyr Arg Ala Gly Pro His Ser Thr Ser Asp Asp Pro Ser Lys
305 310 315 320
Tyr Arg Pro Ala Asp Asp Trp Ser His Phe Pro Leu Gly Asp Pro Ile
325 330 335
Ala Arg Leu Lys Gln His Leu Ile Lys Ile Gly His Trp Ser Glu Glu
340 345 350
Glu His Gln Ala Thr Thr Ala Glu Phe Glu Ala Ala Val Ile Ala Ala
355 360 365
Gln Lys Glu Ala Glu Gln Tyr Gly Thr Leu Ala Asn Gly His Ile Pro
370 375 380
Ser Ala Ala Ser Met Phe Glu Asp Val Tyr Lys Glu Met Pro Asp His
385 390 395 400
Leu Arg Arg Gln Arg Gln Glu Leu Gly Val
405 410
<210>215
<211>1059
<212>DNA
<213>恶臭假单胞菌
<400>215
atgaacgacc acaacaacag catcaacccg gaaaccgcca tggccaccac taccatgacc 60
atgatccagg ccctgcgctc ggccatggat gtcatgcttg agcgcgacga caatgtggtg 120
gtgtacggcc aggacgtcgg ctacttcggc ggcgtgttcc gctgcaccga aggcctgcag 180
accaagtacg gcaagtcccg cgtgttcgac gcgcccatct ctgaaagcgg catcgtcggc 240
accgccgtgg gcatgggtgc ctacggcctg cgcccggtgg tggaaatcca gttcgctgac 300
tacttctacc cggcctccga ccagatcgtt tctgaaatgg cccgcctgcg ctaccgttcg 360
gccggcgagt tcatcgcccc gctgaccctg cgtatgccct gcggtggcgg tatctatggc 420
ggccagacac acagccagag cccggaagcg atgttcactc aggtgtgcgg cctgcgcacc 480
gtaatgccat ccaacccgta cgacgccaaa ggcctgctga ttgcctcgat cgaatgcgac 540
gacccggtga tcttcctgga gcccaagcgc ctgtacaacg gcccgttcga cggccaccat 600
gaccgcccgg ttacgccgtg gtcgaaacac ccgcacagcg ccgtgcccga tggctactac 660
accgtgccac tggacaaggc cgccatcacc cgccccggca atgacgtgag cgtgctcacc 720
tatggcacca ccgtgtacgt ggcccaggtg gccgccgaag aaagtggcgt ggatgccgaa 780
gtgatcgacc tgcgcagcct gtggccgcta gacctggaca ccatcgtcga gtcggtgaaa 840
aagaccggcc gttgcgtggt agtacacgag gccacccgta cttgtggctt tggcgcagaa 900
ctggtgtcgc tggtgcagga gcactgcttc caccacctgg aggcgccgat cgagcgcgtc 960
accggttggg acacccccta ccctcacgcg caggaatggg cttacttccc agggccttcg 1020
cgggtaggtg cggcattgaa aaaggtcatg gaggtctga 1059
<210>216
<211>352
<212>PRT
<213>恶臭假单胞菌
<400>216
Met Asn Asp His Asn Asn Ser Ile Asn Pro Glu Thr Ala Met Ala Thr
1 5 10 15
Thr Thr Met Thr Met Ile Gln Ala Leu Arg Ser Ala Met Asp Val Met
20 25 30
Leu Glu Arg Asp Asp Asn Val Val Val Tyr Gly Gln Asp Val Gly Tyr
35 40 45
Phe Gly Gly Val Phe Arg Cys Thr Glu Gly Leu Gln Thr Lys Tyr Gly
50 55 60
Lys Ser Arg Val Phe Asp Ala Pro Ile Ser Glu Ser Gly Ile Val Gly
65 70 75 80
Thr Ala Val Gly Met Gly Ala Tyr Gly Leu Arg Pro Val Val Glu Ile
85 90 95
Gln Phe Ala Asp Tyr Phe Tyr Pro Ala Ser Asp Gln Ile Val Ser Glu
100 105 110
Met Ala Arg Leu Arg Tyr Arg Ser Ala Gly Glu Phe Ile Ala Pro Leu
115 120 125
Thr Leu Arg Met Pro Cys Gly Gly Gly Ile Tyr Gly Gly Gln Thr His
130 135 140
Ser Gln Ser Pro Glu Ala Met Phe Thr Gln Val Cys Gly Leu Arg Thr
145 150 155 160
Val Met Pro Ser Asn Pro Tyr Asp Ala Lys Gly Leu Leu Ile Ala Ser
165 170 175
Ile Glu Cys Asp Asp Pro Val Ile Phe Leu Glu Pro Lys Arg Leu Tyr
180 185 190
Asn Gly Pro Phe Asp Gly His His Asp Arg Pro Val Thr Pro Trp Ser
195 200 205
Lys His Pro His Ser Ala Val Pro Asp Gly Tyr Tyr Thr Val Pro Leu
210 215 220
Asp Lys Ala Ala Ile Thr Arg Pro Gly Asn Asp Val Ser Val Leu Thr
225 230 235 240
Tyr Gly Thr Thr Val Tyr Val Ala Gln Val Ala Ala Glu Glu Ser Gly
245 250 255
Val Asp Ala Glu Val Ile Asp Leu Arg Ser Leu Trp Pro Leu Asp Leu
260 265 270
Asp Thr Ile Val Glu Ser Val Lys Lys Thr Gly Arg Cys Val Val Val
275 280 285
His Glu Ala Thr Arg Thr Cys Gly Phe Gly Ala Glu Leu Val Ser Leu
290 295 300
Val Gln Glu His Cys Phe His His Leu Glu Ala Pro Ile Glu Arg Val
305 310 315 320
Thr Gly Trp Asp Thr Pro Tyr Pro His Ala Gln Glu Trp Ala Tyr Phe
325 330 335
Pro Gly Pro Ser Arg Val Gly Ala Ala Leu Lys Lys Val Met Glu Val
340 345 350
<210>217
<211>1272
<212>DNA
<213>恶臭假单胞菌
<400>217
atgggcacgc acgtcatcaa gatgccggac attggcgaag gcatcgcgca ggtcgaattg 60
gtggaatggt tcgtcaaggt gggcgacatc atcgccgagg accaagtggt agccgacgtc 120
atgaccgaca aggccaccgt ggaaatcccg tcgccggtca gcggcaaggt gctggccctg 180
ggtggccagc caggtgaagt gatggcggtc ggcagtgagc tgatccgcat cgaagtggaa 240
ggcagcggca accatgtgga tgtgccgcaa gccaagccgg ccgaagtgcc tgcggcaccg 300
gtagccgcta aacctgaacc acagaaagac gttaaaccgg cggcgtacca ggcgtcagcc 360
agccacgagg cagcgcccat cgtgccgcgc cagccgggcg acaagccgct ggcctcgccg 420
gcggtgcgca aacgcgccct cgatgccggc atcgaattgc gttatgtgca cggcagcggc 480
ccggccgggc gcatcctgca cgaagacctc gacgcgttca tgagcaaacc gcaaagcgct 540
gccgggcaaa cccccaatgg ctatgccagg cgcaccgaca gcgagcaggt gccggtgatc 600
ggcctgcgcc gcaagatcgc ccagcgcatg caggacgcca agcgccgggt cgcgcacttc 660
agctatgtgg aagaaatcga cgtcaccgcc ctggaagccc tgcgccagca gctcaacagc 720
aagcacggcg acagccgcgg caagctgaca ctgctgccgt tcctggtgcg cgccctggtc 780
gtggcactgc gtgacttccc gcagataaac gccacctacg atgacgaagc gcagatcatc 840
acccgccatg gcgcggtgca tgtgggcatc gccacccaag gtgacaacgg cctgatggta 900
cccgtgctgc gccacgccga agcgggcagc ctgtgggcca atgccggtga gatttcacgc 960
ctggccaacg ctgcgcgcaa caacaaggcc agccgcgaag agctgtccgg ttcgaccatt 1020
accctgacca gcctcggcgc cctgggcggc atcgtcagca cgccggtggt caacaccccg 1080
gaagtggcga tcgtcggtgt caaccgcatg gttgagcggc ccgtggtgat cgacggccag 1140
atcgtcgtgc gcaagatgat gaacctgtcc agctcgttcg accaccgcgt ggtcgatggc 1200
atggacgccg ccctgttcat ccaggccgtg cgtggcctgc tcgaacaacc cgcctgcctg 1260
ttcgtggagt ga 1272
<210>218
<211>423
<212>PRT
<213>恶臭假单胞菌
<400>218
Met Gly Thr His Val Ile Lys Met Pro Asp Ile Gly Glu Gly Ile Ala
1 5 10 15
Gln Val Glu Leu Val Glu Trp Phe Val Lys Val Gly Asp Ile Ile Ala
20 25 30
Glu Asp Gln Val Val Ala Asp Val Met Thr Asp Lys Ala Thr Val Glu
35 40 45
Ile Pro Ser Pro Val Ser Gly Lys Val Leu Ala Leu Gly Gly Gln Pro
50 55 60
Gly Glu Val Met Ala Val Gly Ser Glu Leu Ile Arg Ile Glu Val Glu
65 70 75 80
Gly Ser Gly Asn His Val Asp Val Pro Gln Ala Lys Pro Ala Glu Val
85 90 95
Pro Ala Ala Pro Val Ala Ala Lys Pro Glu Pro Gln Lys Asp Val Lys
100 105 110
Pro Ala Ala Tyr Gln Ala Ser Ala Ser His Glu Ala Ala Pro Ile Val
115 120 125
Pro Arg Gln Pro Gly Asp Lys Pro Leu Ala Ser Pro Ala Val Arg Lys
130 135 140
Arg Ala Leu Asp Ala Gly Ile Glu Leu Arg Tyr Val His Gly Ser Gly
145 150 155 160
Pro Ala Gly Arg Ile Leu His Glu Asp Leu Asp Ala Phe Met Ser Lys
165 170 175
Pro Gln Ser Ala Ala Gly Gln Thr Pro Asn Gly Tyr Ala Arg Arg Thr
180 185 190
Asp Ser Glu Gln Val Pro Val Ile Gly Leu Arg Arg Lys Ile Ala Gln
195 200 205
Arg Met Gln Asp Ala Lys Arg Arg Val Ala His Phe Ser Tyr Val Glu
210 215 220
Glu Ile Asp Val Thr Ala Leu Glu Ala Leu Arg Gln Gln Leu Asn Ser
225 230 235 240
Lys His Gly Asp Ser Arg Gly Lys Leu Thr Leu Leu Pro Phe Leu Val
245 250 255
Arg Ala Leu Val Val Ala Leu Arg Asp Phe Pro Gln Ile Asn Ala Thr
260 265 270
Tyr Asp Asp Glu Ala Gln Ile Ile Thr Arg His Gly Ala Val His Val
275 280 285
Gly Ile Ala Thr Gln Gly Asp Asn Gly Leu Met Val Pro Val Leu Arg
290 295 300
His Ala Glu Ala Gly Ser Leu Trp Ala Asn Ala Gly Glu Ile Ser Arg
305 310 315 320
Leu Ala Asn Ala Ala Arg Asn Asn Lys Ala Ser Arg Glu Glu Leu Ser
325 330 335
Gly Ser Thr Ile Thr Leu Thr Ser Leu Gly Ala Leu Gly Gly Ile Val
340 345 350
Ser Thr Pro Val Val Asn Thr Pro Glu Val Ala Ile Val Gly Val Asn
355 360 365
Arg Met Val Glu Arg Pro Val Val Ile Asp Gly Gln Ile Val Val Arg
370 375 380
Lys Met Met Asn Leu Ser Ser Ser Phe Asp His Arg Val Val Asp Gly
385 390 395 400
Met Asp Ala Ala Leu Phe Ile Gln Ala Val Arg Gly Leu Leu Glu Gln
405 410 415
Pro Ala Cys Leu Phe Val Glu
420
<210>219
<211>1380
<212>DNA
<213>恶臭假单胞菌
<400>219
atgcaacaga ctatccagac aaccctgttg atcatcggcg gcggccctgg cggctatgtg 60
gcggccatcc gcgccgggca actgggcatc cctaccgtgc tggtggaagg ccaggcgctg 120
ggcggtacct gcctgaacat cggctgcatt ccgtccaagg cgctgatcca tgtggccgag 180
cagttccacc aggcctcgcg ctttaccgaa ccctcgccgc tgggcatcag cgtggcttcg 240
ccacgcctgg acatcggcca gagcgtggcc tggaaagacg gcatcgtcga tcgcctgacc 300
actggtgtcg ccgccctgct gaaaaagcac ggggtgaagg tggtgcacgg ctgggccaag 360
gtgcttgatg gcaagcaggt cgaggtggat ggccagcgca tccagtgcga gcacctgttg 420
ctggccacgg gctccagcag tgtcgaactg ccgatgctgc cgttgggtgg gccggtgatt 480
tcctcgaccg aggccctggc accgaaagcc ctgccgcaac acctggtggt ggtgggcggt 540
ggctacatcg gcctggagct gggtatcgcc taccgcaagc tcggcgcgca ggtcagcgtg 600
gtggaagcgc gcgagcgcat cctgccgact tacgacagcg aactgaccgc cccggtggcc 660
gagtcgctga aaaagctggg tatcgccctg caccttggcc acagcgtcga aggttacgaa 720
aatggctgcc tgctggccaa cgatggcaag ggcggacaac tgcgcctgga agccgaccgg 780
gtgctggtgg ccgtgggccg ccgcccacgc accaagggct tcaacctgga atgcctggac 840
ctgaagatga atggtgccgc gattgccatc gacgagcgct gccagaccag catgcacaac 900
gtctgggcca tcggcgacgt ggccggcgaa ccgatgctgg cgcaccgggc catggcccag 960
ggcgagatgg tggccgagat catcgccggc aaggcacgcc gcttcgaacc cgctgcgata 1020
gccgccgtgt gcttcaccga cccggaagtg gtcgtggtcg gcaagacgcc ggaacaggcc 1080
agtcagcaag gcctggactg catcgtcgcg cagttcccgt tcgccgccaa cggccgggcc 1140
atgagcctgg agtcgaaaag cggtttcgtg cgcgtggtcg cgcggcgtga caaccacctg 1200
atcctgggct ggcaagcggt tggcgtggcg gtttccgagc tgtccacggc gtttgcccag 1260
tcgctggaga tgggtgcctg cctggaggat gtggccggta ccatccatgc ccacccgacc 1320
ctgggtgaag cggtacagga agcggcactg cgtgccctgg gccacgccct gcatatctga 1380
<210>220
<211>459
<212>PRT
<213>恶臭假单胞菌
<400>220
Met Gln Gln Thr Ile Gln Thr Thr Leu Leu Ile Ile Gly Gly Gly Pro
1 5 10 15
Gly Gly Tyr Val Ala Ala Ile Arg Ala Gly Gln Leu Gly Ile Pro Thr
20 25 30
Val Leu Val Glu Gly Gln Ala Leu Gly Gly Thr Cys Leu Asn Ile Gly
35 40 45
Cys Ile Pro Ser Lys Ala Leu Ile His Val Ala Glu Gln Phe His Gln
50 55 60
Ala Ser Arg Phe Thr Glu Pro Ser Pro Leu Gly Ile Ser Val Ala Ser
65 70 75 80
Pro Arg Leu Asp Ile Gly Gln Ser Val Ala Trp Lys Asp Gly Ile Val
85 90 95
Asp Arg Leu Thr Thr Gly Val Ala Ala Leu Leu Lys Lys His Gly Val
100 105 110
Lys Val Val His Gly Trp Ala Lys Val Leu Asp Gly Lys Gln Val Glu
115 120 125
Val Asp Gly Gln Arg Ile Gln Cys Glu His Leu Leu Leu Ala Thr Gly
130 135 140
Ser Ser Ser Val Glu Leu Pro Met Leu Pro Leu Gly Gly Pro Val Ile
145 150 155 160
Ser Ser Thr Glu Ala Leu Ala Pro Lys Ala Leu Pro Gln His Leu Val
165 170 175
Val Val Gly Gly Gly Tyr Ile Gly Leu Glu Leu Gly Ile Ala Tyr Arg
180 185 190
Lys Leu Gly Ala Gln Val Ser Val Val Glu Ala Arg Glu Arg Ile Leu
195 200 205
Pro Thr Tyr Asp Ser Glu Leu Thr Ala Pro Val Ala Glu Ser Leu Lys
210 215 220
Lys Leu Gly Ile Ala Leu His Leu Gly His Ser Val Glu Gly Tyr Glu
225 230 235 240
Asn Gly Cys Leu Leu Ala Asn Asp Gly Lys Gly Gly Gln Leu Arg Leu
245 250 255
Glu Ala Asp Arg Val Leu Val Ala Val Gly Arg Arg Pro Arg Thr Lys
260 265 270
Gly Phe Asn Leu Glu Cys Leu Asp Leu Lys Met Asn Gly Ala Ala Ile
275 280 285
Ala Ile Asp Glu Arg Cys Gln Thr Ser Met His Asn Val Trp Ala Ile
290 295 300
Gly Asp Val Ala Gly Glu Pro Met Leu Ala His Arg Ala Met Ala Gln
305 310 315 320
Gly Glu Met Val Ala Glu Ile Ile Ala Gly Lys Ala Arg Arg Phe Glu
325 330 335
Pro Ala Ala Ile Ala Ala Val Cys Phe Thr Asp Pro Glu Val Val Val
340 345 350
Val Gly Lys Thr Pro Glu Gln Ala Ser Gln Gln Gly Leu Asp Cys Ile
355 360 365
Val Ala Gln Phe Pro Phe Ala Ala Asn Gly Arg Ala Met Ser Leu Glu
370 375 380
Ser Lys Ser Gly Phe Val Arg Val Val Ala Arg Arg Asp Asn His Leu
385 390 395 400
Ile Leu Gly Trp Gln Ala Val Gly Val Ala Val Ser Glu Leu Ser Thr
405 410 415
Ala Phe Ala Gln Ser Leu Glu Met Gly Ala Cys Leu Glu Asp Val Ala
420 425 430
Gly Thr Ile His Ala His Pro Thr Leu Gly Glu Ala Val Gln Glu Ala
435 440 445
Ala Leu Arg Ala Leu Gly His Ala Leu His Ile
450 455
<210>221
<211>1407
<212>DNA
<213>拜氏梭菌
<400>221
atgaataaag acacactaat acctacaact aaagatttaa aattaaaaac aaatgttgaa 60
aacattaatt taaagaacta caaggataat tcttcatgtt tcggagtatt cgaaaatgtt 120
gaaaatgcta taaacagcgc tgtacacgcg caaaagatat tatcccttca ttatacaaaa 180
gaacaaagag aaaaaatcat aactgagata agaaaggccg cattagaaaa taaagaggtt 240
ttagctacca tgattctgga agaaacacat atgggaaggt atgaagataa aatattaaag 300
catgaattag tagctaaata tactcctggt acagaagatt taactactac tgcttggtca 360
ggtgataatg gtcttacagt tgtagaaatg tctccatatg gcgttatagg tgcaataact 420
ccttctacga atccaactga aactgtaata tgtaatagca tcggcatgat agctgctgga 480
aatgctgtag tatttaacgg acacccaggc gctaaaaaat gtgttgcttt tgctattgaa 540
atgataaata aagcaattat ttcatgtggc ggtcctgaga atttagtaac aactataaaa 600
aatccaacta tggaatccct agatgcaatt attaagcatc ctttaataaa acttctttgc 660
ggaactggag gtccaggaat ggtaaaaacc ctcttaaatt ctggcaagaa agctataggt 720
gctggtgctg gaaatccacc agttattgta gatgataccg ctgatataga aaaggctggt 780
aagagtatca ttgaaggctg ttcttttgat aataatttac cttgtattgc agaaaaagaa 840
gtatttgttt ttgagaatgt tgcagatgat ttaatatcta acatgctaaa aaataatgct 900
gtaattataa atgaagatca agtatcaaaa ttaatagatt tagtattaca aaaaaataat 960
gaaactcaag aatactttat aaacaaaaaa tgggtaggaa aagatgcaaa attattctca 1020
gatgaaatag atgttgagtc tccttcaaat attaaatgca tagtctgcga agtaaatgca 1080
aatcatccat ttgtcatgac agaactcatg atgccaatat taccaattgt aagagttaaa 1140
