CA2466340C - Farnesyl dibenzodiazepinone, processes for its production and its use as a pharmaceutical - Google Patents

Abstract

This invention relates to a novel farnesylated dibenzodiazepinone, named ECO-04601, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining such compounds. One method of obtaining the ECO-04601 compound is by cultivation of a novel strain of Micromonospora sp., 046-ECO11; another method involves expression of biosynthetic pathway genes in transformed host cells.
The present invention further relates to Micromonospora sp. strain 046-ECO11, to the use of ECO-04601 and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, bacterial cell growth, mammalian lipoxygenase, and to pharmaceutical compositions comprising ECO-04601 or a pharmaceutically acceptable salt or derivative thereof.
Finally, the invention relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

Description

TITLE OF INVENTION: FARNESYL DIBENZODIAZEPINONE, PROCESSES FOR
ITS PRODUCTION AND ITS USE AS A PHARMACEUTICAL

FIELD OF THE INVENTION
This invention relates to a novel farnesylated dibenzodiazepinone, named ECO-04601, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining the compound. One method of obtaining the compound is by cultivation of a novel strain of Micromonospora sp., i.e., 046-ECO11 or [SO1]046;
another method involves expression of biosynthetic pathway genes in transformed host cells. The present invention further relates to Micromonospora sp.
strains 046-ECO11 and [S01]046, to the use of ECO-04601 and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, bacterial cell growth, mammalian Iipoxygenase, and for treating acute and chronic inflammation, and to pharmaceutical compositions comprising ECO-04601 or a pharmaceutically acceptable salt or derivative thereof.
Finally, the invention relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

BACKGROUND OF THE INVENTION
The euactinomycetes are a subset of a large and complex group of Gram-positive bacteria known as actinomycetes. Over the past few decades these organisms, which are abundant in soil, have generated significant commercial and scientific interest as a result of the large number of therapeutically useful compounds, particularfy antibiotics, produced as secondary metabolites. The intensive search for'strains able to produce new antibiotics has led to the identification of hundreds of new species.
Many of the euactinomycetes, particularly Streptomyces and the closely related Saccharopolyspora genera, have been extensively studied. Both of these genera produce a notable diversity of biologically active metabolites. Because of the commercial significance of these compounds, much is known about the genetics and physiology of these organisms.
Another representative genus of euactinomycetes, Micromonospora, has also generated commercial interest. For example, U.S. Patent No. 5,541,181 (Ohkuma et al.) discloses a dibenzodiazepinone compound, specifically 5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-l1-one (named "BU-4664L"), produced by a known euactinomycetes strain, Micromonospora sp. M990-6 (ATCC 55378). The Ohkuma et al. patent reports that BU-4664L and its chemically synthesized di- and tri-alkoxy and acyloxy derivatives possess anti-inflammatory and anti-tumor cell activities.
Although many biologically active compounds have been identified from bacteria, there remains the need to obtain novel naturally occurring compounds with enhanced properties. Current methods of obtaining such compounds include screening of natural isolates and chemical modification of existing compounds, both of which are costly and time consuming. Current screening methods are based on general biological properties of the compound, which require prior knowledge of the structure of the molecules. Methods for chemically modifying known active compounds exist, but still suffer from practical limitations as to the type of compounds obtainable.
Thus, there exists a considerable need to obtain pharmaceutically active compounds in a cost-effective manner and with high yield. The present invention solves these problems by providing a novel strain of Micromonospora capable of producing a potent new therapeutic compound, as well as reagents (e.g., polynucleotides, vectors comprising the polynucleotides and host cells comprising the vectors) and methods to generate novel compounds by de novo biosynthesis rather than by chemical synthesis.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a compound of the formula N~~~

OH
OH i HO

(Formula !I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the formula o N~/
( / / \
OH
N

oH I
HO
or a pharmaceutically acceptable salt thereof, together with a pharmaceuticaily acceptable carrier.
In a further aspect, the invention relates to a class of compounds represented by Formula I:

1 ~/ w V ~ 3- CH3 Nw w R2o b--Formula I
wherein, W 1, W2 and W3 is each independently selected from H2 I__ _Ij H_I 1. ;or -~-~-H ~ ' -~'HC ' c\

the chain from the tricycle may terminate at W3, W2 or W1 with W3, W2 or W 1 respectively being either -CH=O or -CH2OH;

A is selected from -NH--, -NCH2R1, -NC(O)R1;
Ri is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;
R2, R3, and R4 is each independently selected from H, R5, -C(O)R6 R5 is each independently selected from Cl-6 alkyl, C2-7 alkalene, aryl or heteroaryl;
R6 is each independently selected from H, Cl-6 alkyl, C2-7 alkalene, aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.
In one embodiment, A is NH.
In another embodiment, A is -NCH2R'.
In another embodiment, A is -NC(O)R1.
In another embodiment, R2 is H.
In another embodiment, R3 is H.
In another embodiment, R4 is H.
In another embodiment, R2, R3 and R4 are each H.
In another embodiment, R2, R3 and R4 are each H, and W' is -CH =CH-.
In another embodiment, R2, R3 and R4 are each H, and W2 is -CH =CH-.
In another embodiment, R2, R3 and R4 are each H, and W3 is -CH =CH-.
In another embodiment, A is NH and R2, R3 and R4 are each H.
In another embodiment, A is NH, each of W', W2, and W3 is -CH =CH-.
The invention further encompasses a compound selected from the group consisting of:
0 CH, CHs H, O CH, CH, CHs N" / CH
/ N / / , HO CHz ~..
0=C OH HO H,C HO ; I /

o H CH CH0 PNt O CHa CH~ CH, N N CH3 O / --'l ~
/ 0 O , CH

N---" 0~

H'C HO O

O CH, CH, Cti, 0 CH, CH3 CH, /
\
/ N CH3 N / 'Y~~~CH3 0 N ~ N , ~ P
HO
H ~. OH HO H OH
HO ~ HO

O CH3 CH3 CFI3 0 CH3 CH, CHa N CH3 PNt' N~ CH3 ~ I 0 0 N / ' 1 HO
HO
H ~ OH H ~.~ OH
HO HO
O CH, CH, CH3 0 CH,3 CH3 CH, O
PNt' N CH' CH' \ N ~ \
HO
HO
H -~ OH H ' OH HO y HO

0 CH, CH, CH, 0 CH3 CH3 CH, o p p 3 PNt' N CH, CH
N
HO
H OH HO H _ OH
HO HO

O CH3 CH, CH0 0 CH, CH3 CHa N / CH, o PN-HO HO
H OH H OH HO ~ HO

CH3 CH, CH.0 0 CH;, CH, CH3 PNt' PNt \ h HO H _ OH HO H ~ OH
HO y HO

O CH3 CH, CH;0 0 H;, CH~, CH~
PN I N CH, N CH, O O \ I 0 HO H / \ OH HO H ~\ OH HO 9 HO

O CH, CH3 CH0 0 CH;, CHa CH3 P
i ~ \
HO HO
H OH H _ OH
HO HO
O CH, CH, CH3 0 CH;, i H,a CH3 PN-- N CH, N CH, HO H / OH HO H OH
HO ~ HO ~

O CH, CH3 CH3 0 CH3 CH3 CH3 q / CH, p\'~ ~ \
HO H OH Y" O H _ OH
HO H,C/ Hp O CHa CH, CH3 N CH, 0 CH, CH, CH, I i / CH, N \ \
HO
0 H -~. pH N ~ \ 0 p HO /
O-H3C H ~ HO CH3 0 CH3 CH3 CH, 0 CH, CH, CH3 \ / N CHa PNt/ CH3 O O p CHJ
0 H / 1 O,~CH~ HO H 1 -k H''C / O
O~,\CH, --,\CH3 qNt, CH, CH, CH, NCHa PNt, N CH3 OO 1 O HsC H O_ OH
p \
O /
H ~ O CH, 0 H2C HO CHa CH3 CH, CH3 O CH3 ; H3 CH, PNt' N N'pH
N / \

H'C~-O H ~ OH H ~ OH
HO e H,C-O ~ .

0 CHa CHa CHa 0 CH3 CHa CHa N CHa / H3C'0 H ~ \ 0-CHa N \
HO O-CHa H
HO ~ CHa f 0 CHa CHa CH, ' N CHa 0 CHa CHa CHaCH

~ PNt' a N / HO J O-CHa ' H i HaC,~O H _ -O.--CHa .
CHa ~ HO y O CHa CHa CHa PNt/ 0 CH3 CH3 H3 CHa N CH PNt H'C-O H OH HO / ~ OH

CHa HO ~

~&N)CHg PNt' CH3 % /
HO H a OH HO H OH
HO ~ HO

0 CHs CH3 CH3 0 CH3 CH3 CH3 \ / .
J CH3 'I- CH3 HO H ~' OH HO H ~' OH
HO HO

N

PNt' i ~ ~
HO H _ OH HO H ~ OH
HO HO
O CH3 CH3 0 CH3 CH, N //0 \ I N i OH

N b HO HO
H OH H OH
HO HO ~
0 CHs 0 Chh N
OH
P N ~ /O PNt, HO H / \ OH HO H \ OH
HO

HO H OH HO H ~\ OH
HO ~ HO

0 CH, CH3 CH, 0 CH3 CH3 CHa ~CH3 CH3 OH OH
)OH OH
H H OH
HO OH HO

HO HO

O CH3 CH, CH3 O Chi, CH, CH3 ~ CH, N CH, \ / OH \ I OH OH
OH OH OH
HO H OH HO H ~' OH
HO ; HO

0 CH, CH3 CH3 0 CH3 CH3 CH3 OH OH OH OH

HO HO ; and 0 CH3 CH, CH3 qN N
OH OH OH HO OH
H
HO

In one embodiment, the invention relates to compositions of the compounds of Formula I together with a pharmaceutically acceptable carrier.
The invention further encompasses a farnesyl dibenzodiazepinone obtained by a method comprising: a) cultivating Micromonospora sp. strain [S01 ]046, wherein the cultivation is performed under aerobic conditions in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms; and b) isolating a farnesyl dibenzodiazepinone from the bacteria cultivated in step (a). In one embodiment the farnesyl dibenzodiazapinone is the compound of Formula II.
In one embodiment, the farnesyl dibenzodiazepinone generates NMR spectra essentially as shown in Figure 3, 4, 5, 6 and 7. In another embodiment, the farnesyl dibenzodiazepinone generates an'H NMR spectrum of Figure 3.
The invention further encompasses a process for making a farnesyl dibenzodiazapinone compound, comprising cultivation of Micromonospora sp.
strain 046-ECO11, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of the compound.
The invention further encompasses a process for making a farnesyl dibenzodiazepinone compound comprising cultivation of Micromonospora sp.
strain [S01 ]046 in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of the compound.
In one embodiment, the cultivation occurs under aerobic conditions.
In another embodiment, the carbon atom and nitrogen atom sources are chosen from the components shown in Table 16.
In another embodiment, the cultivation is carried out at a temperature ranging from 18 C to 40 C. In a further embodiment, the temperature range is 18 C to 29 C.
In another embodiment, the cultivation is carried out at a pH ranging from 6 to 9.
The invention further encompasses the Micromonospora sp. having IDAC
Accession No. 231203-01.
The invention further encompasses a method of inhibiting the growth of a cancer cell, the method comprising contacting the cancer cell with a compound of Formula I, such that growth of the cancer cell is inhibited.
In one embodiment, the compound is ECO-04601.
The invention further encompasses a method of inhibiting the growth of a cancer cell in a mammal, the method comprising administering a compound of Formula I to a mammal comprising a cancer cell, such that growth of the cancer cell is inhibited in the mammal.
In one embodiment, the compound is ECO-04601.
The invention further encompasses a method of treating a pre-cancerous or cancerous condition in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a compound of Formula I, such that a pre-cancerous or cancerous condition is treated.
In one embodiment, the compound is ECO-04601.
The invention further encompasses a method of treating a bacterial infection in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I to a mammal having a bacterial infection, such that the bacterial infection is treated.

In one embodiment, the compound is ECO-04601.
The invention further encompasses a method of reducing inflammation in a mammal, comprising administering to a mammal having inflammation a therapeutically effective amount of a compound of Formula I, such that the inflammation is reduced.
In one embodiment, the compound is ECO-04601.
The invention further encompasses an isolated polynucleotide comprising one or more of SEQ ID NOs. 1, 64 and 73, wherein the polynucleotide encodes a polypeptide that participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.
The invention further encompasses an isolated polynucleotide comprising SEQ ID NOs. 1, 64 and 73, wherein the polynucleotide encodes a polypeptide that participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.
The invention further encompasses an isolated polynucleotide that encodes a polypeptide selected from the group consisting of SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89.
In one embodiment, the isolated polynucleotide comprising SEQ ID No. 1 encodes a polypeptide selected from the group consisting of SEQ ID Nos. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63.
In another embodiment, the isolated polynucleotide comprising SEQ ID No.
64 encodes a polypeptide selected from the group consisting of SEQ ID NOS: 66, 68, 70 and 72.
In another embodiment, the isolated polynucleotide comprising SEQ ID No.
73, encodes a polypeptide selected from the group consisting of SEQ ID NOS:
75, 77, 79, 81, 83, 85, 87 and 89.
The invention further encompasses an isolated polypeptide of SEQ ID NO. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 or 89.
In one embodiment, the polypeptide participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.
The invention further encompasses an expression vector comprising one or more of the polynucleotides described herein.
The invention further encompasses a recombinant prokaryotic organism comprising one or more such expression vectors.
In one embodiment, the organism is an actinomycete.
In another embodiment, the organism requires the expression vector to synthesize a farnesyl dibenzodiazepinone. That is, the organism is deficient in the ability to synthesize a farnesyl dibenzodiazepinone before transformation with a polynucleotide as described herein.
The invention further encompasses a method of rnaking a farnesyl dibenzodiazepinone de novo in a prokaryote, comprising the steps of: (a) providing a prokaryote that is incapable of synthesizing a farnesyl dibenzodiazepinone;
(b) transforming the prokaryote with an expression vector as described herein; and (c) culturing the prokaryote; wherein the culturing results in the synthesis of a farnesyl dibenzodiazepinone in the prokaryote.
In one embodiment, the prokaryote is an actinomycete.
In another embodiment, the vector expresses a polypeptide of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 or 89.

Brief Description of the Figures FIGURE 1 shows the mass of ECO-04601 determined by electrospray mass spectrometry to be 462.6.
FIGURE 2 shows the absorption spectrum of purified ECO-04601 with a UVmax at 230 nm and a shoulder at 290nm.
FIGURE 3 shows proton NMR data for the compound dissolved in MeOH-d4.
FIGURE 4 shows multidimensional pulse sequences gDQCOSY.
FIGURE 5 shows multidimensional pulse sequences gHSQC.
FIGURE 6 shows multidimensional pulse sequences gHMBC.
FIGURE 7 shows multidimensional pulse sequences NOESY.
FIGURE 8 shows the in vitro anti-inflammatory activity of ECO-04601. Graph shows percent inhibition of 5-lipoxygenase activity plotted against the Log pM concentration of ECO-04601 and NDGA. Graph shows the EC50 of ECO-04601 to be 0.93pM.