gatatagatg aagctgttaa atatacaaag atagcagaac aaaatagaaa acatagtgcc 1200
tatatttatt ctaaaaatat agacaaccta aatagatttg aaagagaaat tgatactact 1260
atttttgtaa agaatgctaa atcttttgct ggtgttggtt atgaagctga aggatttaca 1320
actttcacta ttgctggatc tactggtgaa ggcataacct ctgcaagaaa ttttacaaga 1380
caaagaagat gtgtacttgc cggctaa 1407
<210>222
<211>468
<212>PRT
<213>拜氏梭菌
<400>222
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys
340 345 350
Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210>223
<211>2589
<212>DNA
<213>丙醇丁酮梭菌
<400>223
atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60
aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120
gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180
ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240
aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300
gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360
atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420
agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480
ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540
ttaatgcaaa aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600
tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660
gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720
ggtgttatat gtgcttctga acaatctgta atagtcttaa aatccatata taacaaggta 780
aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840
cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900
actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960
gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020
atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080
ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140
gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200
ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260
ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320
accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380
aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440
tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500
cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560
aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620
ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680
gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740
ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800
tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860
gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920
ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980
acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040
tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100
atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160
cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220
ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280
tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340
aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400
ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460
ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520
aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580
caaccttaa 2589
<210>224
<211>862
<212>PRT
<213>丙醇丁酮梭菌
<400>224
Met Lys Val Thr Thr Val Lys Glu Leu Asp Glu Lys Leu Lys Val Ile
1 5 10 15
Lys Glu Ala Gln Lys Lys Phe Ser Cys Tyr Ser Gln Glu Met Val Asp
20 25 30
Glu Ile Phe Arg Asn Ala Ala Met Ala Ala Ile Asp Ala Arg Ile Glu
35 40 45
Leu Ala Lys Ala Ala Val Leu Glu Thr Gly Met Gly Leu Val Glu Asp
50 55 60
Lys Val Ile Lys Asn His Phe Ala Gly Glu Tyr Ile Tyr Asn Lys Tyr
65 70 75 80
Lys Asp Glu Lys Thr Cys Gly Ile Ile Glu Arg Asn Glu Pro Tyr Gly
85 90 95
Ile Thr Lys Ile Ala Glu Pro Ile Gly Val Val Ala Ala Ile Ile Pro
100 105 110
Val Thr Asn Pro Thr Ser Thr Thr Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125
Lys Thr Arg Asn Gly Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys
130 135 140
Ser Thr Ile Leu Ala Ala Lys Thr Ile Leu Asp Ala Ala Val Lys Ser
145 150 155 160
Gly Ala Pro Glu Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu
165 170 175
Leu Thr Gln Tyr Leu Met Gln Lys Ala Asp Ile Thr Leu Ala Thr Gly
180 185 190
Gly Pro Ser Leu Val Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ala Ile
195 200 205
Gly Val Gly Pro Gly Asn Thr Pro Val Ile Ile Asp Glu Ser Ala His
210 215 220
Ile Lys Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn
225 230 235 240
Gly Val Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Lys Ser Ile
245 250 255
Tyr Asn Lys Val Lys Asp Glu Phe Gln Glu Arg Gly Ala Tyr Ile Ile
260 265 270
Lys Lys Asn Glu Leu Asp Lys Val Arg Glu Val Ile Phe Lys Asp Gly
275 280 285
Ser Val Asn Pro Lys Ile Val Gly Gln Ser Ala Tyr Thr Ile Ala Ala
290 295 300
Met Ala Gly Ile Lys Val Pro Lys Thr Thr Arg Ile Leu Ile Gly Glu
305 310 315 320
Val Thr Ser Leu Gly Glu Glu Glu Pro Phe Ala His Glu Lys Leu Ser
325 330 335
Pro Val Leu Ala Met Tyr Glu Ala Asp Asn Phe Asp Asp Ala Leu Lys
340 345 350
Lys Ala Val Thr Leu Ile Asn Leu Gly Gly Leu Gly His Thr Ser Gly
355 360 365
Ile Tyr Ala Asp Glu Ile Lys Ala Arg Asp Lys Ile Asp Arg Phe Ser
370 375 380
Ser Ala Met Lys Thr Val Arg Thr Phe Val Asn Ile Pro Thr Ser Gln
385 390 395 400
Gly Ala Ser Gly Asp Leu Tyr Asn Phe Arg Ile Pro Pro Ser Phe Thr
405 410 415
Leu Gly Cys Gly Phe Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly
420 425 430
Pro Lys His Leu Leu Asn Ile Lys Thr Val Ala Glu Arg Arg Glu Asn
435 440 445
Met Leu Trp Phe Arg Val Pro His Lys Val Tyr Phe Lys Phe Gly Cys
450 455 460
Leu Gln Phe Ala Leu Lys Asp Leu Lys Asp Leu Lys Lys Lys Arg Ala
465 470 475 480
Phe Ile Val Thr Asp Ser Asp Pro Tyr Asn Leu Asn Tyr Val Asp Ser
485 490 495
Ile Ile Lys Ile Leu Glu His Leu Asp Ile Asp Phe Lys Val Phe Asn
500 505 510
Lys Val Gly Arg Glu Ala Asp Leu Lys Thr Ile Lys Lys Ala Thr Glu
515 520 525
Glu Met Ser Ser Phe Met Pro Asp Thr Ile Ile Ala Leu Gly Gly Thr
530 535 540
Pro Glu Met Ser Ser Ala Lys Leu Met Trp Val Leu Tyr Glu His Pro
545 550 555 560
Glu Val Lys Phe Glu Asp Leu Ala Ile Lys Phe Met Asp Ile Arg Lys
565 570 575
Arg Ile Tyr Thr Phe Pro Lys Leu Gly Lys Lys Ala Met Leu Val Ala
580 585 590
Ile Thr Thr Ser Ala Gly Ser Gly Ser Glu Val Thr Pro Phe Ala Leu
595 600 605
Val Thr Asp Asn Asn Thr Gly Asn Lys Tyr Met Leu Ala Asp Tyr Glu
610 615 620
Met Thr Pro Asn Met Ala Ile Val Asp Ala Glu Leu Met Met Lys Met
625 630 635 640
Pro Lys Gly Leu Thr Ala Tyr Ser Gly Ile Asp Ala Leu Val Asn Ser
645 650 655
Ile Glu Ala Tyr Thr Ser Val Tyr Ala Ser Glu Tyr Thr Asn Gly Leu
660 665 670
Ala Leu Glu Ala Ile Arg Leu Ile Phe Lys Tyr Leu Pro Glu Ala Tyr
675 680 685
Lys Asn Gly Arg Thr Asn Glu Lys Ala Arg Glu Lys Met Ala His Ala
690 695 700
Ser Thr Met Ala Gly Met Ala Ser Ala Asn Ala Phe Leu Gly Leu Cys
705 710 715 720
His Ser Met Ala Ile Lys Leu Ser Ser Glu His Asn Ile Pro Ser Gly
725 730 735
Ile Ala Asn Ala Leu Leu Ile Glu Glu Val Ile Lys Phe Asn Ala Val
740 745 750
Asp Asn Pro Val Lys Gln Ala Pro Cys Pro Gln Tyr Lys Tyr Pro Asn
755 760 765
Thr Ile Phe Arg Tyr Ala Arg Ile Ala Asp Tyr Ile Lys Leu Gly Gly
770 775 780
Asn Thr Asp Glu Glu Lys Val Asp Leu Leu Ile Asn Lys Ile His Glu
785 790 795 800
Leu Lys Lys Ala Leu Asn Ile Pro Thr Ser Ile Lys Asp Ala Gly Val
805 810 815
Leu Glu Glu Asn Phe Tyr Ser Ser Leu Asp Arg Ile Ser Glu Leu Ala
820 825 830
Leu Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Phe Pro Leu Thr Ser
835 840 845
Glu Ile Lys Glu Met Tyr Ile Asn Cys Phe Lys Lys Gln Pro
850 855 860
<210>225
<211>2577
<212>DNA
<213>丙醇丁酮梭菌
<400>225
atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60
aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120
gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180
cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240
aaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300
gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360
attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420
cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480
ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540
ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600
tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660
gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720
ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780
aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840
aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900
ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960
gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020
atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080
ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140
aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200
ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260
acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320
agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380
aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440
tttatagtaa cagataaaga tctttttaaa cttggatatg ttaataaaat aacaaaggta 1500
ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560
gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620
attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680
gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740
ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800
tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860
acttcttatg aattgacccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920
cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980
gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040
tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100
atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160
cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220
gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280
ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340
aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400
ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460
ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520
aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaa 2577
<210>226
<211>800
<212>PRT
<213>丙醇丁酮梭菌
<400>226
Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu
1 5 10 15
Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp
20 25 30
Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn
35 40 45
Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp
50 55 60
Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr
65 70 75 80
Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly
85 90 95
Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro
100 105 110
Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125
Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys
130 135 140
Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala
145 150 155 160
Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu
165 170 175
Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly
180 185 190
Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile
195 200 205
Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp
210 215 220
Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn
225 230 235 240
Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile
245 250 255
Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu
260 265 270
Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly
275 280 285
Ala Ile Asn Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys
290 295 300
Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu
305 310 315 320
Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser
325 330 335
Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys
340 345 350
Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser
355 360 365
Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly
370 375 380
Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln
385 390 395 400
Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr
405 410 415
Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu
420 425 430
Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn
435 440 445
Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys
450 455 460
Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala
465 470 475 480
Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys
485 490 495
Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr
500 505 510
Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys
515 520 525
Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly
530 535 540
Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro
545 550 555 560
Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys
565 570 575
Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala
580 585 590
Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val
595 600 605
Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu
610 615 620
Leu Thr Pro Asn Met Ala Ile Ile Asp Thr Glu Leu Met Leu Asn Met
625 630 635 640
Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala
645 650 655
Ile Glu Ala Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu
660 665 670
Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr
675 680 685
Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala
690 695 700
Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys
705 710 715 720
His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly
725 730 735
Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr
740 745 750
Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn
755 760 765
Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly
770 775 780
Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys
785 790 795 800
<210>227
<211>924
<212>DNA
<213>恶臭假单胞菌
<400>227
atgagcaaga aactcaaggc ggccatcata ggccccggca atatcggtac cgatctggtg 60
atgaagatgc tccgttccga gtggattgag ccggtgtgga tggtcggcat cgaccccaac 120
tccgacggcc tcaaacgcgc ccgcgatttc ggcatgaaga ccacagccga aggcgtcgac 180
ggcctgctcc cgcacgtgct ggacgacgac atccgcatcg ccttcgacgc cacctcggcc 240
tatgtgcatg ccgagaatag ccgcaagctc aacgcgcttg gcgtgctgat ggtcgacctg 300
accccggcgg ccatcggccc ctactgcgtg ccgccggtca acctcaagca gcatgtcggc 360
cgcctggaaa tgaacgtcaa catggtcacc tgcggcggcc aggccaccat ccccatggtc 420
gccgcggtgt cccgcgtgca gccggtggcc tacgccgaga tcgtcgccac cgtctcctcg 480
cgctcggtcg gcccgggcac gcgcaagaac atcgacgagt tcacccgcac caccgccggc 540
gccatcgagc aggtcggcgg cgccagggaa ggcaaggcga tcatcgtcat caacccggcc 600
gagccgccgc tgatgatgcg cgacaccatc cactgcctga ccgacagcga gccggaccag 660
gctgcgatca ccgcttcggt tcacgcgatg atcgccgagg tgcagaaata cgtgcccggc 720
taccgcctga agaacggccc ggtgttcgac ggcaaccgcg tgtcgatctt catggaagtc 780
gaaggcctgg gcgactacct gcccaagtac gccggcaacc tcgacatcat gaccgccgcc 840
gcgctgcgta ccggcgagat gttcgccgag gaaatcgccg ccggcaccat tcaactgccg 900
cgtcgcgaca tcgcgctggc ttga 924
<210>228
<211>307
<212>PRT
<213>恶臭假单胞菌
<400>228
Met Ser Lys Lys Leu Lys Ala Ala Ile Ile Gly Pro Gly Asn Ile Gly
1 5 10 15
Thr Asp Leu Val Met Lys Met Leu Arg Ser Glu Trp Ile Glu Pro Val
20 25 30
Trp Met Val Gly Ile Asp Pro Asn Ser Asp Gly Leu Lys Arg Ala Arg
35 40 45
Asp Phe Gly Met Lys Thr Thr Ala Glu Gly Val Asp Gly Leu Leu Pro
50 55 60
His Val Leu Asp Asp Asp Ile Arg Ile Ala Phe Asp Ala Thr Ser Ala
65 70 75 80
Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Ala Leu Gly Val Leu
85 90 95
Met Val Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro Pro
100 105 110
Val Asn Leu Lys Gln His Val Gly Arg Leu Glu Met Asn Val Asn Met
115 120 125
Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val Ser
130 135 140
Arg Val Gln Pro Val Ala Tyr Ala Glu Ile Val Ala Thr Val Ser Ser
145 150 155 160
Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr Arg
165 170 175
Thr Thr Ala Gly Ala Ile Glu Gln Val Gly Gly Ala Arg Glu Gly Lys
180 185 190
Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp
195 200 205
Thr Ile His Cys Leu Thr Asp Ser Glu Pro Asp Gln Ala Ala Ile Thr
210 215 220
Ala Ser Val His Ala Met Ile Ala Glu Val Gln Lys Tyr Val Pro Gly
225 230 235 240
Tyr Arg Leu Lys Asn Gly Pro Val Phe Asp Gly Asn Arg Val Ser Ile
245 250 255
Phe Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala Gly
260 265 270
Asn Leu Asp Ile Met Thr Ala Ala Ala Leu Arg Thr Gly Glu Met Phe
275 280 285
Ala Glu Glu Ile Ala Ala Gly Thr Ile Gln Leu Pro Arg Arg Asp Ile
290 295 300
Ala Leu Ala
305
<210>229
<211>924
<212>DNA
<213>嗜热栖热菌
<400>229
atgtccgaaa gggttaaggt agccatcctg ggctccggca acatcgggac ggacctgatg 60
tacaagctcc tgaagaaccc gggccacatg gagcttgtgg cggtggtggg gatagacccc 120
aagtccgagg gcctggcccg ggcgcgggcc ttagggttag aggcgagcca cgaagggatc 180
gcctacatcc tggagaggcc ggagatcaag atcgtctttg acgccaccag cgccaaggcc 240
cacgtgcgcc acgccaagct cctgagggag gcggggaaga tcgccataga cctcacgccg 300
gcggcccggg gcccttacgt ggtgcccccg gtgaacctga aggaacacct ggacaaggac 360
aacgtgaacc tcatcacctg cggggggcag gccaccatcc ccctggtcta cgcggtgcac 420
cgggtggccc ccgtgctcta cgcggagatg gtctccacgg tggcctcccg ctccgcgggc 480
cccggcaccc ggcagaacat cgacgagttc accttcacca ccgcccgggg cctggaggcc 540
atcggggggg ccaagaaggg gaaggccatc atcatcctga acccggcgga accccccatc 600
ctcatgacca acaccgtgcg ctgcatcccc gaggacgagg gctttgaccg ggaggccgtg 660
gtggcgagcg tccgggccat ggagcgggag gtccaggcct acgtgcccgg ctaccgcctg 720
aaggcggacc cggtgtttga gaggcttccc accccctggg gggagcgcac cgtggtctcc 780
atgctcctgg aggtggaggg ggcgggggac tatttgccca aatacgccgg caacctggac 840
atcatgacgg cttctgcccg gagggtgggg gaggtcttcg cccagcacct cctggggaag 900
cccgtggagg aggtggtggc gtga 924
<210>230
<211>307
<212>PRT
<213>嗜热栖热菌
<400>230
Met Ser Glu Arg Val Lys Val Ala Ile Leu Gly Ser Gly Asn Ile Gly
1 5 10 15
Thr Asp Leu Met Tyr Lys Leu Leu Lys Asn Pro Gly His Met Glu Leu
20 25 30
Val Ala Val Val Gly Ile Asp Pro Lys Ser Glu Gly Leu Ala Arg Ala
35 40 45
Arg Ala Leu Gly Leu Glu Ala Ser His Glu Gly Ile Ala Tyr Ile Leu
50 55 60
Glu Arg Pro Glu Ile Lys Ile Val Phe Asp Ala Thr Ser Ala Lys Ala
65 70 75 80
His Val Arg His Ala Lys Leu Leu Arg Glu Ala Gly Lys Ile Ala Ile
85 90 95
Asp Leu Thr Pro Ala Ala Arg Gly Pro Tyr Val Val Pro Pro Val Asn
100 105 110
Leu Lys Glu His Leu Asp Lys Asp Asn Val Asn Leu Ile Thr Cys Gly
115 120 125
Gly Gln Ala Thr Ile Pro Leu Val Tyr Ala Val His Arg Val Ala Pro
130 135 140
Val Leu Tyr Ala Glu Met Val Ser Thr Val Ala Ser Arg Ser Ala Gly
145 150 155 160
Pro Gly Thr Arg Gln Asn Ile Asp Glu Phe Thr Phe Thr Thr Ala Arg
165 170 175
Gly Leu Glu Ala Ile Gly Gly Ala Lys Lys Gly Lys Ala Ile Ile Ile
180 185 190
Leu Asn Pro Ala Glu Pro Pro Ile Leu Met Thr Asn Thr Val Arg Cys
195 200 205
Ile Pro Glu Asp Glu Gly Phe Asp Arg Glu Ala Val Val Ala Ser Val
210 215 220
Arg Ala Met Glu Arg Glu Val Gln Ala Tyr Val Pro Gly Tyr Arg Leu
225 230 235 240
Lys Ala Asp Pro Val Phe Glu Arg Leu Pro Thr Pro Trp Gly Glu Arg
245 250 255
Thr Val Val Ser Met Leu Leu Glu Val Glu Gly Ala Gly Asp Tyr Leu
260 265 270
Pro Lys Tyr Ala Gly Asn Leu Asp Ile Met Thr Ala Ser Ala Arg Arg
275 280 285
Val Gly Glu Val Phe Ala Gln His Leu Leu Gly Lys Pro Val Glu Glu
290 295 300
Val Val Ala
305
<210>231
<211>1254
<212>DNA
<213>大肠杆菌
<400>231
atgacattct ccctttttgg tgacaaattt acccgccact ccggcattac gctgttgatg 60
gaagatctga acgacggttt acgcacgcct ggcgcgatta tgctcggcgg cggtaatccg 120
gcgcagatcc cggaaatgca ggactacttc cagacgctac tgaccgacat gctggaaagt 180
ggcaaagcga ctgatgcact gtgtaactac gacggtccac aggggaaaac ggagctactc 240
acactgcttg ccggaatgct gcgcgagaag ttgggttggg atatcgaacc acagaatatt 300
gcactaacaa acggcagcca gagcgcgttt ttctacttat ttaacctgtt tgccggacgc 360
cgtgccgatg gtcgggtcaa aaaagtgctg ttcccgcttg caccggaata cattggctat 420
gctgacgccg gactggaaga agatctgttt gtctctgcgc gtccgaatat tgaactgctg 480
ccggaaggcc agtttaaata ccacgtcgat tttgagcatc tgcatattgg cgaagaaacc 540
gggatgattt gcgtctcccg gccgacgaat ccaacaggca atgtgattac tgacgaagag 600
ttgctgaagc ttgacgcgct ggcgaatcaa cacggcattc cgctggtgat tgataacgct 660
tatggcgtcc cgttcccggg tatcatcttc agtgaagcgc gcccgctatg gaatccgaat 720
atcgtgctgt gcatgagtct ttccaagctg ggtctacctg gctcccgctg cggcattatc 780
atcgccaatg aaaaaatcat caccgccatc accaatatga acggcattat cagcctggca 840
cctggcggta ttggtccggc gatgatgtgt gaaatgatta agcgtaacga tctgctgcgc 900
ctgtctgaaa cagtcatcaa accgttttac taccagcgtg ttcaggaaac tatcgccatc 960
attcgccgct atttaccgga aaatcgctgc ctgattcata aaccggaagg agccattttc 1020
ctctggctat ggtttaagga tttgcccatt acgaccaagc agctctatca gcgcctgaaa 1080
gcacgcggcg tgctgatggt gccggggcac aacttcttcc cagggctgga taaaccgtgg 1140
ccgcatacgc atcaatgtat gcgcatgaac tacgtaccag agccggagaa aattgaggcg 1200
ggggtgaaga ttctggcgga agagatagaa agagcctggg ctgaaagtca ctaa 1254
<210>232
<211>417
<212>PRT
<213>大肠杆菌
<400>232
Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile
1 5 10 15
Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala
20 25 30
Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp
35 40 45
Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr
50 55 60
Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu
65 70 75 80
Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu
85 90 95
Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr
100 105 110
Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys
115 120 125
Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly
130 135 140
Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu
145 150 155 160
Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile
165 170 175
Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr
180 185 190
Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala
195 200 205
Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro
210 215 220
Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn
225 230 235 240
Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg
245 250 255
Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn
260 265 270
Met Asn Gly Ile Ile Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met
275 280 285
Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr
290 295 300
Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile
305 310 315 320
Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu
325 330 335
Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr
340 345 350
Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro
355 360 365
Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His
370 375 380
Gln Gys Met Arg Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala
385 390 395 400
Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser
405 410 415
His
<210>233
<211>1278
<212>DNA
<213>地衣芽孢杆菌
<400>233
ttataagtat tcaacctgtt tctcatatac acccttcgca attttagcta aaacatcgat 60
tccccttata atatcttcat ccgccgcggt taggctgatt cgtatacact ggtgtgaatg 120
cgccaggcgc cgggattgac ggtgaaagaa agatgatccg ggaacgataa tgactccatc 180
cgctttcata tactcataca gcgctgcatc ggtcaccggc aggtcttcaa accacagcca 240
tccgaaaagc gatccttccc cttgatgcag ataccatttg atgtcttcag gcatcttgca 300
taaaagcgtt tccttgagca gcatgaattt attgcggtaa tatggcctga cttcattcag 360
cgacacgtcg gcgaggcgcc cgtcattcaa tactgatgca gccatatact gccccagcct 420
tgaagaatgg atcgccgcat tcgactgaaa agcttccatt gcctgaatat accgggacgg 480
cccgatggcg attccgatcc tttcgccagg caggccggct tttgaaaggc tcatacagtg 540
aatgatctgc tcgttgaaaa tcggttccat gtcgataaag tgaatcgccg gaaaaggcgg 600
agcatatgcg gaatcaatga acagcggaac attcgcttct cggcatgcgt ctgaaatgaa 660
tgctacatct tctttaggca agatgtttcc gcaaggattg ttcgggcgcg atagcaagac 720
agcaccgatg cgcatcctct ctaaaaaccc cttacggtcg agctcatatc gaaacgtatg 780
atcatccaat ttcgatatga gcggagggat cccctcaatc atctcccgct ccagtgccgc 840
cccgctgtat cccgaatagt caggcagcat cgggatcaag gcttttttca tcacagatcc 900
gcttcccatt ccgcaaaacg aattgatcgc cagaaaaaac agctgctggc ttccggctgt 960
aatcaacacg ttctcttttc gaatgccggc gctataccgc tctgaaaaga agcggacaac 1020
acttgcaatc agttcatcgg ttccatagct cgatccgtat tggccgatca ccgaagaaaa 1080
cctgtcatcg tcaaggagat cggcaagagc cgacttccac atggctgaca cgccgggcaa 1140
aatcatcgga ttgcccgcac ttaaattaat gtatgaccgt tcaccgccgg ccaggacttc 1200
ctgaatatcg ctcatcacag ccctgacccc tgttttctca atcattttct ctccgatttt 1260
gcttaatggc ggcttcac 1278
<210>234
<211>425
<212>PRT
<213>地衣芽孢杆菌
<400>234
Met Lys Pro Pro Leu Ser Lys Ile Gly Glu Lys Met Ile Glu Lys Thr
1 5 10 15
Gly Val Arg Ala Val Met Ser Asp Ile Gln Glu Val Leu Ala Gly Gly
20 25 30
Glu Arg Ser Tyr Ile Asn Leu Ser Ala Gly Asn Pro Met Ile Leu Pro
35 40 45
Gly Val Ser Ala Met Trp Lys Ser Ala Leu Ala Asp Leu Leu Asp Asp
50 55 60
Asp Arg Phe Ser Ser Val Ile Gly Gln Tyr Gly Ser Ser Tyr Gly Thr
65 70 75 80
Asp Glu Leu Ile Ala Ser Val Val Arg Phe Phe Ser Glu Arg Tyr Ser
85 90 95
Ala Gly Ile Arg Lys Glu Asn Val Leu Ile Thr Ala Gly Ser Gln Gln
100 105 110
Leu Phe Phe Leu Ala Ile Asn Ser Phe Cys Gly Met Gly Ser Gly Ser
115 120 125
Val Met Lys Lys Ala Leu Ile Pro Met Leu Pro Asp Tyr Ser Gly Tyr
130 135 140
Ser Gly Ala Ala Leu Glu Arg Glu Met Ile Glu Gly Ile Pro Pro Leu
l45 150 155 160
Ile Ser Lys Leu Asp Asp His Thr Phe Arg Tyr Glu Leu Asp Arg Lys
165 170 175
Gly Phe Leu Glu Arg Met Arg Ile Gly Ala Val Leu Leu Ser Arg Pro
180 185 190
Asn Asn Pro Cys Gly Asn Ile Leu Pro Lys Glu Asp Val Ala Phe Ile
195 200 205
Ser Asp Ala Cys Arg Glu Ala Asn Val Pro Leu Phe Ile Asp Ser Ala
210 215 220
Tyr Ala Pro Pro Phe Pro Ala Ile His Phe Ile Asp Met Glu Pro Ile
225 230 235 240
Phe Asn Glu Gln Ile Ile His Cys Met Ser Leu Ser Lys Ala Gly Leu
245 250 255
Pro Gly Glu Arg Ile Gly Ile Ala Ile Gly Pro Ser Arg Tyr Ile Gln