FIGURE 9 shows inhibition of tumor growth resulting from administration of 10 to 30 mg/kg of ECO-04601 to glioblastoma-bearing mice beginning one day after tumor cell inoculation.
FIGURE 10 shows inhibition of tumor growth resulting from administration of 20-mg/kg of ECO-04601 to glioblastoma-bearing mice beginning ten days after tumor cell inoculation.
FIGURE 11 shows micrographs of tumor sections from mice bearing glioblastoma tumors and treated with saline or ECO-04601. The cell density of tumor treated with ECO-04601 appears decreased and nuclei from ECO-04601-treated tumor cells are larger and pynotic suggesting a cytotoxic effect.
FIGURE 12 shows the biosynthetic locus of ECO-04601, isolated from Micromonospora sp. strain 046-ECO11, including the positions of cosmids 046KM and 046KQ.
FIGURE 13 shows a schematic diagram of the biosynthetic pathway for the production of the farnesyl-diphosphate group of ECO-04601 with biosynthetic enzymes indicated by their ORF number and family designation.
FIGURE 14 shows a schematic diagram of the biosynthetic pathway for the production of (a) 3-hyd roxy-anth ran ilate-adenylate, and (b) 2-amino-6-hydroxy-[1,4]benzoquinone components as specified by ORFs present in the locus encoding ECO-04601. Biosynthetic enzymes are indicated by their ORF number and family designation.
FIGURE 15 shows a schematic diagram of the biosynthetic pathway for the assembly of the ECO-04601 precursors, farnesyl-diphosphate, 3-hydroxy-anthranilate-adenylate and 2-amino-6-hydroxy-[1,4]benzoquinone. Biosynthetic enzymes are indicated by their ORF
number and family designation.
FIGURE 16 shows a sequence listing table indicating the SEQ ID NO. and function for each of the open reading frames (ORFs) of the 046D biosynthetic locus and the corresponding gene product.
FIGURE 17 shows results of the fatty acid analysis of Micromonospora sp.
strain 046ECO11 (Accession No. IDAC 070303-01). Analysis was conducted using gas chromatography on fatty acid methyl esters (FAME).
FIGURE 18 illustrates the 16S ribosomal RNA analysis of Micromonospora sp.
strain 046EC011 (Accession No. IDAC 070303-01). Alignment of 16S
ribosomal RNA sequences demonstrates the phylogenetic relatedness of Micromonospora sp. strain 046EC011 (indicated as MID352 ECOPIA#1 con) to Micromonospora chalcea.
FIGURE 19 shows the complete'H and 13C NMR assignments for ECO-04601 when measured in MeOH-d4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel farnesyl dibenzodiazepinone, referred to herein as "ECO-04601," which was isolated from novel strains of actinomycetes, Micromonospora sp. strain 046-ECO11 and strain [S01]046. These microorganisms were analysed using gas chromatography as Fatty acid methyl esters (FAME) (Figure 17) 6S ribosomal RNA determination (Figure 18) and were found to belong to the genus of Micromonospora. These organisms were deposited on March 7, 2003, and December 23, 2003, respectively, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession Nos. IDAC 070303-01 and IDAC 231203-01, respectively.
The invention further relates to pharmaceutically acceptable salts and derivatives of ECO-04601, and to methods for obtaining such compounds. One method of obtaining the compound is by cultivating Micromonospora sp. strain ECO11, or a mutant or a variant thereof, under suitable Micromonospora culture conditions, preferably using the fermentation protocol described hereinbelow.
The invention also relates to a method for producing novel farnesyl dibenzodiazepinones by selectively altering the genetic information of an organism.
The present invention further provides isolated and purified polynucleotides that encode farnesyl dibenzodiazepinone domains, i.e., polypeptides from farnesyl dibenzodiazepinone-producing microorganisms, fragments thereof, vectors containing those polynucleotides, and host cells transformed with those vectors.
These polynucleotides, fragments thereof, and vectors comprising the polynucleotides can be used as reagents in the above described method. Portions of the polynucleotide sequences disclosed herein are also useful as primers for the amplification of DNA or as probes to identify related domains from other farnesyl dibenzodiazepinone producing microorganisms.
The present invention also relates to pharmaceutical compositions comprising ECO-04601 and its pharmaceutically acceptable salts and derivatives. ECO-04601 is useful as a pharmaceutical, in particular for. use as an inhibitor of cancer cell growth, bacterial cell growth, and mammalian lipoxygenase. The invention also relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.
The following detailed description discloses how to make and use ECO-04601 and compositions containing this compound to inhibit microbial growth and/or specific disease pathways.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the farnesylated dibenzodiazepinone compounds of the present invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit bacterial growth, and methods of using the pharmaceutical compositions to treat diseases, including cancer, and chronic and acute inflammation.

l. Definitions For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.
As used herein, the term "farnesyl dibenzodiazepinone" refers to a class of dibenzodiazepinone compounds containing a farnesyl moiety. The term includes, but is not limited to, the exemplified compound of the present invention, 1 0-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-1 1 -one, which is referred to herein as "ECO-04601." As used herein, the term "farnesyl dibenzodiazepinone" includes compounds of this class that can be used as intermediates in chemical syntheses.
As used herein, the term "alkyl" refers to linear or branched hydrocarbon groups.
Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexymethyl, and the like. Alkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.
The term "alkenyl" refers to linear, branched or cyclic hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and the like. Alkenyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino.
The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.
The terms "cycloalkyl" and "cycloalkyl ring" refer to a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The terms "heterocyclyl" and "heterocyclic" refer to a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from 0, N, NH, NRx, P02, S, SO or S02 in a single or fused Iheterocyclic ring system having from three to fifteen ring members. Examples of a heterocyclyl or heterocyclic ring include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl.
Heterocyclyl, heterocyclic or heterocyclyl ring may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term "amino acid" refers to any natural amino acid, all natural amino acids are well known to a person skilled in the art.

The term "halo" refers to a halogen atom, e.g., bromine, chlorine, fluorine and iodine.
The terms "aryl" and "aryl ring" refer to aromatic groups in a single or fused ring system, having from five to fifteen ring members. Examples of aryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl. Aryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkythio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The terms "heteroaryl" and "heteroaryl ring" refer to aromatic groups in a single or fused ring system, having from five to fifteen ririg members and containing at least one hetero atom such as 0, N, S, SO and S02. Examples of heteroaryl groups include, without limitation, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyi, oxazolyl, oxadiazoyl, triazolyi, and pyrrolyl groups. Heteroaryl groups may opitionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl.
The terms "aralkyl" and "heteroaralkyl" refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkyl group, such as benzyl.
Aralkyl and heteroaralkyl may be optionally substituted as the aryl and heteroaryl groups.
Similarly, the terms "aralkenyl" and "heteroaralkenyl" refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkene group, such as benzyl. Aralkenyl and heteroaralkenyl may be optionally substituted as the aryl and heteroaryl groups.
The compounds of the present invention can possess one or more asymmetric carbon atoms and can exist as optical isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention are useful as single isomers or as a mixture of stereochemical isomeric forms.
Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes.

The invention encompasses isolated or purified compounds. An "isolated" or "purified" compound refers to a compound which represents at least 10%, 20%, 50%, 80% or 90% of the compound of the present invention present in a mixture, provided that the mixture comprising the compound of the invention has demonstrable (i.e. statistically significant) biological activity including antibacterial, cytostatic, cytotoxic, antiinflammatory or enzyme inhibitory action when tested in conventional biological assays known to a person skilled in the art.
The terms "farnesyl dibenzodiazepinone-producing microorganism" and "producer of farnesyl dibenzodiazepinone," as used herein, refer to a microorganism that carries genetic information necessary to produce a farnesyl dibenzodiazepinone compound, whether or not the organism naturally produces the compound. The terms apply equally to organisms in which the genetic information to produce the farnesyl dibenzodiazepinone compound is found in the organism as it exists in its natural environment, and to organisms in which the genetic information is introduced by recombinant techniques.
Specific organisms contemplated herein include, without limitation, organisms of the family Micromonosporaceae, of which preferred genera include Micromonospora, Actinoplanes and Dactylosporangium; the family Streptomycetaceae, of which preferred genera include Streptomyces and Kitasatospora; the family Pseudonocardiaceae, of which preferred genera are Amycolatopsis and Saccharopolyspora; and the family Actinosynnemataceae, of which preferred genera include Saccharothrix and Actinosynnema; however the terms are intended to encompass all organisms containing genetic information necessary to produce a farnesyl dibenzodiazepinone compound. A preferred producer of a farnesyl dibenzodiazepinone compound includes microbial strain ECO11, a deposit of which was made on March 7, 2003, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No.
IDAC 070303-01.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening regions (introns) between individual coding segments (exons).