260 265 270
Ala Met Glu Ala Phe Gln Ser Asn Ala Ala Ile His Ser Ser Arg Leu
275 280 285
Gly Gln Tyr Met Ala Ala Ser Val Leu Asn Asp Gly Arg Leu Ala Asp
290 295 300
Val Ser Leu Asn Glu Val Arg Pro Tyr Tyr Arg Asn Lys Phe Met Leu
305 310 315 320
Leu Lys Glu Thr Leu Leu Cys Lys Met Pro Glu Asp Ile Lys Trp Tyr
325 330 335
Leu His Gln Gly Glu Gly Ser Leu Phe Gly Trp Leu Trp Phe Glu Asp
340 345 350
Leu Pro Val Thr Asp Ala Ala Leu Tyr Glu Tyr Met Lys Ala Asp Gly
355 360 365
Val Ile Ile Val Pro Gly Ser Ser Phe Phe His Arg Gln Ser Arg Arg
370 375 380
Leu Ala His Ser His Gln Cys Ile Arg Ile Ser Leu Thr Ala Ala Asp
385 390 395 400
Glu Asp Ile Ile Arg Gly Ile Asp Val Leu Ala Lys Ile Ala Lys Gly
405 410 415
Val Tyr Glu Lys Gln Val Glu Tyr Leu
420 425
<210>235
<211>930
<212>DNA
<213>大肠杆菌
<400>235
atgaccacga agaaagctga ttacatttgg ttcaatgggg agatggttcg ctgggaagac 60
gcgaaggtgc atgtgatgtc gcacgcgctg cactatggca cttcggtttt tgaaggcatc 120
cgttgctacg actcgcacaa aggaccggtt gtattccgcc atcgtgagca tatgcagcgt 180
ctgcatgact ccgccaaaat ctatcgcttc ccggtttcgc agagcattga tgagctgatg 240
gaagcttgtc gtgacgtgat ccgcaaaaac aatctcacca gcgcctatat ccgtccgctg 300
atcttcgtcg gtgatgttgg catgggagta aacccgccag cgggatactc aaccgacgtg 360
attatcgctg ctttcccgtg gggagcgtat ctgggcgcag aagcgctgga gcaggggatc 420
gatgcgatgg tttcctcctg gaaccgcgca gcaccaaaca ccatcccgac ggcggcaaaa 480
gccggtggta actacctctc ttccctgctg gtgggtagcg aagcgcgccg ccacggttat 540
caggaaggta tcgcgctgga tgtgaacggt tatatctctg aaggcgcagg cgaaaacctg 600
tttgaagtga aagatggtgt gctgttcacc ccaccgttca cctcctccgc gctgccgggt 660
attacccgtg atgccatcat caaactggcg aaagagctgg gaattgaagt acgtgagcag 720
gtgctgtcgc gcgaatccct gtacctggcg gatgaagtgt ttatgtccgg tacggcggca 780
gaaatcacgc cagtgcgcag cgtagacggt attcaggttg gcgaaggccg ttgtggcccg 840
gttaccaaac gcattcagca agccttcttc ggcctcttca ctggcgaaac cgaagataaa 900
tggggctggt tagatcaagt taatcaataa 930
<210>236
<211>309
<212>PRT
<213>大肠杆菌
<400>236
Met Thr Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val
1 5 10 15
Arg Trp Glu Asp Ala Lys Val His Val Met Ser His Ala Leu His Tyr
20 25 30
Gly Thr Ser Val Phe Glu Gly Ile Arg Cys Tyr Asp Ser His Lys Gly
35 40 45
Pro Val Val Phe Arg His Arg Glu His Met Gln Arg Leu His Asp Ser
50 55 60
Ala Lys Ile Tyr Arg Phe Pro Val Ser Gln Ser Ile Asp Glu Leu Met
65 70 75 80
Glu Ala Cys Arg Asp Val Ile Arg Lys Asn Asn Leu Thr Ser Ala Tyr
85 90 95
Ile Arg Pro Leu Ile Phe Val Gly Asp Val Gly Met Gly Val Asn Pro
100 105 110
Pro Ala Gly Tyr Ser Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly
115 120 125
Ala Tyr Leu Gly Ala Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val
130 135 140
Ser Ser Trp Asn Arg Ala Ala Pro Asn Thr Ile Pro Thr Ala Ala Lys
145 150 155 160
Ala Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg
165 170 175
Arg His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Val Asn Gly Tyr Ile
180 185 190
Ser Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Asp Gly Val Leu
195 200 205
Phe Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp
210 215 220
Ala Ile Ile Lys Leu Ala Lys Glu Leu Gly Ile Glu Val Arg Glu Gln
225 230 235 240
Val Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser
245 250 255
Gly Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln
260 265 270
Val Gly Glu Gly Arg Cys Gly Pro Val Thr Lys Arg IIe Gln Gln Ala
275 280 285
Phe Phe Gly Leu Phe Thr Gly Glu Thr Glu Asp Lys Trp Gly Trp Leu
290 295 300
Asp Gln Val Asn Gln
305
<210>237
<211>1131
<212>DNA
<213>酿酒酵母
<400>237
atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 60
aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 120
gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 180
tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 240
gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 300
atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 360
ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 420
ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480
agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 540
gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 600
aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 660
ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 720
atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 780
ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 840
gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 900
actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 960
gtttctccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1020
ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1080
ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a 1131
<210>238
<211>376
<212>PRT
<213>酿酒酵母
<400>238
Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr
1 5 l0 15
Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys
20 25 30
Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly
35 40 45
Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro
50 55 60
Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys
65 70 75 80
Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met
85 90 95
Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr
100 105 110
Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln
115 120 125
Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg
130 135 140
Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp
145 150 155 160
Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys
165 170 175
Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg
180 185 190
Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala
195 200 205
Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln
210 215 220
Asn Leu Trp Leu Phe Gly Pro Asn Asn Asn Ile Thr Glu Val Gly Thr
225 230 235 240
Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu
245 250 255
Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg
260 265 270
Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp
275 280 285
Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser
290 295 300
Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile
305 310 3l5 320
Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile
325 330 335
Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala
340 345 350
Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp
355 360 365
Ser Arg Val Val Thr Asp Leu Asn
370 375
<210>239
<211>993
<212>DNA
<213>嗜热碱甲烷杆菌
<400>239
tcagatgtag gtgagccatc cgaagctgtc ctctgtctct gccctgatta tcctgaagaa 60
ctcatcctgc agcagctttg taacgggacc ccttcgcccg gcacctatct ctataccatc 120
aactgatctg atgggtgtta tctctgcggc tgtacctgtg aagaaggcct catctgcgat 180
gtagagcatc tccctggtta tgggttcctc atgcacggta acaccctcgg tcctggctat 240
ctttattacg gagtcccttg ttatccccct cagaagggat gatgaaacag ggggggtgta 300
aatttcaccc tcactgacga ggaatatgtt ctccccgcta ccctcactta tgtagccatg 360
gtagtccagc attatggcct catcatagcc gtgtctcaca gcctccatct tggcaagctg 420
tgagttgagg tagttaccgc cggcctttgc catgttgggc attgtgtttg gtgccatcct 480
ccgccaggtt gaaacaccag catcgacacc aacctcaagg gcctctgcac ccagataggc 540
cccccattcc caggcagcca cagcgacgtc cactgggcag ttcaccgggt gaacacccat 600
ctcaccgtat cccctgaata ccacgggtct tatatagcac tcctcaagtc cgttctccct 660
gacggtctca actatggcat cacatatctg ctcctgggtg tagggtatgt ccatccggta 720
tatctttgca gaatcaaaaa ggcgtttaac atgctcccgc aaacggaaga tggctgaccc 780
cttactgttc ctgtagcacc ttattccctc aaagacagat gatccataat gcacaacatg 840
tgagagtacg tggacggtgg cttcttccca ttcaaccatt tcaccgttta accatatctt 900
tccactggct tcgcatgaca tgataataac ctcaggtgat ttactaggat aggttatggt 960
tggaggccta tataatgctc tccataaccg caa 993
<210>240
<211>330
<212>PRT
<213>嗜热碱甲烷杆菌
<400>240
Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile Thr Tyr Pro
1 5 10 15
Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys
20 25 30
Ile Trp Leu Asn Gly Glu Met Val Glu Trp Glu Glu Ala Thr Val His
35 40 45
Val Leu Ser His Val Val His Tyr Gly Ser Ser Val Phe Glu Gly Ile
50 55 60
Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg Leu Arg Glu
65 70 75 80
His Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile
85 90 95
Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg
100 105 110
Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg Pro Val Val Phe Arg Gly
115 120 125
Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp Val Ala
130 135 140
Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu
145 150 155 160
Val Gly Val Asp Ala Gly Val Ser Thr Trp Arg Arg Met Ala Pro Asn
165 170 175
Thr Met Pro Asn Met Ala Lys Ala Gly Gly Asn Tyr Leu Asn Ser Gln
180 185 190
Leu Ala Lys Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile Met
195 200 205
Leu Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe
210 215 220
Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu
225 230 235 240
Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala Arg Thr Glu
245 250 255
Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu Tyr Ile
260 265 270
Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu Ile Thr Pro Ile
275 280 285
Arg Ser Val Asp Gly Ile Glu Ile Gly Ala Gly Arg Arg Gly Pro Val
290 295 300
Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg Ile Ile Arg Ala Glu Thr
305 310 315 320
Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile
325 330
<210>241
<211>1095
<212>DNA
<213>天蓝色链霉菌
<400>241
tcacggccgg ggacgggcct ccgccatccg ctgctcggcg atccggtcgg ccgccgcggc 60
cggcggaata ccgtcctcct tcgcacgtgc gaatatggcc agcgtggtgt cgtagatctt 120
cgaggccttc gccttgcacc ggtcgaagtc gaacccgtgc agctcgtcgg cgacctggat 180
gacaccgccg gcgttcacca catagtccgg cgcgtagagg atcccgcggt cggcgaggtc 240
cttctcgacg cccgggtggg cgagctggtt gttggccgcg ccgcacacca ccttggcggt 300
cagcaccggc acggtgtcgt cgttcagcgc gccgccgagc gcgcagggcg cgtagatgtc 360
caggttctcc acccggatca gcgcgtcggt gtcggcgacg gcgaccaccg acgggtgccg 420
ctccgtgatc ccgcgcacca cgtccttgcg cacgtccgtg acgacgacgt gggcgccctc 480
ggcgagcagg tgctcgacca ggtggtggcc gaccttgccg acgcccgcga tgccgacggt 540
gcggtcgcgc agcgtcgggt cgccccacag gtgctgggcg gcggcccgca tgccctggta 600
gacgccgaag gaggtgagca cggaggagtc gcccgcgccg ccgttctccg gggaacgccc 660
ggtcgtccag cggcactcgc gggccacgac gtccatgtcg gcgacgtagg tgccgacgtc 720
gcacgcggtg acgtagcggc cgcccagcga ggcgacgaac cggccgtagg cgaggagcag 780
ctcctcgctc ttgatctgct ccggatcgcc gatgatcacg gccttgccgc caccgtggtc 840
cagaccggcc atggcgttct tgtacgacat cccgcgggcg aggttcagcg cgtcggcgac 900
ggcctccgcc tcgctcgcgt acgggtagaa gcgggtaccg ccgagcgccg ggcccagggc 960
ggtggagtgg agggcgatca cggccttgag gccgctggca cggtcctggc agagcacgac 1020
ttgctcatgt cccccctgat ccgagtggaa cagggtgtgc agtacatcag caggtgcgcc 1080
gtttacgtcg gtcac 1095
<210>242
<211>364
<212>PRT
<213>天蓝色链霉菌
<400>242
Met Thr Asp Val Asn Gly Ala Pro Ala Asp Val Leu His Thr Leu Phe
1 5 10 15
His Ser Asp Gln Gly Gly His Glu Gln Val Val Leu Cys Gln Asp Arg
20 25 30
Ala Ser Gly Leu Lys Ala Val Ile Ala Leu His Ser Thr Ala Leu Gly
35 40 45
Pro Ala Leu Gly Gly Thr Arg Phe Tyr Pro Tyr Ala Ser Glu Ala Glu
50 55 60
Ala Val Ala Asp Ala Leu Asn Leu Ala Arg Gly Met Ser Tyr Lys Asn
65 70 75 80
Ala Met Ala Gly Leu Asp His Gly Gly Gly Lys Ala Val Ile Ile Gly
85 90 95
Asp Pro Glu Gln Ile Lys Ser Glu Glu Leu Leu Leu Ala Tyr Gly Arg
100 105 110
Phe Val Ala Ser Leu Gly Gly Arg Tyr Val Thr Ala Cys Asp Val Gly
115 120 125
Thr Tyr Val Ala Asp Met Asp Val Val Ala Arg Glu Cys Arg Trp Thr
130 135 140
Thr Gly Arg Ser Pro Glu Asn Gly Gly Ala Gly Asp Ser Ser Val Leu
145 150 155 160
Thr Ser Phe Gly Val Tyr Gln Gly Met Arg Ala Ala Ala Gln His Leu
165 170 175
Trp Gly Asp Pro Thr Leu Arg Asp Arg Thr Val Gly Ile Ala Gly Val
180 185 190
Gly Lys Val Gly His His Leu Val Glu His Leu Leu Ala Glu Gly Ala
195 200 205
His Val Val Val Thr Asp Val Arg Lys Asp Val Val Arg Gly Ile Thr
210 215 220
Glu Arg His Pro Ser Val Val Ala Val Ala Asp Thr Asp Ala Leu Ile
225 230 235 240
Arg Val Glu Asn Leu Asp Ile Tyr Ala Pro Cys Ala Leu Gly Gly Ala
245 250 255
Leu Asn Asp Asp Thr Val Pro Val Leu Thr Ala Lys Val Val Cys Gly
260 265 270
Ala Ala Asn Asn Gln Leu Ala His Pro Gly Val Glu Lys Asp Leu Ala
275 280 285
Asp Arg Gly Ile Leu Tyr Ala Pro Asp Tyr Val Val Asn Ala Gly Gly
290 295 300
Val Ile Gln Val Ala Asp Glu Leu His Gly Phe Asp Phe Asp Arg Cys
305 310 315 320
Lys Ala Lys Ala Ser Lys Ile Tyr Asp Thr Thr Leu Ala Ile Phe Ala
325 330 335
Arg Ala Lys Glu Asp Gly Ile Pro Pro Ala Ala Ala Ala Asp Arg Ile
340 345 350
Ala Glu Gln Arg Met Ala Glu Ala Arg Pro Arg Pro
355 360
<210>243
<211>1095
<212>DNA
<213>枯草芽孢杆菌
<400>243
atggaacttt ttaaatatat ggagaaatac gattacgaac aattggtatt ctgccaggat 60
gaacaatctg gattaaaagc gattatcgcc attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga tgcgctcaga 180
ttggcaagag gcatgaccta taagaacgcg gcggcaggct taaaccttgg cggcggaaaa 240
acagtcatta tcggcgatcc gcgcaaagac aaaaatgagg aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa tggcagatac atcacggctg aagatgtggg cacaacggtc 360
gaggatatgg atatcattca tgatgagaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccgtc cccagtcaca gcgtacgggg tgtacagagg aatgaaggca 480
gcagctaaag ctgctttcgg aaccgattct cttgaaggaa aaaccattgc tgtacagggt 540
gttgggaacg tagcctataa cctttgccgc cacctgcatg aagaaggagc aaacttaatc 600
gttacggata tcaacaaaca atctgtacag cgtgcagttg aagattttgg cgcccgtgcg 660
gtagatcctg atgacattta ttcacaagac tgcgatattt atgcgccgtg tgcccttggt 720
gcgactatta acgacgacac cattaaacag ctgaaggcga aagtgatcgc aggtgcggct 780
aacaaccaat taaaagagac acgccatggt gatcaaattc acgaaatggg catcgtttat 840
gcaccggatt acgtgattaa cgcgggcggt gtcatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat cgagcgtgta 960
cttgagattt ctcagcgtga cggcattcct gcatatttag cggctgaccg cttagcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210>244
<211>364
<212>PRT
<213>枯草芽孢杆菌
<400>244
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp
210 215 220
Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210>245
<211>1785
<212>DNA
<213>生绿链霉菌
<400>245
gtgtcaactt cctccgcttc ttccgggccg gacctcccct tcgggcccga ggacacgcca 60
tggcagaagg ccttcagcag gctgcgggcg gtggatggcg tgccgcgcgt caccgcgccg 120
tccagtgatc cgcgtgaggt ctacatggac atcccggaga tccccttctc caaggtccag 180
atccccccgg acggaatgga cgagcagcag tacgcagagg ccgagagcct cttccgccgc 240
tacgtagacg cccagacccg caacttcgcg ggataccagg tcaccagcga cctcgactac 300
cagcacctca gtcactatct caaccggcat ctgaacaacg tcggcgatcc ctatgagtcc 360
agctcctaca cgctgaactc caaggtcctt gagcgagccg ttctcgacta cttcgcctcc 420
ctgtggaacg ccaagtggcc ccatgacgca agcgatccgg aaacgtactg gggttacgtg 480
ctgaccatgg gctccagcga aggcaacctg tacgggttgt ggaacgcacg ggactatctg 540
tcgggcaagc tgctgcggcg ccagcaccgg gaggccggcg gcgacaaggc ctcggtcgtc 600
tacacgcaag cgctgcgaca cgaagggcag agtccgcatg cctacgagcc ggtggcgttc 660
ttctcgcagg acacgcacta ctcgctcacg aaggccgtgc gggttctggg catcgacacc 720
ttccacagca tcggcagcag tcggtatccg gacgagaacc cgctgggccc cggcactccg 780
tggccgaccg aagtgccctc ggttgacggt gccatcgatg tcgacaaact cgcctcgttg 840
gtccgcttct tcgccagcaa gggctacccg atactggtca gcctcaacta cgggtcaacg 900
ttcaagggcg cctacgacga cgtcccggcc gtggcacagg ccgtgcggga catctgcacg 960
gaatacggtc tggatcggcg gcgggtatac cacgaccgca gtaaggacag tgacttcgac 1020
gagcgcagcg gcttctggat ccacatcgat gccgccctgg gggcgggcta cgctccctac 1080
ctgcagatgg cccgggatgc cggcatggtc gaggaggcgc cgcccgtttt cgacttccgg 1140
ctcccggagg tgcactcgct gaccatgagc ggccacaagt ggatgggaac accgtgggca 1200
tgcggtgtct acatgacacg gaccgggctg cagatgaccc cgccgaagtc gtccgagtac 1260
atcggggcgg ccgacaccac cttcgcgggc tcccgcaacg gcttctcgtc actgctgctg 1320
tgggactacc tgtcccggca ttcgtatgac gatctggtgc gcctggccgc cgactgcgac 1380
cggctggccg gctacgccca cgaccggttg ctgaccttgc aggacaaact cggcatggat 1440
ctgtgggtcg cccgcagccc gcagtccctc acggtgcgct tccgtcagcc atgtgcagac 1500
atcgtccgca agtactcgct gtcgtgtgag acggtctacg aagacaacga gcaacggacc 1560
tacgtacatc tctacgccgt tccccacctc actcgggaac tcgtggatga gctcgtgcgc 1620
gatctgcgcc agcccggagc cttcaccaac gctggtgcac tggaggggga ggcctgggcc 1680
ggggtgatcg atgccctcgg ccgcccggac cccgacggaa cctatgccgg cgccttgagc 1740
gctccggctt ccggcccccg ctccgaggac ggcggcggga gctga 1785
<210>246
<211>594
<212>PRT
<213>生绿链霉菌
<400>246
Met Ser Thr Ser Ser Ala Ser Ser Gly Pro Asp Leu Pro Phe Gly Pro
1 5 10 15
Glu Asp Thr Pro Trp Gln Lys Ala Phe Ser Arg Leu Arg Ala Val Asp
20 25 30
Gly Val Pro Arg Val Thr Ala Pro Ser Ser Asp Pro Arg Glu Val Tyr
35 40 45
Met Asp Ile Pro Glu Ile Pro Phe Ser Lys Val Gln Ile Pro Pro Asp
50 55 60
Gly Met Asp Glu Gln Gln Tyr Ala Glu Ala Glu Ser Leu Phe Arg Arg
65 70 75 80
Tyr Val Asp Ala Gln Thr Arg Asn Phe Ala Gly Tyr Gln Val Thr Ser
85 90 95
Asp Leu Asp Tyr Gln His Leu Ser His Tyr Leu Asn Arg His Leu Asn
100 105 110
Asn Val Gly Asp Pro Tyr Glu Ser Ser Ser Tyr Thr Leu Asn Ser Lys
115 120 125
Val Leu Glu Arg Ala Val Leu Asp Tyr Phe Ala Ser Leu Trp Asn Ala
130 135 140
Lys Trp Pro His Asp Ala Ser Asp Pro Glu Thr Tyr Trp Gly Tyr Val
145 150 155 160
Leu Thr Met Gly Ser Ser Glu Gly Asn Leu Tyr Gly Leu Trp Asn Ala
165 170 175
Arg Asp Tyr Leu Ser Gly Lys Leu Leu Arg Arg Gln His Arg Glu Ala
180 185 190
Gly Gly Asp Lys Ala Ser Val Val Tyr Thr Gln Ala Leu Arg His Glu
195 200 205
Gly Gln Ser Pro His Ala Tyr Glu Pro Val Ala Phe Phe Ser Gln Asp
210 215 220
Thr His Tyr Ser Leu Thr Lys Ala Val Arg Val Leu Gly Ile Asp Thr
225 230 235 240
Phe His Ser Ile Gly Ser Ser Arg Tyr Pro Asp Glu Asn Pro Leu Gly
245 250 255
Pro Gly Thr Pro Trp Pro Thr Glu Val Pro Ser Val Asp Gly Ala Ile
260 265 270
Asp Val Asp Lys Leu Ala Ser Leu Val Arg Phe Phe Ala Ser Lys Gly
275 280 285
Tyr Pro Ile Leu Val Ser Leu Asn Tyr Gly Ser Thr Phe Lys Gly Ala
290 295 300
Tyr Asp Asp Val Pro Ala Val Ala Gln Ala Val Arg Asp Ile Cys Thr
305 310 315 320
Glu Tyr Gly Leu Asp Arg Arg Arg Val Tyr His Asp Arg Ser Lys Asp
325 330 335
Ser Asp Phe Asp Glu Arg Ser Gly Phe Trp Ile His Ile Asp Ala Ala
340 345 350
Leu Gly Ala Gly Tyr Ala Pro Tyr Leu Gln Met Ala Arg Asp Ala Gly
355 360 365
Met Val Glu Glu Ala Pro Pro Val Phe Asp Phe Arg Leu Pro Glu Val
370 375 380
His Ser Leu Thr Met Ser Gly His Lys Trp Met Gly Thr Pro Trp Ala
385 390 395 400
Cys Gly Val Tyr Met Thr Arg Thr Gly Leu Gln Met Thr Pro Pro Lys
405 410 415
Ser Ser Glu Tyr Ile Gly Ala Ala Asp Thr Thr Phe Ala Gly Ser Arg
420 425 430
Asn Gly Phe Ser Ser Leu Leu Leu Trp Asp Tyr Leu Ser Arg His Ser
435 440 445
Tyr Asp Asp Leu Val Arg Leu Ala Ala Asp Cys Asp Arg Leu Ala Gly
450 455 460
Tyr Ala His Asp Arg Leu Leu Thr Leu Gln Asp Lys Leu Gly Met Asp
465 470 475 480
Leu Trp Val Ala Arg Ser Pro Gln Ser Leu Thr Val Arg Phe Arg Gln
485 490 495
Pro Cys Ala Asp Ile Val Arg Lys Tyr Ser Leu Ser Cys Glu Thr Val
500 505 510
Tyr Glu Asp Asn Glu Gln Arg Thr Tyr Val His Leu Tyr Ala Val Pro
515 520 525
His Leu Thr Arg Glu Leu Val Asp Glu Leu Val Arg Asp Leu Arg Gln
530 535 540
Pro Gly Ala Phe Thr Asn Ala Gly Ala Leu Glu Gly Glu Ala Trp Ala
545 550 555 560
Gly Val Ile Asp Ala Leu Gly Arg Pro Asp Pro Asp Gly Thr Tyr Ala
565 570 575
Gly Ala Leu Ser Ala Pro Ala Ser Gly Pro Arg Ser Glu Asp Gly Gly
580 585 590
Gly Ser
<210>247
<211>1323
<212>DNA
<213>反硝化产碱菌
<400>247
atgagcgctg ccaaactgcc cgacctgtcc cacctctgga tgccctttac cgccaaccgg 60
cagttcaagg cgaacccccg cctgctggcc tcggccaagg gcatgtacta cacgtctttc 120
gacggccgcc agatcctgga cggcacggcc ggcctgtggt gcgtgaacgc cggccactgc 180
cgcgaagaaa tcgtctccgc catcgccagc caggccggcg tcatggacta cgcgccgggg 240
ttccagctcg gccacccgct ggccttcgag gccgccaccg ccgtggccgg cctgatgccg 300
cagggcctgg accgcgtgtt cttcaccaat tcgggctccg aatcggtgga caccgcgctg 360
aagatcgccc tggcctacca ccgcgcgcgc ggcgaggcgc agcgcacccg cctcatcggg 420
cgcgagcgcg gctaccacgg cgtgggcttc ggcggcattt ccgtgggcgg catctcgccc 480
aaccgcaaga ccttctccgg cgcgctgctg ccggccgtgg accacctgcc gcacacccac 540
agcctggaac acaacgcctt cacgcgcggc cagcccgagt ggggcgcgca cctggccgac 600
gagttggaac gcatcatcgc cctgcacgac gcctccacca tcgcggccgt gatcgtcgag 660
cccatggccg gctccaccgg cgtgctcgtc ccgcccaagg gctatctcga aaaactgcgc 720
gaaatcaccg cccgccacgg cattctgctg atcttcgacg aagtcatcac cgcgtacggc 780
cgcctgggcg aggccaccgc cgcggcctat ttcggcgtaa cgcccgacct catcaccatg 840
gccaagggcg tgagcaacgc cgccgttccg gccggcgccg tcgcggtgcg ccgcgaagtg 900
catgacgcca tcgtcaacgg accgcaaggc ggcatcgagt tcttccacgg ctacacctac 960
tcggcccacc cgctggccgc cgccgccgtg ctcgccacgc tggacatcta ccgccgcgaa 1020
gacctgttcg cccgcgcccg caagctgtcg gccgcgttcg aggaagccgc ccacagcctc 1080
aagggcgcgc cgcacgtcat cgacgtgcgc aacatcggcc tggtggccgg catcgagctg 1140
tcgccgcgcg aaggcgcccc gggcgcgcgc gccgccgaag ccttccagaa atgcttcgac 1200
accggcctca tggtgcgcta cacgggcgac atcctcgcgg tgtcgcctcc gctcatcgtc 1260
gacgaaaacc agatcggcca gatcttcgag ggcatcggca aggtgctcaa ggaagtggct 1320
tag 1323
<210>248
<211>440
<212>PRT
<213>ω氨基酸:丙酮酸转氨酶
<400>248
Met Ser Ala Ala Lys Leu Pro Asp Leu Ser His Leu Trp Met Pro Phe
1 5 10 15
Thr Ala Asn Arg Gln Phe Lys Ala Asn Pro Arg Leu Leu Ala Ser Ala
20 25 30
Lys Gly Met Tyr Tyr Thr Ser Phe Asp Gly Arg Gln Ile Leu Asp Gly
35 40 45
Thr Ala Gly Leu Trp Cys Val Asn Ala Gly His Cys Arg Glu Glu Ile
50 55 60
Val Ser Ala Ile Ala Ser Gln Ala Gly Val Met Asp Tyr Ala Pro Gly
65 70 75 80
Phe Gln Leu Gly His Pro Leu Ala Phe Glu Ala Ala Thr Ala Val Ala
85 90 95
Gly Leu Met Pro Gln Gly Leu Asp Arg Val Phe Phe Thr Asn Ser Gly
100 105 110
Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr His Arg
115 120 125
Ala Arg Gly Glu Ala Gln Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly
130 135 140
Tyr His Gly Val Gly Phe Gly Gly Ile Ser Val Gly Gly Ile Ser Pro
145 150 155 160
Asn Arg Lys Thr Phe Ser Gly Ala Leu Leu Pro Ala Val Asp His Leu
165 170 175
Pro His Thr His Ser Leu Glu His Asn Ala Phe Thr Arg Gly Gln Pro
180 185 190
Glu Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Ile Ala Leu
195 200 205
His Asp Ala Ser Thr Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly
210 215 220
Ser Thr Gly Val Leu Val Pro Pro Lys Gly Tyr Leu Glu Lys Leu Arg
225 230 235 240
Glu Ile Thr Ala Arg His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile
245 250 255
Thr Ala Tyr Gly Arg Leu Gly Glu Ala Thr Ala Ala Ala Tyr Phe Gly
260 265 270
Val Thr Pro Asp Leu Ile Thr Met Ala Lys Gly Val Ser Asn Ala Ala
275 280 285
Val Pro Ala Gly Ala Val Ala Val Arg Arg Glu Val His Asp Ala Ile
290 295 300
Val Asn Gly Pro Gln Gly Gly Ile Glu Phe Phe His Gly Tyr Thr Tyr
305 310 315 320
Ser Ala His Pro Leu Ala Ala Ala Ala Val Leu Ala Thr Leu Asp Ile
325 330 335
Tyr Arg Arg Glu Asp Leu Phe Ala Arg Ala Arg Lys Leu Ser Ala Ala
340 345 350
Phe Glu Glu Ala Ala His Ser Leu Lys Gly Ala Pro His Val Ile Asp
355 360 365
Val Arg Asn Ile Gly Leu Val Ala Gly Ile Glu Leu Ser Pro Arg Glu
370 375 380
Gly Ala Pro Gly Ala Arg Ala Ala Glu Ala Phe Gln Lys Cys Phs Asp
385 390 395 400
Thr Gly Leu Met Val Arg Tyr Thr Gly Asp Ile Leu Ala Val Ser Pro
405 410 415
Pro Leu Ile Val Asp Glu Asn Gln Ile Gly Gln Ile Phe Glu Gly Ile
420 425 430
Gly Lys Val Leu Lys Glu Val Ala
435 440
<210>249
<211>1332
<212>DNA
<213>真养雷氏菌
<400>249
atggacgccg cgaagaccgt gattcccgat ctcgatgccc tgtggatgcc ctttaccgcg 60
aaccgccagt acaaggcggc gccgcgcctg ctggcctcgg ccagcggcat gtactacacc 120
acccacgacg gacgccagat cctcgacggt tgcgcgggcc tctggtgcgt agcggccggc 180
cactgccgca aggagattgc cgaggccgtg gcccgccagg ccgccacgct cgactacgcg 240
ccgccgttcc agatgggcca tccgctgtcg ttcgaagccg ccaccaaggt ggccgcgatc 300
atgccgcagg gactggaccg catcttcttc acgaattccg gttcggaatc ggtggacacc 360
gcgctgaaga ttgcgctggc ctaccaccgt gcgcgcggcg agggccagcg cacccgcttc 420
atcgggcgcg aacgcggtta ccacggcgtg ggctttggcg gcatggctgt cggtggcatc 480
gggccgaacc gcaaggcgtt ctcggccaac ctgatgccgg gcaccgacca tctgccggcg 540
acgctgaata tcgccgaagc ggcgttctcc aagggtcagc cgacatgggg cgcgcacctt 600
gccgacgaac tcgagcgcat cgtcgcgctg catgatccgt ccacgattgc cgccgtcatc 660
gtggaaccgc tggcgggctc cgccggggtg ctggtgccgc cggtcggcta cctcgacaag 720
ctgcgcgaga tcacgaccaa gcacggcatc ctgctgatct tcgacgaggt catcacggcc 780
tttggtcgcc tgggtaccgc caccgcggcg gaacgcttca aggtcacgcc ggacctgatc 840
accatggcca aggccatcaa caacgccgcc gtgccgatgg gtgccgtggc cgtgcgccgc 900
gaagtccatg acaccgtggt caactcggcc gcgccgggcg cgatcgaact cgcgcatggc 960
tacacctact cgggccaccc gctggccgcc gccgctgcca tcgccacgct ggacctgtat 1020
cagcgcgaga acctgttcgg ccgtgccgcg gagctgtcgc cggtgttcga agcggccgtt 1080
cacagcgtac gcagcgcgcc gcatgtgaag gacatccgca acctcggcat ggtggccggc 1140
atcgagctgg agccgcgtcc gggccagccc ggcgcacgcg cctacgaagc cttcctcaaa 1200
tgccttgagc gtggcgtgct ggtgcgctac accggcgata tcctcgcgtt ctcgccgccg 1260
ctgatcatca gcgaggcgca gattgccgag ctgttcgata cggtcaagca ggccttgcag 1320
gaagtgcagt aa 1332
<210>250
<211>443
<212>PRT
<213>真养雷氏菌
<400>250
Met Asp Ala Ala Lys Thr Val Ile Pro Asp Leu Asp Ala Leu Trp Met
1 5 10 15
Pro Phe Thr Ala Asn Arg Gln Tyr Lys Ala Ala Pro Arg Leu Leu Ala
20 25 30
Ser Ala Ser Gly Met Tyr Tyr Thr Thr His Asp Gly Arg Gln Ile Leu
35 40 45
Asp Gly Cys Ala Gly Leu Trp Cys Val Ala Ala Gly His Cys Arg Lys
50 55 60
Glu Ile Ala Glu Ala Val Ala Arg Gln Ala Ala Thr Leu Asp Tyr Ala
65 70 75 80
Pro Pro Phe Gln Met Gly His Pro Leu Ser Phe Glu Ala Ala Thr Lys
85 90 95
Val Ala Ala Ile Met Pro Gln Gly Leu Asp Arg Ile Phe Phe Thr Asn
100 105 110
Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr
115 120 125
His Arg Ala Arg Gly Glu Gly Gln Arg Thr Arg Phe Ile Gly Arg Glu
130 135 140
Arg Gly Tyr His Gly Val Gly Phe Gly Gly Met Ala Val Gly Gly Ile
145 150 155 160