The terms "gene locus, "gene cluster," and "biosynthetic locus" refer to a group of genes or variants thereof involved in the biosynthesis of a farnesyl benzodiazepinone compound. The biosynthetic locus iri strain 046-ECO11 that directs the production of ECO-04601 is often referred to herein, in both the written description and Figures, as "046D." Genetic modification of gene locus, gene cluster or biosynthetic locus refers to any genetic recombinant techniques known in the art including mutagenesis, inactivation, or replacement of nucleic acids that can be applied to generate variants of ECO-04601.
A DNA or nucleotide "coding sequence" or "sequence encoding" a particular polypeptide or protein, is a DNA sequence which is transcribed and translated into a polypeptide or protein when placed under the control of an appropriate regulatory sequence.
"Oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably 15 and more preferably at least 20 nucleotides in length, preferably no more than 100 nucleotides in length, that are hybridizable to a genomic DNA molecule, a cDNA
molecule, or an mRNA molecule encoding a gene, mRNA, cDNA or other nucleic acid of interest.
A promoter sequence is "operably linked to" a coding sequence recognized by RNA polymerase which initiates transcription at the promoter and transcribes the coding sequence into mRNA.
The term "replicon" as used herein means any genetic element, such as a plasmid, cosmid, chromosome or virus, that behaves as an autonomous unit of polynucleotide replication within a cell. A "expression vector" or "vector" is a replicon in which another polynucleotide fragment is attached, such as to bring about the replication and/or expression of the attached fragment. "Plasmids" are designated herein by a lower case "p" preceded or followed by capital letters and/or numbers.
The starting plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described herein are known in the art and will be apparent to the skilled artisan.
The terms "express" and "expression" means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved iri transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intraceliular, extracellular or secreted.
"Digestion" of DNA refers to enzymatic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinary skilled artisan. For analytical purposes, typically 1 pg of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 pi of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 pg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular enzymes are specified by the manufacturer.
Incubation times of about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the gel electrophoresis may be performed to isolate the desired fragment.
The term "isolated" as used herein means that the material is removed from its original environment (e.g. the natural environment where the material is naturally occurring). For example, a naturaliy occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, which is separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that the vector or composition is not part of the natural environment.
The term "restriction fragment" as used herein refers to any linear DNA
generated by the action of one or more restriction enzymes.
The term "transformation" means the introduction of a foreign gene, foreign nucleic acid, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone" or "recombinant". The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
The terms "recombinant polynucleotide" and "recombinant polypeptide" as used herein mean a polynucleotide or polypeptide which by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide or polypeptide with which it is associated in nature and/or is linked to a polynucleotide or polypeptide other than that to which it is linked in nature.
The term "host cell" as used herein, refer to both prokaryotic and eukaryotic cells which are used as recipients of the recombinant polynucleotides and vectors provided herein. In one embodiment, the host cell is a prokaryote.
The terms "open reading frame" and "ORF" as used herein refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
As used herein and as known in the art, the term "identity" is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A.M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993)). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M.
Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48:1073. "Substantially identical,"
as used herein, means there is a very high degree of homology (preferably 100%
sequence identity) between subject polynucleotide sequences. However, polynucleotides having greater than 90%, or 95% sequence identity may be used in the present inventiori, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated.
As used herein, the term "treatment" refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alieviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a farnesyl dibenzodiazepinone and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount"
refers to that amount of a farnesyl dibenzodiazepinone effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

The term "pharmaceutically acceptable salt" refers to both acid addition salts and base addition salts. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Exemplary acid addition salts include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic,,B-hydroxybutyric, malonic, galactaric, galacturonic acid and the like. Suitable pharmaceutically acceptable base addition salts include, without limitation, metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine and the like. Additional examples of pharmaceutically acceptable salts are listed in Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1), 1-19. All of these salts may be prepared by conventional means from a farnesyl dibenzodiazepinone by treating the compound with the appropriate acid or base.

H. Farnesylated Dibenzodiazepinone Compounds In one aspect, the invention relates to a novel #arnesyl dibenzodiazepinone, referred to herein as "ECO-04601" and having the chemical structure represented by the following formula:

0 N/~/
I / / \
OH
OH
HO

ECO-04601 may be described as a new dibenzodiazepinone having a 10-farnesyl substituent located on the nitrogen atom in the 10 position of the dibenzodiazepine ring (i.e., the amide nitrogen in the diazepinone ring), and three phenolic hydroxy substituents in the 4,6 and 8 positions of the dibenzodiazepinone ring. ECO-04601 may be characterized by any one or more of its physicochemical and spectral properties given below, such as its mass, UV, and NMR
spectroscopic data. Mass was determined by electrospray mass spectrometry to be 462.6 (FIGURE 1); UV = 230 nm with a shoulder at 290nm (FIGURE 2). NMR data were collected using MeOH- d4, including proton (FIGURE 3), and multidimensional pulse sequences gDQCOSY (FIGURE 4), gHSQC (FIGURE 5), gHMBC (FIGURE 6), and NOESY (FIGURE 7).

In another aspect, the invention relates to a novel class of farnesyl dibenzodiazepinone compounds represented by Formula I:

CHg ~/ 1~ 3 N/\w w A /
P

Formula I.
wherein, W 15 W2 and W3 is each independently selected -from H

H2 ( I I
.~~C-H-~- ; -~-H=C-~ \ or the chain from the tricycle may terminate at W3, 1N2 or W1 with W3, W2 or W 1 respectively being either -CH=O or -CH2OH;

A is selected from -NH--, -NCH2R1, -NC(O)Rl;

R1 is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;

R2, R3, and R4 is each independently selected from H, R5, -C(O)R6 R5 is each independently selected from C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl;

R6 is each independently selected from H, C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.

In other embodiments, the invention provides compounds of Formula I, wherein A is selected from the group consisting of NH, NCH2R1, and NC(O)R1;
wherein R2 is H; R3 is H; and R4 is H. In another embodiment, R2, R3 and R4 are each H; and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H; and W1 is -CH =CH- and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H, and W2 is -CH =CH- and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H; and W3 is -CH =CH- ; and all other groups are as previously defined. In a further embodiment, A is NH; R2, R3 and R4 are each H;
and all other groups are as previously defined. In a further embodiment, A is NH;
each of W1, W2, and W3 is -CH =CH-; and all other groups are as previously defined. The invention encompasses all pharmaceutically acceptable salts of the foregoing compounds.
The following are exemplary compounds of the invention:

O

I ~ I \ OH
N
i CH
HO
Formula II

O CH, CH3 CHy O CH, CH, CH3 HO OH
CHz HO O-C pH HO
/ HO
H~C

Formula III Formula IV

0 CH3 CH, CH, N CH
Hs CH, CH, \ /

a / \r\
õO
PN- ~ CHy H,C
~ ' CH,I
HO / OH O" \
~S.
~
H'C HO CH

Formula V Formula VI

p CH~ CHs CN, 0 Ci, CH3 H3 N tlo CH, HO H OH HO H OH
HO HO

Formula VII Formula VIII

O Hs CHs CH0 0 CH3 XH, H9 PN N CH, CH3 O O O
~ i ~ ~
HO H _ OH HO H ~ OH
HO ~ HO

Formula VIX Formula X

O CH, CH, CH3 O CH~ CH3 CH3 Ph N CH3 0 N /
HO I \ OH HO H OH
H ~
HO HO

Formula XI Formula XII

0 CH3 CH3 CH3 0 CH, CHs OH, PNt' N O 0 0 HO N
HO F/ OH H OHHO HO

Formula XIII Formula XIV

O CH, CH3 CH3 0 CH3 CH3 CHs N

qNt' \ HO j HO
H _ OH H OH
HO HO

Formula XV Formula XVI

O CH3 CH, CH0 0 CH3 CH3 CH3 N O / O
PN /

HO H _ OH HO H OH
~ 1 ~ b-HO HO

Formula XVII Formula XVIII

0 CH3 CH3 CH0 0 CH, CH3 CH3 /
O ~ CH N CH, N O
PNtNt HO H / OH HO H OH
HO HO

Formula XIX Formula XX

O CH, CH, Chi, O CH3 CH3 CH3 P N CH3 N CH, N , HO
N
HO / H _ OH H OH
HO HO O ~

Formula XXI Formula XXII

0 CH, CH, CH, 0 CH~ CH3 CH, N CH, N
\ ~ ~ ~ \
PNt H H H HO H _ OH
HO HO ~

Formula XXIII Formula XXIV

O CH, CH3 CH, O CH3 CH, CH3 HO / CH, P N CH, PN-N ~ 1 \~, ~ \
H _ OH J H _ OH

HO H,C/ HO

Formula XXV Formula XXVI

0 CH, CH, CH3 N CH3 0 CFi3 CH3 CHs N P CH, Hp 0 H OH N ' O
p HO
r H
NaC Chi, HO

Formula XXVII Formula XXVIII

0 CH3 CH, CH, O CH3 CH3 CH3 N CH, PN- CH, 0 H ' O~CH3 0~,,.~
H,C OH3 ~ O~
O CH, CHa Formula XXIX Formula XXX

0 CH, CH, CH, ~ ' PN N CH3 0~ / ' O OH
O / \ ~-HaC H O_''-O ~
H ~ O CH~
H'C HO

Formula XXXI Formula XXXII

O CH, CH, CH0 0 CI-I, CH, CH3 N / / N~\\V~ ~% ~/ ~CH3 N / \ HO / \
HaC-O OH H ~~ OH
H 7 ~
HO HaC-O

Formula XXXIII Formula XXXIV

0 CH, CH3 CH3 0 CH, CH, CH, N CHa / 3 .
' N ~ \ H'C'O H '~. O-CH' H O\
HO CH, Formula XXXV Formula XXXVI

N CH 0 CH3 CH, CH3 a b~O-CH, HO / /
N P
H N
i H3C-O H -..O-CH3 CHa ~ HO

Formula XXXVII Formula XXXVIII

0 CH, H3 CH3 /

HO OH
H
CH, HO e Formula XXXIX Formula XL

0 CH3 CH3 CH3 0 CH, CH3 CH3 /

HO pH HO H _ OH
H HO HO

Formula XLI Formula XLII

N CH3 I N'CH3 qNt' i \ N
HO HO
H OH H OH
HO HO

Formula XLIII Formula XLIV

\ \ ~
HO y % HO
H OH H -OH
HO ~ HO

Formula XLV Formula XLVI

q CH3 CH3 0 CHa CH3 OH
N PN-b-HO NH OH HO H HO OH
HO

Formula XLVII Formula XLVIII

H
O N~=~~~~OH
q N

HO H OH HO H b Ohl HO HO

Formula XLIX Formula L

NO P N,,,,,_,,Ori HO

Formula L9 Formula LII

O CH, CH3 CH0 0 CH3 CH3 CH, N CHa CH, OH / OH
OH OH

HO / H ' OH HO H OH
HO HO

Formula LIII Formula LIV

O CH, CH3 CH0 0 CH3 CH3 CH3 ' / N / CH, N / CH3 ~ / OH OH OH
OH )OH OH
HO H OH HO H OH
i ) i /
HO ; HO

Formula LV Formula LVI

O CH, CH, CH3 0 CH, CH3 CH, OH OH OH OH
/ 'OH OH J OH OH
PNt/ PNt, CH, HO OH HO H OH
H. ; Ho ; and Formula LVII Formula LVIII

H~ H3 H, OH OH OH
OH OH OH
HO OH
H
HO

Formula LIX

Certain embodiments expressly exclude one or more of the compounds of Formula I. In one embodiment, the compound of Formula II is excluded.
The compounds of this invention may be formuiated into pharmaceutical compositions comprised of compounds of Formula I in combination with a pharmaceutical acceptable carrier, as discussed in Section V below.

III. Method of Making a Farnesyl Dibenzodiazepinone by Fermentation In one embodiment, ECO-04601 is obtained by cultivating a novel strain of Micromonospora, namely Micromonospora sp. strain 046-ECO11. Strain 046-ECO11 was deposited on March 7, 2003, with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No. 070303-01. The deposit of the strain was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those skilled in the art and are not an admission that a deposit is required for enablement.

It is to be understood that the present invention is not limited to use of the particular strain 046-EC011. Rather, the present invention contemplates the use of other ECO-04601 producing organisms, such as mutants or variants of 046-EC011 that can be derived from this organism by known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, antibiotic selection and the like; or through the use of recombinant genetic engineering techniques, as described in Section IV below.

The farnesyl dibenzodiazepinone compounds of the present invention may be .,~..~.,.... ~.~.,,~.

biosynthesized by various microorganisms. Microorganisms that may synthesize the compounds of the present invention include but are not limited to bacteria of the order Actinomycetales, also referred to as actinomycetes. Non-limiting examples of members belonging to the genera of Actinomycetes include Nocardia, Geodermatophilus, Actinoplanes, Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria, Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces, Microbispora, Streptosporangium, and Actinomadura. The taxonomy of actinomycetes is complex and reference is made to Goodfellow, Suprageneric Classitrcation of Actinomycetes (1989); Bergey's Manual of Systematic Bacteriology, Vol. 4 (Williams and Wilkins, Baltimore, pp. 2322-2339); and to Embley and Stackebrandt, "The molecular phylogeny and systematics of the actinomycetes,"
Annu. Rev. Microbiol. (1994) 48:257-289, for genera that may synthesize the compounds of the invention.
Farnesyl dibenzodiazepinone-producing microorganisms are cultivated in culture medium containing known nutritional sources for actinomycetes. Such media having assimilable sources of carbon, nitrogen, plus optional inorganic salts and other known growth factors at a pH of about 6 to about 9. Suitable media include, without limitation, the growth media provided in Table 16. Microorganisms are cultivated at incubation temperatures of about 18 C to about 40 C for about 3 to about 40 days.
The culture media inoculated with the farnesyl dibenzodiazepinone-producing microorganisms may be aerated by incubating the inoculated culture media with agitation, for example, shaking on a rotary shaker, or a shaking water bath.
Aeration may also be achieved by the injection of air, oxygen or an appropriate gaseous mixture to the inoculated culture media during incubation. Following cultivation, the farnesyl dibenzodiazepinone compounds can be extracted and isolated from the cultivated culture media by techniques known to a skilled person in the art and/or disclosed herein, including for example centrifugation, chromatography, adsorption, filtration. For example, the cultivated culture media can be mixed with a suitable organic solvent such as n-butanol, n-butyl acetate or 4-methyl-2-pentanone, the organic layer can be separated for example, by centrifugation followed by the removal of the solvent, by evaporation to dryness or by evaporation to dryness under vacuum. The resulting residue can optionally be reconstituted with for example water, ethanol, ethyl acetate, methanol or a mixture thereof, and re-extracted with a suitable organic solvent such as hexane, carbon tetrachloride, methylene chloride or a mixture thereof. Following removal of the solvent, the compounds may be further purified by the use of standard techniques, such as chromatography.
The farnesyl dibenzodiapezinones biosynthesized by microorganisms may optionally be subjected to random and/or directed chemical modifications to form compounds that are derivatives or structural analogs. Such derivatives or structural analogs having similar functional activities are within the scope of the present invention. Farnesyl dibenzodiapezinone compounds may optionally be modified using methods known in the art and described herein.

IV. Method of Makinci a Farnesyl Dibenzodiazepinone by Recombinant Technology In another embodiment, the present invention relates to nucleic acid molecules that encode proteins useful in the production of farnesyl benzodiazepinones. Specifically, the present invention provides recombinant DNA
vectors and nucleic acid molecules that encode all or part of the biosynthetic locus in strain 046-ECO11, which directs the production of ECO-04601, and is referred to herein as "046D." The invention further includes genetic modification of 046D
using conventional genetic recombinant techniques, such as mutagenesis, inactivation, or replacement of nucleic acids, to produce chemical variants of ECO-04601.
The invention thus provides a method for making a farnesyl benzodiazepinone compound using a transformed host cell comprising a recombinant DNA vector that encodes one or more of the polypeptides of the present invention, and culturing the host cell under conditions such that farnesyl benzodiazepinone is produced. The host cell is a prokaryote. In one embodiment, the host cell is an actinomycete. In another embodiment, the host cell is a Streptomyces host cell.
The invention provides recombinant nucleic acids that produce a variety of farnesyl dibenzodiazepinone compounds that cannot be readily synthesized by chemical methodology alone. The invention allows direct manipulation of 046D
biosynthetic locus via genetic engineering of the enzymes involved in the biosynthesis of a farnesyl benzodiazepinone according to the invention. The biosynthetic locus is described in Example 11.

Recombinant DNA Vectors Vectors of the invention typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA
into another segment of DNA involves the use of specific enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A"cassette" refers to a DNA coding sequence or segment of DNA
that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are desigried to ensure insertion of the cassette in the proper reading frame. Generally, a nucleic acid molecule that encodes a protein useful in the production of a farnesyl benzodiazepinone is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a prokaryote e.g. actinomycte, by transformation (see below). A
segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid"
which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. In one embodiment of the invention, the coding DNA encodes for polypeptides of SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 or 89 that are required for the biosynthesis of a farnesyl benzodiazepinone.
Promoter DNA of a recombinant vector is a DNA sequence that initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.
Promoter DNA and coding may be from the same or different organisms.
Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g.
antibiotic resistance, and one or more expression cassettes. Vector constructs may be produced using conventional molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Examples of promoters that function in actinomycetes, e.g. Streptomyces, are taught in US Patent Nos. 5,830,695 and 5,466,590. Another example of a transcription promoter useful in Actinomycetes expression vectors is tipA, a promoter inducible by the antibiotic thiostrepton [c.f. Murakami, T., et al., (1989), J.
Bacteriol, 171, 1459].

Transformation of Actinomycetes A suitable transformation method for use with an actinomycete comprises forming the actinomycete culture into spheroplasts using lysozyme. A buffer solution containing recombinant DNA vectors and polyethylene glycol is then added, in order to introduce the vector into the host cells, by using either of the methods of Thompson or Keiser [c. f. Thompson, C. J., et al., (1982), J. Bacteriol., 151, or Keiser, T. et al. (2000), "Practical Streptomyces Genetics", The John Innes Foundation, Norwich], for example. A thiostrepton-resistance gene is frequently used as a selective marker in the transformation plasmid [c.f. Hopwood, D. A., et al., (1987), "Methods in Enzymology" 153, 116, Academic Press, New York], but the present invention is not limited thereto. Additional methods for the transformation of actinomycetes are taught in US 5,393,665.

Assay for farnesyl dibenzodiazepinone or biosynthetic intermediates Actinomycetes defective in farnesyl dibenzodiazepinone biosynthesis are transformed with one or more expression vectors encoding one or more proteins in the farnesyl benzodiazepinone biosynthetic pathway, thus restoring farnesyl benzodiazepinone biosynthesis by genetic complementation of the specific defect.

The presence or absence of farnesyl dibenzodiazepinone or intermediates in the biosynthetic pathway (see Figures 13, 14 and 15) in a recombinant actinomycete can be determined using methodologies that are well known to persons of skill in the art. For example, ethyl acetate extracts of fermentation media used for the culture of a recombinant actinomycete are processed as described in Example 2 and fractions containing farnesyl dibenzodiazepinone or intermediates detected by TLC on commercial Kieselgel 60F254 plates. Farnesyl dibenzodiazepinone and intermediate compounds are visualized by inspection of dried plates under UV light or by spraying the plates with a spray containing vanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently heating the plate. The exact identity of the compounds separated by TLC is then determined using gas chromatography-mass spectroscopy. Methods of mass spectroscopy are taught in the published U.S.
Patent No. 6,683,300.

Mutagenesis The invention allows direct manipulation of 046D biosynthetic locus via genetic engineering of the enzymes involved in the biosynthesis of a farnesyl benzodiazepinone according to the invention.
A number of methods are known in the art that permit the random as well as targeted mutation of the DNA sequences of the invention (see for example, Ausubel et. aI. Short Protocols in Molecular Biology (1995) 3rd Ed. John Wiley & Sons, Inc.).
In addition, there are a number of of commercially available kits for site-directed mutagenesis, including both conventional and PCR-based methods. Examples include the EXSITETM PCR-Based Site-directed Mutagenesis Kit available from Stratagene (Catalog No. 200502) and the QUIKCHANGETM Site-directed mutagenesis Kit from Stratagene (Catalog No. 200518), and the CHAMELEON
double-stranded Site-directed mutagenesis kit, also from Stratagene (Catalog No.
200509).
In addition the nucleotides of the invention may be generated by insertional mutation or truncation (N-terminal, internal or C-terminal) according to methodology known to a person skilled in the art.
Older methods of site-directed mutagenesis known in the art rely on sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3' end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation.
More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-strarided template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.
The protocol described beloW accommodates these considerations through the following steps. First, the template concentration used is approximately fold higher than that used in conventional PCR reactions, allowing a reduction in the number of cycles from 25-30 down to 5-10 without dramatically reducing product yield. Second, the restriction endonuclease Dpn I (recognition target sequence: 5-Gm6ATC-3, where the A residue is methylated) is used to select against parental DNA, since most common strains of E. coli Dam methylate their DNA at the sequence 5-GATC-3. Third, Taq Extender is used in the PCR mix in order to increase the proportion of long (i.e., full plasmid length) PCR products.
Finally, Pfu DNA polymerase is used to polish the ends of the PCR product prior to intramolecular ligation using T4 DNA ligase.
A non-limiting example for the isolation of mutant polynucleotides is described in detail as follows:
Plasmid template DNA (approximately 0.5 pmole) is added to a PCR cocktail containing: 1x mutagenesis buffer (20 mM Tris HCI, pH 7.5; 8 mM MgC12;
4.0,ug/mI
BSA); 12-20 pmole of each primer (one of skill in the art may design a mutagenic primer as necessary, giving consideration to those factors such as base composition, primer length and intended buffer salt concentrations that affect the annealing characteristics of oligonucleotide primers; one primer must contain the desired mutation, and one (the same or the other) must contain a 5' phosphate to facilitate later ligation), 250 pM each dNTP, 2.5 U Taq DNA polymerase, and, 2.5 U
of Taq Extender (Available from Stratagene; See Nielson et al. (1994) Strategies 7:
27, and U.S. Patent No. 5,556,772). Primers can be prepared using the triester method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185-3191.
Alternatively automated synthesis may be preferred, for example, on a Biosearch 8700 DNA
Synthesizer using cyanoethyl phosphoramidite chemistry.
The PCR cycling is performed as follows: 1 cycle of 4 min at 94 C, 2 min at 50 C and 2 min at 72 C; followed by 5-10 cycles of 1 min at 94 C, 2 min at 54 C
and 1 min at 72 C. The parental template DNA and the linear, PCR-generated DNA
incorporating the mutagenic primer are treated with Dpnl (10 U) and Pfu DNA
polymerase (2.5U). This results in the Dpnl digestion of the in vivo methylated parental template and hybrid DNA and the removal, by Pfu DNA polymerase, of the non-template-directed Taq DNA polymerase-extended base(s) on the linear PCR
product. The reaction is incubated at 37 C for 30 min and then transferred to for an additional 30 min. Mutagenesis buffer (115NI of 1x) containing 0.5 mM
ATP
is added to the Dpn{-digested, Pfu DNA polymerase-polished PCR products. The solution is mixed and 10 ul are removed to a new microfuge tube and T4 DNA
ligase (2-4 U) is added. The ligation is incubated for greater than 60 min at 37 C.
Finally, the treated solution is transformed into competent E. coli according to standard methods.
Methods of random mutagenesis, which will result in a panel of mutants bearing one or more randomly situated mutations, exist in the art. Such a panel of mutants may then be screened for those exhibiting reduced uracil detection activity relative to the wild-type polymerase (e.g., by measuring the incorporation of 10nmoles of dNTPs into polymeric form in 30 minutes in the presence of 200,1M
dUTP and at the optimal temperature for a given DNA polymerase). An example of a method for random mutagenesis is the so-called "error-prone PCR method". As the name implies, the method amplifies a given sequence under conditions in which the DNA polymerase does not support high fidelity incorporation. The conditions encouraging error-prone incorporation for different DNA polymerases vary, however one skilled in the art may determine such conditions for a given enzyme. A key variable for many DNA polymerases in the fidelity of amplification is, for example, the type and concentration of divalent metal ion in the buffer. The use of manganese ion and/or variation of the magnesium or manganese ion concentration may therefore be applied to influence the error rate of the polymerase.
Genes for desired mutant polypeptides generated by mutagenesis may be sequenced to identify the sites and number of mutations. For those mutants comprising more than one mutation, the effect of a given mutation may be evaluated by introduction of the identified mutation to the wild-type gene by site-directed mutagenesis in isolation from the other mutations borne by the particular mutant.
Screening assays of the single mutant thus produced will then allow the determination of the effect of that mutation alone.