Gly Pro Asn Arg Lys Ala Phe Ser Ala Asn Leu Met Pro Gly Thr Asp
165 170 175
His Leu Pro Ala Thr Leu Asn Ile Ala Glu Ala Ala Phe Ser Lys Gly
180 185 190
Gln Pro Thr Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Val
195 200 205
Ala Leu His Asp Pro Ser Thr Ile Ala Ala Val Ile Val Glu Pro Leu
210 215 220
Ala Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly Tyr Leu Asp Lys
225 230 235 240
Leu Arg Glu Ile Thr Thr Lys His Gly Ile Leu Leu Ile Phe Asp Glu
245 250 255
Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Ala Thr Ala Ala Glu Arg
260 265 270
Phe Lys Val Thr Pro Asp Leu Ile Thr Met Ala Lys Ala Ile Asn Asn
275 280 285
Ala Ala Val Pro Met Gly Ala Val Ala Val Arg Arg Glu Val His Asp
290 295 300
Thr Val Val Asn Ser Ala Ala Pro Gly Ala Ile Glu Leu Ala His Gly
305 310 315 320
Tyr Thr Tyr Ser Gly His Pro Leu Ala Ala Ala Ala Ala Ile Ala Thr
325 330 335
Leu Asp Leu Tyr Gln Arg Glu Asn Leu Phe Gly Arg Ala Ala Glu Leu
340 345 350
Ser Pro Val Phe Glu Ala Ala Val His Ser Val Arg Ser Ala Pro His
355 360 365
Val Lys Asp Ile Arg Asn Leu Gly Met Val Ala Gly Ile Glu Leu Glu
370 375 380
Pro Arg Pro Gly Gln Pro Gly Ala Arg Ala Tyr Glu Ala Phe Leu Lys
385 390 395 400
Cys Leu Glu Arg Gly Val Leu Val Arg Tyr Thr Gly Asp Ile Leu Ala
405 410 415
Phe Ser Pro Pro Leu Ile Ile Ser Glu Ala Gln Ile Ala Glu Leu Phe
420 425 430
Asp Thr Val Lys Gln Ala Leu Gln Glu Val Gln
435 440
<210>251
<211>1341
<212>DNA
<213>Shewanella oneidensis
<400>251
atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60
ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120
atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180
tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240
atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300
ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360
tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420
cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480
gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540
cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600
ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660
gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720
tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780
gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840
ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900
tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960
ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020
tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080
gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140
gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200
ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260
tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320
gcaattcacg ccgttggatg a 1341
<210>252
<211>446
<212>PRT
<213>β丙氨酸-丙酮酸转氨酶
<400>252
Met Ala Asp Ser Pro Asn Asn Leu Ala His Glu His Pro Ser Leu Glu
1 5 10 15
His Tyr Trp Met Pro Phe Thr Ala Asn Arg Gln Phe Lys Ala Ser Pro
20 25 30
Arg Leu Leu Ala Gln Ala Glu Gly Met Tyr Tyr Thr Asp Ile Asn Gly
35 40 45
Asn Lys Val Leu Asp Ser Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly
50 55 60
His Gly Arg Arg Glu Ile Ser Glu Ala Val Ser Lys Gln Ile Arg Gln
65 70 75 80
Met Asp Tyr Ala Pro Ser Phe Gln Met Gly His Pro Ile Ala Phe Glu
85 90 95
Leu Ala Glu Arg Leu Thr Glu Leu Ser Pro Glu Gly Leu Asn Lys Val
100 105 110
Phe Phe Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Met
115 120 125
Ala Leu Cys Tyr His Arg Ala Asn Gly Gln Ala Ser Arg Thr Arg Phe
130 135 140
Ile Gly Arg Glu Met Gly Tyr His Gly Val Gly Phe Gly Gly Ile Ser
145 150 155 160
Val Gly Gly Leu Ser Asn Asn Arg Lys Ala Phe Ser Gly Gln Leu Leu
165 170 175
Gln Gly Val Asp His Leu Pro His Thr Leu Asp Ile Gln His Ala Ala
180 185 190
Phe Ser Arg Gly Leu Pro Ser Leu Gly Ala Glu Lys Ala Glu Val Leu
195 200 205
Glu Gln Leu Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile
210 215 220
Val Glu Pro Met Ser Gly Ser Ala Gly Val Ile Leu Pro Pro Gln Gly
225 230 235 240
Tyr Leu Lys Arg Leu Arg Glu Ile Thr Lys Lys His Gly Ile Leu Leu
245 250 255
Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Val Gly Ala Ala Phe
260 265 270
Ala Ser Gln Arg Trp Gly Val Ile Pro Asp Ile Ile Thr Thr Ala Lys
275 280 285
Ala Ile Asn Asn Gly Ala Ile Pro Met Gly Ala Val Phe Val Gln Asp
290 295 300
Tyr Ile His Asp Thr Cys Met Gln Gly Pro Thr Glu Leu Ile Glu Phe
305 310 315 320
Phe His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Ala Ala
325 330 335
Leu Ala Thr Leu Ser Ile Tyr Gln Asn Glu Gln Leu Phe Glu Arg Ser
340 345 350
Phe Glu Leu Glu Arg Tyr Phe Glu Glu Ala Val His Ser Leu Lys Gly
355 360 365
Leu Pro Asn Val Ile Asp Ile Arg Asn Thr Gly Leu Val Ala Gly Phe
370 375 380
Gln Leu Ala Pro Asn Ser Gln Gly Val Gly Lys Arg Gly Tyr Ser Val
385 390 395 400
Phe Glu His Cys Phe His Gln Gly Thr Leu Val Arg Ala Thr Gly Asp
405 410 415
Ile Ile Ala Met Ser Pro Pro Leu Ile Val Glu Lys His Gln Ile Asp
420 425 430
Gln Met Val Asn Ser Leu Ser Asp Ala Ile His Ala Val Gly
435 440 445
<210>253
<211>1347
<212>DNA
<213>恶臭假单胞菌
<400>253
atgaacatgc ccgaaactgg tcctgccggt atcgccagcc agctcaagct ggacgcccac 60
tggatgccct acaccgccaa ccgcaacttc cagcgcgacc cacgcctgat cgtggcggcc 120
gaaggcaact acctggtcga tgaccacggg cgcaagatct tcgacgccct gtccggcctg 180
tggacctgcg gcgcagggca cactcgcaag gaaatcgctg acgcggtgac ccgtcaactg 240
agtacgctgg actactcccc agcgttccag ttcggccacc cgctgtcgtt ccagctggcg 300
gaaaagatcg ccgagctggt tccgggcaat ctgaatcacg tcttctatac caactccggt 360
tccgagtgcg ccgataccgc actgaagatg gtgcgtgcct actggcgcct gaaaggccag 420
gcaaccaaga ccaagatcat cggccgtgcc cgtggttacc atggcgtgaa catcgccggt 480
accagcctgg gtggcgtcaa cggtaaccgc aagatgtttg gccagctgct ggacgtcgac 540
cacctgcctc acactgtatt gccggtgaac gccttctcga aaggcttgcc ggaagagggc 600
ggtatcgcgc tggctgacga aatgctcaag ctgatcgagc tgcacgatgc ctccaacatc 660
gcagcagtca tcgtcgagcc gctggccggt tcggccggtg tgctgccgcc gccaaagggt 720
tacctgaagc gcctgcgtga aatctgcacc cagcacaaca ttctgctgat cttcgacgaa 780
gtgatcacag gcttcggccg catgggcgcg atgaccggct cggaagcctt cggcgttacc 840
ccggacctga tgtgcatcgc caagcaggtg accaacggcg ccatcccgat gggcgcagtg 900
attgccagca gcgagatcta ccagaccttc atgaaccagc cgaccccgga atacgccgtg 960
gaattcccac acggctacac ctattcggcg cacccggtag cctgtgccgc cggtctcgcc 1020
gcgctggacc tgctgcagaa ggaaaacctg gtgcagtccg cggctgaact ggcgccgcat 1080
ttcgagaagc tgctgcacgg cgtgaagggc accaagaata tcgtcgatat ccgcaactac 1140
ggcctggccg gcgccatcca gatcgccgcc cgtgacggtg atgccatcgt tcgcccttac 1200
gaagcggcca tgaagctgtg gaaagcgggc ttctatgtac gctttggtgg cgacaccctg 1260
cagttcggcc caaccttcaa taccaagccg caggaactgg accgcttgtt cgatgctgtt 1320
ggcgaaaccc tgaacctgat cgactga 1347
<210>254
<211>448
<212>PRT
<213>恶臭假单胞菌
<400>254
Met Asn Met Pro Glu Thr Gly Pro Ala Gly Ile Ala Ser Gln Leu Lys
1 5 10 15
Leu Asp Ala His Trp Met Pro Tyr Thr Ala Asn Arg Asn Phe Gln Arg
20 25 30
Asp Pro Arg Leu Ile Val Ala Ala Glu Gly Asn Tyr Leu Val Asp Asp
35 40 45
His Gly Arg Lys Ile Phe Asp Ala Leu Ser Gly Leu Trp Thr Cys Gly
50 55 60
Ala Gly His Thr Arg Lys Glu Ile Ala Asp Ala Val Thr Arg Gln Leu
65 70 75 80
Ser Thr Leu Asp Tyr Ser Pro Ala Phe Gln Phe Gly His Pro Leu Ser
85 90 95
Phe Gln Leu Ala Glu Lys Ile Ala Glu Leu Val Pro Gly Asn Leu Asn
100 105 110
His Val Phe Tyr Thr Asn Ser Gly Ser Glu Cys Ala Asp Thr Ala Leu
115 120 125
Lys Met Val Arg Ala Tyr Trp Arg Leu Lys Gly Gln Ala Thr Lys Thr
130 135 140
Lys Ile Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Ile Ala Gly
l45 150 155 160
Thr Ser Leu Gly Gly Val Asn Gly Asn Arg Lys Met Phe Gly Gln Leu
165 170 175
Leu Asp Val Asp His Leu Pro His Thr Val Leu Pro Val Asn Ala Phe
180 185 190
Ser Lys Gly Leu Pro Glu Glu Gly Gly Ile Ala Leu Ala Asp Glu Met
195 200 205
Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile
210 215 220
Val Glu Pro Leu Ala Gly Ser Ala Gly Val Leu Pro Pro Pro Lys Gly
225 230 235 240
Tyr Leu Lys Arg Leu Arg Glu Ile Cys Thr Gln His Asn Ile Leu Leu
245 250 255
Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Met Gly Ala Met Thr
260 265 270
Gly Ser Glu Ala Phe Gly Val Thr Pro Asp Leu Met Cys Ile Ala Lys
275 280 285
Gln Val Thr Asn Gly Ala Ile Pro Met Gly Ala Val Ile Ala Ser Ser
290 295 300
Glu Ile Tyr Gln Thr Phe Met Asn Gln Pro Thr Pro Glu Tyr Ala Val
305 310 315 320
Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala
325 330 335
Ala Gly Leu Ala Ala Leu Asp Leu Leu Gln Lys Glu Asn Leu Val Gln
340 345 350
Ser Ala Ala Glu Leu Ala Pro His Phe Glu Lys Leu Leu His Gly Val
355 360 365
Lys Gly Thr Lys Asn Ile Val Asp Ile Arg Asn Tyr Gly Leu Ala Gly
370 375 380
Ala Ile Gln Ile Ala Ala Arg Asp Gly Asp Ala Ile Val Arg Pro Tyr
385 390 395 400
Glu Ala Ala Met Lys Leu Trp Lys Ala Gly Phe Tyr Val Arg Phe Gly
405 410 415
Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Thr Lys Pro Gln Glu
420 425 430
Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Thr Leu Asn Leu Ile Asp
435 440 445
<210>255
<211>1701
<212>DNA
<213>肉桂地链霉菌
<400>255
atggacgctg acgcgatcga ggaaggccgc cgacgctggc aggcccgtta cgacaaggcc 60
cgcaagcgcg acgcggactt caccacgctc tccggggacc ccgtcgaccc cgtctacggc 120
ccccggcccg gggacacgta cgacgggttc gagcggatcg gctggccggg ggagtacccc 180
ttcacccgcg ggctctacgc caccgggtac cgcggccgca cctggaccat ccgccagttc 240
gccggcttcg gcaacgccga gcagacgaac gagcgctaca agatgatcct ggccaacggc 300
ggcggcggcc tctccgtcgc cttcgacatg ccgaccctca tgggccgcga ctccgacgac 360
ccgcgctcgc tcggcgaggt cggccactgc ggtgtcgcca tcgactccgc cgccgacatg 420
gaggtcctct tcaaggacat cccgctcggc gacgtcacga cgtccatgac catcagcggg 480
cccgccgtgc ccgtcttctg catgtacctc gtcgcggccg agcgccaggg cgtcgacccg 540
gccgtcctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600
tggctcttcc agcccgagcc gcacctgcgc ctcatcggcg acctgatgga gcactgcgcg 660
cgcgacatcc ccgcgtacaa gccgctctcg gtctccggct accacatccg cgaggccggg 720
gcgacggccg cgcaggagct cgcgtacacc ctcgcggacg gcttcgggta cgtggaactg 780
ggcctctcgc gcggcctgga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840
gcgcacgtcg acttcttcga ggagatcgcg aagttccgcg ccgcacgccg catctgggcg 900
cgctggctcc gggacgagta cggagcgaag accgagaagg cacagtggct gcgcttccac 960
acgcagaccg cgggggtctc gctcacggcc cagcagccgt acaacaacgt ggtgcggacg 1020
gcggtggagg ccctcgccgc ggtgctcggc ggcacgaact ccctgcacac caacgctctc 1080
gacgagaccc ttgccctccc cagcgagcag gccgcggaga tcgcgctgcg cacccagcag 1140
gtgctgatgg aggagaccgg cgtcgccaac gtcgcggacc cgctgggcgg ctcctggtac 1200
atcgagcagc tcaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcagg 1260
gagcgggggc ggcgggcctg ccccgacggg cagcacccga tcgggccgat cacctccggc 1320
atcctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccag 1380
taccagcggt ccctggagaa gggcgacaag cgggtcgtcg gcgtcaactg cctcgaaggc 1440
tccgtcaccg gcgacctgga gatcctgcgc gtcagccacg aggtcgagcg cgagcaggtg 1500
cgggagcttg cggggcgcaa ggggcggcgt gacgatgcgc gggtgcgggc ctcgctcgac 1560
gcgatgctcg ccgctgcgcg ggacgggtcg aacatgattg cccccatgct ggaggcggtg 1620
cgggccgagg cgaccctcgg ggagatctgc ggggtgcttc gcgatgagtg gggggtctac 1680
gtggagccgc ccgggttctg a 1701
<210>256
<211>566
<212>PRT
<213>肉桂地链霉菌
<400>256
Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg
1 5 10 15
Tyr Asp Lys Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly
20 25 30
Asp Pro Val Asp Pro Val Tyr Gly Pro Arg Pro Gly Asp Thr Tyr Asp
35 40 45
Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly
50 55 60
Leu Tyr Ala Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe
65 70 75 80
Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile
85 90 95
Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr
100 105 110
Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly
115 120 125
His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe
130 135 140
Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly
145 150 155 160
Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln
165 170 175
Gly Val Asp Pro Ala Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe
180 185 190
Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His
195 200 205
Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Arg Asp Ile Pro
210 215 220
Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly
225 230 235 240
Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly
245 250 255
Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala
260 265 270
Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu
275 280 285
Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg
290 295 300
Asp Glu Tyr Gly Ala Lys Thr Glu Lys Ala Gln Trp Leu Arg Phe His
305 310 315 320
Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn
325 330 335
Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr
340 345 350
Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser
355 360 365
Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu
370 375 380
Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr
385 390 395 400
Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe
405 410 415
Glu Gln Ile Arg Glu Arg Gly Arg Arg Ala Cys Pro Asp Gly Gln His
420 425 430
Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly
435 440 445
Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Arg Ser
450 455 460
Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Cys Leu Glu Gly
465 470 475 480
Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu
485 490 495
Arg Glu Gln Val Arg Glu Leu Ala Gly Arg Lys Gly Arg Arg Asp Asp
500 505 510
Ala Arg Val Arg Ala Ser Leu Asp Ala Met Leu Ala Ala Ala Arg Asp
515 520 525
Gly Ser Asn Met Ile Ala Pro Met Leu Glu Ala Val Arg Ala Glu Ala
530 535 540
Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr
545 550 555 560
Val Glu Pro Pro Gly Phe
565
<210>257
<211>411
<212>DNA
<213>肉桂地链霉菌
<400>257
atgggtgtgg cagccgggcc gatccgcgtg gtggtcgcca agccggggct cgacgggcac 60
gatcgcgggg ccaaggtgat cgcgcgggcg ttgcgtgacg cgggtatgga ggtcatctac 120
accgggctgc accagacgcc cgagcaggtg gtggacaccg cgatccagga ggacgccgac 180
gcgatcggcc tctccatcct ctccggagcg cacaacacgc tgttcgcgcg cgtgttggag 240
ctcttgaagg agcgggacgc ggaggacatc aaggtgtttg gtggcggcat catcccggag 300
gcggacatcg cgccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggggccacc 360
accacgtcga tcgtggagtg ggttcggggg aacgtgcgac aggccgtctg a 411
<210>258
<211>136
<212>PRT
<213>肉桂地链霉菌
<400>258
Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly
1 5 10 15
Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg
20 25 30
Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu
35 40 45
Gln Val Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu
50 55 60
Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Arg Val Leu Glu
65 70 75 80
Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly
85 90 95
Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala
100 105 110
Glu Ile Phe Thr Pro Gly Ala Thr Thr Thr Ser Ile Val Glu Trp Val
115 120 125
Arg Gly Asn Val Arg Gln Ala Val
130 135
<210>259
<211>1701
<212>DNA
<213>天蓝色链霉菌
<400>259
atggacgctc atgccataga ggagggccgc cttcgctggc aggcccggta cgacgcggcg 60
cgcaagcgcg acgcggactt caccacgctc tccggagacc ccgtggagcc ggtgtacggg 120
ccccgccccg gggacgagta cgagggcttc gagcggatcg gctggccggg cgagtacccc 180
ttcacccgcg gcctgtatcc gaccgggtac cgggggcgta cgtggaccat ccggcagttc 240
gccgggttcg gcaacgccga gcagaccaac gagcgctaca agatgatcct ccgcaacggc 300
ggcggcgggc tctcggtcgc cttcgacatg ccgaccctga tgggccgcga ctccgacgac 360
ccgcgctcgc tgggcgaggt cgggcactgc ggggtggcca tcgactcggc cgccgacatg 420
gaagtgctgt tcaaggacat cccgctcggg gacgtgacga cctccatgac gatcagcggg 480
cccgccgtgc ccgtgttctg catgtacctc gtcgccgccg agcgccaggg cgtcgacgca 540
tccgtgctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600
tggctcttcc agcccgagcc ccacctccgg ctcatcggcg acctcatgga gtactgcgcg 660
gccggcatcc ccgcctacaa gccgctctcc gtctccggct accacatccg cgaggcgggc 720
gcgacggccg cgcaggagct ggcgtacacg ctcgccgacg gcttcggata cgtggagctg 780
ggcctcagcc gcgggctcga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840
gcgcacctcg acttcttcga ggagatcgcc aagttccgcg cggcccgcag gatctgggcc 900
cgctggatgc gcgacgtgta cggcgcgcgg accgacaagg cccagtggct gcggttccac 960
acccagaccg ccggagtctc gctcaccgcg cagcagccgt acaacaacgt cgtacgcacc 1020
gcggtggagg cgctggcggc cgtgctcggc ggcaccaact ccctgcacac caacgcgctc 1080
gacgagaccc tcgccctgcc cagcgagcag gccgccgaga tcgccctgcg cacccagcag 1140
gtgctgatgg aggagaccgg cgtcgccaac gtcgccgacc cgctgggcgg ttcctggttc 1200
atcgagcagc tgaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcaag 1260
gagcgggggc tgcgcgccca ccccgacggg cagcaccccg tcggaccgat cacctccggc 1320
ctgctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccgc 1380
taccagcagt ccttggagaa ggacgacaag aaggtggtcg gcgtcaacgt ccacaccggc 1440
tccgtcaccg gcgacctgga gatcctgcgg gtcagccacg aggtcgagcg cgagcaggtg 1500
cgggtcctgg gcgagcgcaa ggacgcccgg gacgacgccg ccgtgcgcgg cgccctggac 1560
gccatgctgg ccgcggcccg ctccggcggc aacatgatcg ggccgatgct ggacgcggtg 1620
cgcgcggagg cgacgctggg cgagatctgc ggtgtgctgc gcgacgagtg gggggtgtac 1680
acggaaccgg cggggttctg a 1701
<210>260
<211>566
<212>PRT
<213>天蓝色链霉菌
<400>260
Met Asp Ala His Ala Ile Glu Glu Gly Arg Leu Arg Trp Gln Ala Arg
1 5 10 15
Tyr Asp Ala Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly
20 25 30
Asp Pro Val Glu Pro Val Tyr Gly Pro Arg Pro Gly Asp Glu Tyr Glu
35 40 45
Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly
50 55 60
Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe
65 70 75 80
Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile
85 90 95
Leu Arg Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr
100 105 110
Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly
115 120 125
His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe
130 135 140
Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly
145 150 155 160
Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln
165 170 175
Gly Val Asp Ala Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe
180 185 190
Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His
195 200 205
Leu Arg Leu Ile Gly Asp Leu Met Glu Tyr Cys Ala Ala Gly Ile Pro
210 215 220
Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly
225 230 235 240
Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly
245 250 255
Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala
260 265 270
Pro Gly Leu Ser Phe Phe Phe Asp Ala His Leu Asp Phe Phe Glu Glu
275 280 285
Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Met Arg
290 295 300
Asp Val Tyr Gly Ala Arg Thr Asp Lys Ala Gln Trp Leu Arg Phe His
305 310 315 320
Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn
325 330 335
Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr
340 345 350
Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser
355 360 365
Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu
370 375 380
Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Phe
385 390 395 400
Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe
405 410 415
Glu Gln Ile Lys Glu Arg Gly Leu Arg Ala His Pro Asp Gly Gln His
420 425 430
Pro Val Gly Pro Ile Thr Ser Gly Leu Leu Arg Gly Ile Glu Asp Gly
435 440 445
Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Arg Tyr Gln Gln Ser
450 455 460
Leu Glu Lys Asp Asp Lys Lys Val Val Gly Val Asn Val His Thr Gly
465 470 475 480
Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu
485 490 495
Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Asp Ala Arg Asp Asp
500 505 510
Ala Ala Val Arg Gly Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Ser
515 520 525
Gly Gly Asn Met Ile Gly Pro Met Leu Asp Ala Val Arg Ala Glu Ala
530 535 540
Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr
545 550 555 560
Thr Glu Pro Ala Gly Phe
565
<210>261
<211>417
<212>DNA
<213>天蓝色链霉菌
<400>261
atgggtgtgg cagccggtcc gatccgcgtg gtggtggcca agccggggct cgacggccac 60
gatcgcgggg ccaaggtgat cgcgagggcc ctgcgtgacg ccggtatgga ggtgatctac 120
accgggctcc accagacgcc cgagcagatc gtcgacaccg cgatccagga ggacgccgac 180
gcgatcgggc tgtccatcct ctccggtgcg cacaacacgc tcttcgccgc cgtgatcgag 240
ctgctccggg agcgggacgc cgcggacatc ctggtcttcg gcggcgggat catccccgag 300
gcggacatcg ccccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggcgccacc 360
acggcgtcca tcgtggactg ggtccgggcg aacgtgcggg agcccgcggg agcatag 417
<210>262
<211>138
<212>PRT
<213>天蓝色链霉菌
<400>262
Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly
1 5 10 15
Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg
20 25 30
Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu
35 40 45
Gln Ile Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu
50 55 60
Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Glu
65 70 75 80
Leu Leu Arg Glu Arg Asp Ala Ala Asp Ile Leu Val Phe Gly Gly Gly
85 90 95
Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala
100 105 110
Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Asp Trp Val
115 120 125
Arg Ala Asn Val Arg Glu Pro Ala Gly Ala
130 135
<210>263
<211>1701
<212>DNA
<213>除虫链霉菌
<400>263
tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60
gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120
gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180
cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240
ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300
cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360
gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420
gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480
ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540
gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600
cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660
cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720
cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780
gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840
ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900
gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960
cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020
cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080
gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140
gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200
gcagaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260
gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320
gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380
ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440
ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500
cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560
gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620
gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680
ctcgatggcg tcagcgtcca t 1701
<210>264
<211>566
<212>PRT
<213>除虫链霉菌
<400>264
Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg
1 5 10 15
Tyr Asp Ala Ser Arg Lys Arg Glu Ala Asp Phe Thr Thr Leu Ser Gly
20 25 30
Asp Pro Val Glu Pro Ala Tyr Gly Pro Arg Pro Gly Asp Ala Tyr Glu
35 40 45
Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly
50 55 60
Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe
65 70 75 80
Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Lys Ile
85 90 95
Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr
100 105 110
Leu Met Gly Arg Asp Ser Asp Asp Arg Arg Ala Leu Gly Glu Val Gly
115 120 125
His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe
130 135 140
Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly
145 150 155 160
Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln
165 170 175
Gly Val Asp Pro Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe
180 185 190
Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His
195 200 205
Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Ser Lys Ile Pro
210 215 220
Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly
225 230 235 240
Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly
245 250 255
Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp ValAsp Val Phe Ala
260 265 270
Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu
275 280 285
Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg
290 295 300
Asp Val Tyr Gly Ala Lys Ser Glu Lys Ala Gln Trp Leu Arg Phe His
305 310 315 320
Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn
325 330 335
Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr
340 345 350
Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser
355 360 365
Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu
370 375 380
Glu Thr Gly Val Ala Agn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr
385 390 395 400
Val Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe
405 410 415
Glu Gln Ile Arg Glu Arg Gly Leu Arg Ala His Pro Asp Gly Arg His
420 425 430
Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly
435 440 445
Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Gln Ala
450 455 460
Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Val His His Gly
465 470 475 480
Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu
485 490 495
Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Ser Gly Arg Asp Asp
500 505 510
Thr Ala Val Thr Ala Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Asp
515 520 525
Gly Ser Asn Met Ile Ala Pro Met Leu Asp Ala Val Arg Ala Glu Ala
530 535 540
Thr Leu Gly Glu Ile Cys Asp Val Leu Arg Glu Glu Trp Gly Val Tyr
545 550 555 560
Thr Glu Pro Ala Gly Phe
565
<210>265
<211>417