V. Genes and proteins for the production of ECO-04601 As discussed in more detail below, the isolated, purified or enriched nucleic acids of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 may be used to prepare one of the polypeptides of SEQ ID
NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, respectively, or fragments comprising at least 50, 75, 100, 200, 300, 500 or more consecutive amino acids of one of the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88.
Accordingly, another aspect of the present invention is an isolated, purified or enriched nucleic acid which encodes one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 or more consecutive amino acids of one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. The coding sequences of these nucleic acids may be identical to one of the coding sequences of one of the nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 or a fragment thereof, or may be different coding sequences which encode one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 consecutive amino acids of one of the polypeptides of SEQ
ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 as a result of the redundancy or degeneracy of the genetic code. The genetic code is well known to those of skill in the art and can be obtained, for example, from Stryer, Biochemistry, 3d edition, W. H. Freeman & Co., New York.
The isolated, purified or enriched nucleic acid which encodes one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 may include, but is not limited to: (1) only the coding sequences of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89; (2) the coding sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 and additional coding sequences, such as leader sequences or proprotein; and (3) the coding sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 and non-coding sequences, such as non-coding sequences 5' and/or 3' of the coding sequence. Thus, as used herein, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only coding sequence for the polypeptide as well as a polynucleotide that includes additional coding and/or non-coding sequence.
The invention relates to polynucleotides based on SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 but having polynucleotide changes that are "silent", for example changes which do not alter the amino acid sequence encoded by the polynucleotides of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The invention also relates to polynucleotides which have nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. Such nucleotide changes may be introduced using techniques such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion, and other recombinant DNA techniques.
The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences complementary thereto, or a fragment comprising at least 100, 150, 200, 300, 400 or more consecutive bases of one of the sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences complementary thereto may be used as probes to identify and isolate DNAs encoding the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 espectively. In such procedures, a genomic DNA library is constructed from a sample microorganism or a sample containing a microorganism capable of producing a farnesyl dibenzodiazepinone. The genomic DNA library is then contacted with a probe comprising a coding sequence or a fragment of the coding sequence, encoding one of the polypeptides of SEQ ID
NOS:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, or a fragment thereof under conditions which permit the probe to specifically hybridize to sequences complementary thereto. In a preferred embodiment, the probe is an oligonucleotide of about 10 to about 30 nucleotides in length designed based on a nucleic acid of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. Genomic DNA clones which hybridize to the probe are then detected and isolated. Procedures for preparing and identifying DNA clones of interest are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997; and Sambrook et aL, Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press, 1989. In another embodiment, the probe is a restriction fragment or a PCR
amplified nucleic acid derived from SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89.
The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences complementary thereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 consecutive bases of one of the sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 or the sequences complementary thereto may be used as probes to identify and isolate related nucleic acids. In some embodiments, the related nucleic acids may be genomic DNAs (or cDNAs) from potential farnesyl dibenzodiazepinone producers. In such procedures, a nucleic acid sample containing nucleic acids from a potential farnesyl dibenzodiazepinone producer is contacted with the probe under conditions that permit the probe to specifically hybridize to related sequences. The nucleic acid sample may be a genomic DNA (or cDNA) library from the potential farnesyl dibenzodiazepinone-producer. Hybridization of the probe to nucleic acids is then detected using any of the methods described above.
Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency. As an example of nucleic acid hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45 C in a solution consisting of 0.9 M NaCI, 50 mM

NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/mi polyriboadenylic acid. Approximately 2 x 10' cpm (specific activity 4-9 x 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution. After hours of incubation, the membrane is washed for 30 minutes at room temperature in 1 X SET (150 mM NaCI, 20 mM Tris hydrochloride, pH 7.8, 1 rnM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh 1 X SET at Tm-10 C
for the oligonucleotide probe where Tm is the melting temperature. The membrane is then exposed to autoradiographic film for detection of hybridization signals.
By varying the stringency of the hybridization conditions used to identify nucleic acids, such as genomic DNAs or cDNAs, which hybridize to the detectable probe, nucleic acids having different levels of homology to the probe can be identified and isolated. Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes. The melting temperature of the probe may be calculated using the following formulas:
For oligonucleotide probes between 14 and 70 nucleotides in length the melting temperature (Tm) in degrees Celcius may be calculated using the formula:
Tm=81.5+16.6(log [Na+]) + 0.41 (fraction G+C)-(600/N) where N is the length of the oligonucleotide.
If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation Tm=81.5+16.6(log [Na +]) + 0.41 (fraction G + C)-(0.63% formamide)-(600/N) where N is the length of the probe.
Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5%
SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA or 6X SSC, 5X
Denhardt's reagent, 0.5% SDS, 0:1 mg/mi denatured fragmented salmon sperm DNA, 50% formamide. The composition of the SSC and Denhardt's solutions are listed in Sambrook et al., supra.
Hybridization is conducted by adding the detectable probe to the hybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured by incubating at elevated temperatures and quickly cooling before addition to the hybridization solution. It may also be desirable to similarly denature single stranded probes to eliminate or diminish formation of secondary structures or oligomerization. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25 C below the Tm.
For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10 C below the Tm. Preferably, the hybridization is conducted in 6X
SSC, for shorter probes. Preferably, the hybridization is conducted in 50%
formamide containing solutions, for longer probes. All the foregoing hybridizations would be considered to be examples of hybridization performed under conditions of high stringency.
Following hybridization, the filter is washed for at least 15 minutes in 2X
SSC, 0.1 la SDS at room temperature or higher, depending on the desired stringency. The filter is then washed with 0.1 X SSC, 0.5% SDS at room temperature (again) for minutes to 1 hour. Nucleic acids which have hybridized to the probe are identified by conventional autoradiography and non-radioactive detection methods.
The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucieic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5 C from 68 C to 42 C in a hybridization buffer having a Na+ concentration of approximately 1 M. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate stringency" conditions above 50 C
and 'low stringency" conditions below 50 C. A specific example of "moderate stringency"
hybridization conditions is when the above hybridization is conducted at 55 C.
A
specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45 C.

Alternatively, the hybridization may be carried out in buffers, such as 6X
SSC, containing formamide at a temperature of 42 C. In this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to the probe.
Following hybridization, the filter may be washed with 6X SSC, 0.5% SDS at 50 C. These conditions are considered to be "moderate stringency" conditions above 25%

formamide and "low stringency" conditions below 25% formamide. A specific example of "moderate stringency" hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of "low stringency"
hybridization conditions is when the above hybridization is conducted at 10%
formamide. Nucleic acids which have hybridized to the probe are identified by conventional autoradiography and non-radioactive detection methods.
The preceding methods may be used to isolate nucleic acids having at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70%
sequence identity to a nucleic acid sequence selected from the group consisting of the sequences of SEQ I D NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, _ 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The isolated nucieic acid may have a coding sequence that is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variant may have a substitution, deletion or addition of one or more nucleotides when compared to the nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences complementary thereto.
Additionally, the above procedures may be used to isolate nucleic acids which encode polypeptides having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% identity to a polypeptide having the sequence of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 consecutive amino acids thereof.
Another aspect of the present invention is an isolated or purified polypeptide comprising the sequence of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. As discussed herein, such polypeptides may be obtained by inserting a nucleic acid encoding the polypeptide into a vector such that the coding sequence is operably linked to a sequence capable of driving the expression of the encoded polypeptide in a suitable host cell. For example, the expression vector may comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for modulating expression levels, an origin of replication and a selectable marker.
Promoters suitable for expressing the polypeptide or fragment thereof in bacteria include the E.coli lac or trp promoters, the laci promoter, the lacZ
promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Fungal promoters include the a factor promoter. Eukaryotic promoters include the CMV
immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metailothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used.
Mammalian expression vectors may also comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donors and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. In some embodiments, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
Vectors for expressing the polypeptide or fragment thereof in eukaryotic;cells may also contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.
In addition, the expression vectors preferably contain one or more selectable marker genes to permit selection of host cells containing the vector. Examples of selectable markers that may be used include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S.
cerevisiae TRP1 gene.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, appropriate restriction enzyme sites can be engineered into a DNA sequence by PCR. A variety of cloning techniques are disclosed in Ausbel et al. Current Protocols in Molecular Biology, John Wiley Sons, Inc. 1997 and Sambrook et aL, Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbour Laboratory Press, 1989. Such procedures and others are deemed to be within the scope of those skilled in the art.
The vector may be, for example, in the form of a plasmid, a viral particle, or a phage. Other vectors include derivatives of chromosomal, nonchromosomal and synthetic DNA sequences, viruses, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the weli known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, WI, USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, phiX174, pBluescriptTM II KS, pNHBA, pNH16a, pNH18A, pNH46A
(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is replicable and stable in the host cell.
The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells or eukaryotic cells. As representative examples of appropriate hosts, there may be mentioned: bacteria cells, such as E. coli, Streptomyces lividans, Streptomyces griseofuscus, Streptomyces ambofaciens, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, Bacillus, and Staphylococcus, fungal cells, such as yeast, insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma, and adenoviruses. The selection of an appropriate host is within the abilities of those skilled in the art.
The vector may be introduced into the host cells using any of a variety of techniques, including electroporation transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, ani n or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts (described by Gluzman, Cell, 23:175(1981)), and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Polypeptides of the invention may or may not also include an initial methionine amino acid residue.