<212>DNA
<213>除虫链霉菌
<400>265
ctacgccccg gcaggctgcc gcacgttcgc ccgcacccac tccacgatcg acgccgtggt 60
cgcccccgga gtgaagatct ccgcgacacc cttctccttc agcggcgcga tgtccgcctc 120
ggggatgatg ccgccaccga acaccttgat gtcctcggca tcgcgctcct tgagcagatc 180
gatgaccgcc gcgaacaacg tgttgtgcgc cccggacagg atcgacagcc cgatcgcgtc 240
ggcgtcctcc tggatggccg tgcccacgat ctgctccggc gtctggtgca gccccgtgta 300
aatgacctcc ataccggcat cgcgcagcgc ccgcgcgatc accttggccc cgcgatcgtg 360
gccatcgagc cccggcttgg ccaccaccac gcggatcgga ccggctgcca cacccat 417
<210>266
<211>138
<212>PRT
<213>除虫链霉菌
<400>266
Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly
1 5 10 15
Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg
20 25 30
Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu
35 40 45
Gln Ile Val Gly Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu
50 55 60
Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Asp
65 70 75 80
Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly
85 90 95
Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala
100 105 110
Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Glu Trp Val
115 120 125
Arg Ala Asn Val Arg Gln Pro Ala Gly Ala
130 135
<210>267
<211>2910
<212>DNA
<213>金黄色葡萄球菌
<400>267
gatcaatttc ttttaagtaa tctaaatccc cattttttaa tttcttttta gcctctttaa 60
ataatcctga ataaactaat acctgtttac ctttaagtga tttataaaat gcatcaaaga 120
ctttttgatt tatttattaa ataatcacta tctttaccag aatacttagc catttcatat 180
aattctttat tattattttg tcttattttt tgaacttgaa cttgtgttat ttctgaaatg 240
cccgttacat cacgccataa atctaaccat tcttgttggc taatataata tcttttatct 300
gtgaaatacg atttatttac tgcaattaac acatgaaaat gaggattata atcatctctt 360
tttttattat atgtaatctc taacttacga acatatccct ttataacact acctactttt 420
tttctcttta taagttttct aaaagaatta ttataacgtt ttatttcatt ttctaattca 480
tcactcatta cattaggtgt agtcaaagtt aaaaagataa actccttttt ctcttgctgc 540
ttaatatatt gcatcatcaa agataaaccc aatgcatctt ttctagcttt tctccaagca 600
cagacaggac aaaatcgatt tttacaagaa ttagctttat ataatttctg tttttctaaa 660
gttttatcag ctacaaaaga cagaaatgta ttgcaatctt caactaaatc catttgattc 720
tctccaatat gacgtttaat aaatttctga aatacttgat ttctttgttt tttctcagta 780
tacttttcca tgttataaca cataaaaaca acttagtttt cacaaactat gacaataaaa 840
aaagttgctt tttccccttt ctatgtatgt tttttactag tcatttaaaa cgatacatta 900
ataggtacga aaaagcaact ttttttgcgc ttaaaaccag tcataccaat aacttaaggg 960
taactagcct cgccggcaat agttaccctt attatcaaga taagaaagaa aaggattttt 1020
cgctacgctc aaatccttta aaaaaacaca aaagaccaca ttttttaatg tggtctttat 1080
tcttcaacta aagcacccat tagttcaaca aacgaaaatt ggataaagtg ggatattttt 1140
aaaatatata tttatgttac agtaatattg acttttaaaa aaggattgat tctaatgaag 1200
aaagcagaca agtaagcctc ctaaattcac tttagataaa aatttaggag gcatatcaaa 1260
tgaactttaa taaaattgat ttagacaatt ggaagagaaa agagatattt aatcattatt 1320
tgaaccaaca aacgactttt agtataacca cagaaattga tattagtgtt ttataccgaa 1380
acataaaaca agaaggatat aaattttacc ctgcatttat tttcttagtg acaagggtga 1440
taaactcaaa tacagctttt agaactggtt acaatagcga cggagagtta ggttattggg 1500
ataagttaga gccactttat acaatttttg atggtgtatc taaaacattc tctggtattt 1560
ggactcctgt aaagaatgac ttcaaagagt tttatgattt atacctttct gatgtagaga 1620
aatataatgg ttcggggaaa ttgtttccca aaacacctat acctgaaaat gctttttctc 1680
tttctattat tccatggact tcatttactg ggtttaactt aaatatcaat aataatagta 1740
attaccttct acccattatt acagcaggaa aattcattaa taaaggtaat tcaatatatt 1800
taccgctatc tttacaggta catcattctg tttgtgatgg ttatcatgca ggattgttta 1860
tgaactctat tcaggaattg tcagataggc ctaatgactg gcttttataa tatgagataa 1920
tgccgactgt actttttaca gtcggttttc taatgtcact aacctgcccc gttagttgaa 1980
gaaggttttt atattacagc tccagatcca tatccttctt tttctgaacc gacttctcct 2040
ttttcgcttc tttattccaa ttgctttatt gacgttgagc ctcggaaccc ttaacaatcc 2100
caaaacttgt cgaatggtcg gcttaatagc tcacgctatg ccgacattcg tctgcaagtt 2160
tagttaaggg ttcttctcaa cgcacaataa attttctcgg cataaatgcg tggtctaatt 2220
tttattttta ataaccttga tagcaaaaaa tgccattcca atacaaaacc acatacctat 2280
aatcgataac cacataacag tcataaaacc actccttttt aacaaacttt atcacaagaa 2340
atatttaaat tttaaatgcc tttattttga attttaaggg gcattttaaa gatttagggg 2400
taaatcatat agttttatgc ctaaaaacct acagaagctt ttaaaaagca aatatgagcc 2460
aaataaatat attctaattc tacaaacaaa aatttgagca aattcagtgt cgatttttta 2520
agacactgcc cagttacatg caaattaaaa ttttcatgat tttttatagt tcctaacagg 2580
gttaaaattt gtataacgaa agtataatgt ttatataacg ttagtataat aaagcatttt 2640
aacattatac ttttgataat cgtttatcgt cgtcatcaca ataactttta aaatactcgt 2700
gcataattca cgctgacctc ccaataacta catggtgtta tcgggaggtc agctgttagc 2760
acttatattt tgttattgtt cttcctcgat ttcgtctatc attttgtgat taatttctct 2820
tttttcttgt tctgttaagt cataaagttc actagctaaa tactcttttt gtttccaaat 2880
ataaaaaatt tgatagatat attacggttg 2910
Claims (82)
1.重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至异丁醛(途径步骤d),和
v)异丁醛至异丁醇(途径步骤e);
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
2.重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f)
v)异丁酰-CoA至异丁醛(途径步骤g),和
vi)异丁醛至异丁醇(途径步骤e);
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
3.重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至缬氨酸(途径步骤h),
v)缬氨酸至异丁胺(途径步骤i),
vi)异丁胺至异丁醛(途径步骤j),和
vii)异丁醛至异丁醇(途径步骤e);
其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且其中所述微生物宿主细胞产生异丁醇。
4.根据权利要求1,2或3任一项的宿主细胞,其中催化丙酮酸至乙酰乳酸的底物至产物转化的多肽是乙酰乳酸合酶。
5.根据权利要求1,2或3任一项的宿主细胞,其中催化乙酰乳酸至2,3-二羟基异戊酸的底物至产物转化的多肽是乙酰醇酸异构还原酶。
6.根据权利要求1,2或3任一项的宿主细胞,其中催化2,3-二羟基异戊酸至α-酮基异戊酸的底物至产物转化的多肽是乙酰醇酸脱水酶。
7.根据权利要求1,2或3任一项的宿主细胞,其中催化异丁醛至异丁醇的底物至产物转化的多肽是支链醇脱氢酶。
8.根据权利要求1的宿主细胞,其中催化α-酮基异戊酸至异丁醛的底物至产物转化的多肽是支链α-酮酸脱羧酶。
9.根据权利要求2的宿主细胞,其中催化α-酮基异戊酸至异丁酰-CoA的底物至产物转化的多肽是支链酮酸脱氢酶。
10.根据权利要求2的宿主细胞,其中催化异丁酰-CoA至异丁醛的底物至产物转化的多肽是酰化醛脱氢酶。
11.根据权利要求3的宿主细胞,其中催化α-酮基异戊酸至缬氨酸的底物至产物转化的多肽是转氨酶。
12.根据权利要求3的宿主细胞,其中催化α-酮基异戊酸至缬氨酸的底物至产物转化的多肽是缬氨酸脱氢酶。
13.根据权利要求3的宿主细胞,其中催化缬氨酸至异丁胺的底物至产物转化的多肽是缬氨酸脱羧酶。
14.根据权利要求3的宿主细胞,其中催化异丁胺至异丁醛的底物至产物转化的多肽是ω转氨酶。
15.根据权利要求1,2或3任一项的宿主细胞,其中细胞选自:细菌、蓝细菌、丝状真菌和酵母。
16.根据权利要求15的宿主细胞,其中细胞是选自梭菌属(Clostridium)、发酵单胞菌属(Zymomonas)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)、红球菌属(Rhodococcus)、假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、产碱杆菌属(Alcaligenes)、克雷伯氏菌属(Klebsiella)、类芽孢杆菌属(Paenibacillus)、节杆菌属(Arthrobacter)、棒状杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)、毕赤氏酵母属(Pichia)、假丝酵母属(Candida)、汉森酵母属(Hansenula)和酵母属(Saccharomyces)的成员。
17.根据权利要求16的宿主细胞,其中细胞是大肠杆菌(Escherichia coli)。
18.根据权利要求16的宿主细胞,其中细胞是真养产碱杆菌(Alcaligenes eutrophus)。
19.根据权利要求16的宿主细胞,其中细胞是地衣芽孢杆菌(Bacillus.licheniformis)。
20.根据权利要求16的宿主细胞,其中细胞是浸麻类芽孢杆菌(Paenibacillus macerans)。
21.根据权利要求16的宿主细胞,其中细胞是红串红球菌(Rhodococcus erythropolis)
22.根据权利要求16的宿主细胞,其中细胞是恶臭假单胞菌(Pseudomonas.putida)。
23.根据权利要求16的宿主细胞,其中细胞是枯草芽孢杆菌(Bacillus subtillis)。
24.根据权利要求16的宿主细胞,其中细胞是植物乳杆菌(Lactobacillus plantarum)。
25.根据权利要求16的宿主细胞,其中细胞选自屎肠球菌(Enterococcus faecium),鹑鸡肠球菌(Enterococcus gallinarium)和粪肠球菌(Enterococcus faecalis)。
26.根据权利要求16的宿主细胞,其中细胞是酿酒酵母(Saccharomyces cerevisiae)。
27.根据权利要求4的宿主细胞,其中乙酰乳酸合酶具有选自SEQID NO:2,SEQ ID NO:178和SEQ ID NO:180的氨基酸序列。
28.根据权利要求5的宿主细胞,其中乙酰醇酸异构还原酶具有选自SEQ ID NO:43,SEQ ID NO:181,SEQ ID NO:183和SEQ ID NO:185的氨基酸序列。
29.根据权利要求6的宿主细胞,其中乙酰醇酸脱水酶具有选自SEQ ID NO:6,SEQ ID NO:186,SEQ ID NO:188和SEQ ID NO:190的氨基酸序列。
30.根据权利要求7的宿主细胞,其中支链醇脱氢酶具有选自SEQID NO:10,SEQ ID NO:199,SEQ ID NO:201,SEQ ID NO:203和SEQ ID NO:204的氨基酸序列。
31.根据权利要求8的宿主细胞,其中支链α-酮酸脱羧酶具有选自SEQ ID NO:8,SEQ ID NO:193,SEQ ID NO:195和SEQ ID NO:197的氨基酸序列。
32.根据权利要求9的宿主细胞,其中支链酮酸脱氢酶包括四个亚基,并且所述亚基的氨基酸序列选自SEQ ID NO:214,SEQ ID NO:216,SEQ ID NO:218,SEQ ID NO:220,SEQ ID NO:210,SEQ IDNO:208,SEQ ID NO:206和SEQ ID NO:212。
33.根据权利要求10的宿主细胞,其中酰化醛脱氢酶具有选自SEQ ID NO:222,SEQ ID NO:224,SEQ ID NO:226,SEQ ID NO:228和SEQ ID NO:230的氨基酸序列。
34.根据权利要求11的宿主细胞,其中转氨酶具有选自SEQ IDNO:232,SEQ ID NO:234,SEQ ID NO:236,SEQ ID NO:238和SEQ ID NO:240的氨基酸序列。
35.根据权利要求12的宿主细胞,其中缬氨酸脱氢酶具有选自SEQ ID NO:242和SEQ ID NO:244的氨基酸序列。
36.根据权利要求13的宿主细胞,其中缬氨酸脱羧酶具有SEQ IDNO:246所示的氨基酸序列。
37.根据权利要求14的宿主细胞,其中ω转氨酶具有选自SEQ IDNO:248,SEQ ID NO:250,SEQ ID NO:252和SEQ ID NO:254的氨基酸序列。
38.根据权利要求1,2或3任一项的宿主细胞,其中宿主细胞是兼性厌氧菌。
39.生产异丁醇的方法,所述方法包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至异丁醛(途径步骤d),和
v)异丁醛至异丁醇(途径步骤e),
其中至少一个DNA分子对所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
40.生产异丁醇的方法,所述方法包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f),
v)异丁酰-CoA至异丁醛(途径步骤g),和
vi)异丁醛至异丁醇(途径步骤e),
其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
41.生产异丁醇的方法,所述方法包括:
1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的转化:
i)丙酮酸至乙酰乳酸(途径步骤a),
ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b),
iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c),
iv)α-酮基异戊酸至缬氨酸(途径步骤h),
v)缬氨酸至异丁胺(途径步骤i),
vi)异丁胺至异丁醛(途径步骤j),和
vii)异丁醛至异丁醇(途径步骤e)
其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和
2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养基中的可发酵碳底物。
42.根据权利要求39,40或41任一项的方法,其中碳底物选自单糖,寡糖和多糖。
43.根据权利要求39,40或41任一项的方法,其中碳底物选自葡萄糖,蔗糖和果糖。
44.根据权利要求39,40或41任一项的方法,其中由此产生异丁醇的条件是厌氧的。
45.根据权利要求39,40或41任一项的方法,其中由此产生异丁醇的条件是微好氧的。
46.根据权利要求39,40或41任一项的方法,其中将宿主细胞接触最小培养基中的碳底物。
47.根据权利要求39,40或41任一项的方法,其中催化丙酮酸至乙酰乳酸的底物至产物转化的多肽是乙酰乳酸合酶。
48.根据权利要求39,40或41任一项的方法,其中催化乙酰乳酸至2,3-二羟基异戊酸的底物至产物转化的多肽是乙酰醇酸异构还原酶。
49.根据权利要求39,40或41任一项的方法,其中催化2,3-二羟基异戊酸至α-酮基异戊酸的底物至产物转化的多肽是乙酰醇酸脱水酶。
50.根据权利要求39,40或41任一项的方法,其中催化异丁醛至异丁醇的底物至产物转化的多肽是支链醇脱氢酶。
51.根据权利要求39的方法,其中催化α-酮基异戊酸至异丁醛的底物至产物转化的多肽是支链α-酮酸脱羧酶。
52.根据权利要求40的方法,其中催化α-酮基异戊酸至异丁酰-CoA的底物至产物转化的多肽是支链酮酸脱氢酶。
53.根据权利要求40的方法,其中催化异丁酰-CoA至异丁醛的底物至产物转化的多肽是酰化醛脱氢酶。
54.根据权利要求41的方法,其中催化α-酮基异戊酸至缬氨酸的底物至产物转化的多肽是转氨酶。
55.根据权利要求41的方法,其中催化α-酮基异戊酸至缬氨酸的底物至产物转化的多肽是缬氨酸脱氢酶。
56.根据权利要求41的方法,其中催化缬氨酸至异丁胺的底物至产物转化的多肽是缬氨酸脱羧酶。
57.根据权利要求41的方法,其中催化异丁胺至异丁醛的底物至产物转化的多肽是ω转氨酶。
58.根据权利要求39,40或41任一项的方法,其中宿主细胞选自:细菌、蓝细菌、丝状真菌和酵母。
59.根据权利要求55的方法,其中宿主细胞是选自梭菌属(Clostridium)、发酵单胞菌属(Zymomonas)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)、红球菌属(Rhodococcus)、假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)、乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、产碱杆菌属(Alcaligenes)、克雷伯氏菌属(Klebsiella)、类芽孢杆菌属(Paenibacillus)、节杆菌属(Arthrobacter)、棒状杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)、毕赤氏酵母属(Pichia)、假丝酵母属(Candida)、汉森酵母属(Hansenula)和酵母属(Saccharomyces)的成员。
60.根据权利要求59的方法,其中宿主细胞是大肠杆菌(Escherichia coli)。
61.根据权利要求59的方法,其中宿主细胞是真养产碱杆菌(Alcaligenes eutrophus)。
62.根据权利要求59的方法,其中宿主细胞是地衣芽孢杆菌(Bacillus.licheniformis)。
63.根据权利要求59的方法,其中宿主细胞是浸麻类芽孢杆菌(Paenibacillus macerans)。
64.根据权利要求59的方法,其中宿主细胞是红串红球菌(Rhodococcus erythropolis)。
65.根据权利要求59的方法,其中宿主细胞是恶臭假单胞菌(Pseudomonas.putida)。
66.根据权利要求59的方法,其中宿主细胞是枯草芽孢杆菌(Bacillus subtillis)。
67.根据权利要求59的方法,其中宿主细胞是植物乳杆菌(Lactobacillus plantarum)。
68.根据权利要求59的方法,其中宿主细胞选自屎肠球菌(Enterococcus faecium)、鹑鸡肠球菌(Enterococcus gallinarium)和粪肠球菌(Enterococcus faecalis)。
69.根据权利要求59的方法,其中宿主细胞是酿酒酵母(Saccharomyces cerevisiae)。
70.根据权利要求47的方法,其中乙酰乳酸合酶具有选自SEQ IDNO:2,SEQ ID NO:178和SEQ ID NO:180的氨基酸序列。
71.根据权利要求48的方法,其中乙酰醇酸异构还原酶具有选自SEQ ID NO:43,SEQ ID NO:181,SEQ ID NO:183和SEQ ID NO:185的氨基酸序列。
72.根据权利要求49的方法,其中乙酰醇酸脱水酶具有选自SEQID NO:6,SEQ ID NO:186,SEQ ID NO:188和SEQ ID NO:190的氨基酸序列。
73.根据权利要求50的方法,其中支链醇脱氢酶具有选自SEQ IDNO:10,SEQ ID NO:199,SEQ ID NO:201,SEQ ID NO:203和SEQ ID NO:204的氨基酸序列。
74.根据权利要求51的方法,其中支链α-酮酸脱羧酶具有选自SEQ ID NO:8,SEQ ID NO:193,SEQ ID NO:195和SEQ ID NO:197的氨基酸序列。
75.根据权利要求52的方法,其中支链酮酸脱氢酶包括四个亚基,并且所述亚基的氨基酸序列选自SEQ ID NO:214,SEQ ID NO:216,SEQ ID NO:218,SEQ ID NO:220,SEQ ID NO:210,SEQ ID NO:208,SEQ ID NO:206和SEQ ID NO:212。
76.根据权利要求53的方法,其中酰化醛脱氢酶具有选自SEQ IDNO:222,SEQ ID NO:224,SEQ ID NO:226,SEQ ID NO:228和SEQ ID NO:230的氨基酸序列。
77.根据权利要求54的方法,其中转氨酶具有选自SEQ ID NO:232,SEQ ID NO:234,SEQ ID NO:236,SEQ ID NO:238和SEQ IDNO:240的氨基酸序列。
78.根据权利要求55的方法,其中缬氨酸脱氢酶具有选自SEQ IDNO:242和SEQ ID NO:244的氨基酸序列。
79.根据权利要求56的方法,其中缬氨酸脱羧酶具有SEQ ID NO:246所示的氨基酸序列。
80.根据权利要求57的方法,其中ω转氨酶具有选自SEQ ID NO:248,SEQ ID NO:250,SEQ ID NO:252和SEQ ID NO:254的氨基酸序列。
81.根据权利要求39,40或41任一项的方法,其中宿主细胞是兼性厌氧菌。
82.通过权利要求39,40或41任一项的方法产生的含有异丁醇的发酵培养基。
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CN103080298A (zh) * | 2010-09-07 | 2013-05-01 | 布特马斯先进生物燃料有限责任公司 | 整合编码催化丙酮酸至乙酰乳酸的转化的多肽的多核苷酸 |
CN105950525A (zh) * | 2010-09-07 | 2016-09-21 | 布特马斯先进生物燃料有限责任公司 | 整合编码催化丙酮酸至乙酰乳酸的转化的多肽的多核苷酸 |
CN104284980A (zh) * | 2011-12-30 | 2015-01-14 | 布特马斯先进生物燃料有限责任公司 | 用于丁醇生产的基因开关 |
CN104321436A (zh) * | 2011-12-30 | 2015-01-28 | 布特马斯先进生物燃料有限责任公司 | 醇类的发酵生产 |
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CN103409332A (zh) * | 2012-12-14 | 2013-11-27 | 北京工商大学 | 产β-苯乙醇的酿酒酵母工程菌构建、菌株及其应用 |
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CN104195125B (zh) * | 2014-07-01 | 2017-01-11 | 中国科学院广州能源研究所 | 一种α-酮酸脱羧酶KIVD-LL及其编码基因和应用 |
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CN109722405B (zh) * | 2017-10-31 | 2023-06-23 | 创享(天津)生物科技发展有限公司 | 利用葡萄糖生产异丙基吡喃酮的重组大肠杆菌及用途 |
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