_ . ~- .,~....,,~,..~..~..,...~.

Alternatively, the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof can be synthetically produced by conventional peptide synthesizers. In other embodiments, fragments or portions of the polynucleotides may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
Cell-free translation systems can also be employed to produce one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof using mRNAs transcribed from a DNA
construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some embodiments, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
The present invention also relates to variants of the polypeptides of SEQ ID
NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. The term "variant" includes derivatives or analogs of these polypeptides. In particular, the variants may differ in amino acid sequence from the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
The variants may be naturally occurring or created in vitro. In particular, such variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures.

Other methods of making variants are also familiar to those skilled in the art.
These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Preferably, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
The variants of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 may be variants in which one or more of the amino acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code..
Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the following replacements: replacements of an aliphatic amino acid such as Ala, Val, Leu and lie with another aliphatic amino acid;
replacement of a Ser with a Thr or vice versa; replacement of an acidic residue such as Asp or Glu with another acidic residue; replacement of a residue bearing an amide group, such as Asn or Gin, with another residue bearing an amide group;
exchange of a basic residue such as Lys or Arg with another basic residue; and replacement of an aromatic residue such as Phe or Tyr with another aromatic residue.
Other variants are those in which one or more of the amino acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 include a substituent group. Still other variants are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Additional variants are those in which additional amino acids are fused to the polypeptide, such as leader sequence, a secretory sequence, a proprotein sequence or a sequence that facilitates purification, enrichment, or stabilization of the polypeptide.
In some embodiments, the fragments, derivatives and analogs retain the same biological function or activity as the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. In other embodiments, the fragment, derivative or analogue includes a fused heterologous sequence that facilitates purification, enrichment, detection, stabilization or secretion of the polypeptide that can be enzymatically cleaved, in whole or in part, away from the fragment, derivative or analogue.
Another aspect of the present invention are polypeptides or fragments thereof which have at least 70%, at least 80%, at least 85%, at least 90%, or more than 95% identity to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. It will be appreciated that amino acid "substantially identity" includes conservative substitutions such as those described above.
The polypeptides or fragments having homology to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof may be obtained by isolating the nucleic acids encoding them using the techniques described above.
Alternatively, the homologous polypeptides or fragments may be obtained through biochemical enrichment or purification procedures. The sequence of potentially homologous polypeptides or fragments may be determined by proteolytic digestion, gel electrophoresis and/or microsequencing. The sequence of the prospective homologous polypeptide or fragment can be compared to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
The polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments, derivatives or analogs thereof comprising at least 40, 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof invention may be used in a variety of applications. For example, the polypeptides or fragments, derivatives or analogs thereof may be used to catalyze biochemical reactions as described elsewhere in the specification.

VI. Pharmaceutical compositions comprising farnesyl dibenzodiazepinones In another embodiment, the invention relates to a pharmaceutical composition comprising a farnesyl dibenzodiazepinone, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the farnesyl dibenzodiazepinone is useful for treating a variety of diseases and disorders, including cancer, inflammation and bacterial infections.
The compounds of the present invention, or pharmaceutically acceptable salts thereof, can be formulated for oral, intravenous, intramuscular, subcutaneous, topical or parenteral administration for the therapeutic or prophylactic treatment of diseases, particularly bacterial infections, acute and chronic inflammation and cancer. For oral or parental administration, compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like.
The compositions comprising a compound of this present invention will contain from about 0.1 % to about 99.9%, about 1 % to about 98%, about 5% to about 95%, about 10% to about 80% or about 15% to about 60% by weight of the active compound.
The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate bacterial infection, cancer or inflammation.
(See, e.g., Remington's Pharmaceutical Sciences 2000, Mack Publishing Company, ~

Easton, PA; and Goodman and Gilman, Pharmaceutical Basis of Therapeutics (2001), Pergamon Press, New York, NY, for a general description of the methods for administering various antimicrobial agents for human therapy). The compositions of the present invention can be delivered using controlled (e.g., capsules) or sustained release delivery systems (e.g., bioerodable matrices). Exemplary delayed release delivery systems for drug delivery that are suitable for administration of the compositions of the invention (preferably of Formula I) are described in U.S.
Patent Nos 4,452,775 (issued to Kent), 5,039,660 (issued to Leonard), 3,854,480 (issued to Zaffaroni).
The pharmaceutically acceptable compositions of the present invention comprise one or more compounds of the present invention in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as "carrie-"
materials, and if desired other active ingredients. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.
The compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.
Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethyicellulose, polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.
Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicon fluid, talc, waxes, oils and colloidal silica.
Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product comprising a compound of the present invention.
For oral use, solid formulations such as tablets and capsules are particularly useful. Sustained released or enterically coated preparations may also be devised.
For pediatric and geriatric applications, suspension, syrups and chewable tablets are especially suitable. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica or talc: disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
For intravenous (iv) use, compounds of the present invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution.
Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration.
The compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers.
For intramuscular preparations, a sterile formulation of compounds of the present invention or suitable soluble salts forming the compound, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
For topical use the compounds of present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.
For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.
For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.
Alternatively, the compound of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of the compound can be a solution of the compound or a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules.
The amount of the compound of the present invention in a unit dosage comprises a therapeutically-effective amount of at least one active compound of the present invention which may vary depending on the recipient subject, route and frequency of administration. A recipient subject refers to a plant, a cell culture or an animal such as an ovine or a mammal including a human.
According to this aspect of the present inventiori, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (including a human subject) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols.
Likewise, the methods for using the claimed composition for treating cells in culture, for example, to eliminate or reduce the level of bacterial contamination of a cell culture, utilize art-recognized protocols for treating cell cultures with antibacterial agent(s) with the only substantial procedural modification being the substitution of the compounds of the present invention for the agents used in the art-recognized protocols.
The compounds of the present invention provide a method for treating bacterial infections, pre-cancerous or cancerous conditions, and acute or chronic inflammatory disease. As used herein, the term "unit dosage" refers to a quantity of a therapeutically effective amount of a compound of the present invention that elicits a desired therapeutic response. As used herein, the phrase "therapeutically effective amount" means an amount of a compound of the present invention that prevents the onset, alleviates the symptoms, or stops the progression of a bacterial infection, inflammatory condition, or pre-cancerous or cancerous condition.
The term "treating" is defined as administering, to a subject, a therapeutically effective amount of at least one compound of the present invention, both to prevent the occurrence of a bacterial infection, inflammation or pre-cancer or cancer condition, or to control or eliminate a bacterial infection, inflammation or pre-cancer or cancer condition. The term "desired therapeutic response" refers to treating a recipient subject with a compound of the present invention such that a bacterial or inflammatory condition or pre-cancer or cancer condition is reversed, arrested or prevented in a recipient subject.
The compounds of the present invention can be administered as a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the disease condition, the age and general health of the recipient subject, the tolerance of the recipient subject to the compound and the type of the bacterial infection, inflammatory disorder, or type of cancer.
A compound according to this invention may also be administered in the diet or feed of a patient or animal. The diet for animals cari be normal foodstuffs to which the compound can be added or it can be added to a premix.
The compounds of the present invention may be taken in combination, together or separately with any known clinically approved antibiotic, inflammation or anti-cancer agent to treat a recipient subject in need of such treatment.

VII. Method of Inhibiting Tumor Growth In another embodiment, the present invention relates to a method of inhibiting tumor growth. Compounds as described herein can possess antitumor activity.
The compounds are effective against mammalian tumor cells such as leukemia cells, melanoma cells, breast carcinoma cells, lung carcinoma cells, pancreatic carcinoma cells, ovarian carcinoma cells, renal carcinoma cells, colon carcinoma cells prostate carcinoma cells and glioma cells. The antitumor method of the invention results in inhibition of tumor cells. The term "inhibition", when used in conjunction with the antitumor method refers to suppression, killing, stasis, or destruction of tumor cells.
The antitumor method preferably results in prevention, reduction or elimination of invasive activity and related metastasis of tumor cells. The term "effective amount"
when used in conjunction with the antitumor cell method refers to the amount of the compound sufficient to result in the inhibition of mammaiian tumor cells.
The inhibition of mammalian tumor growth according to this method can be monitored in several ways. First, tumor cells grown in vitro can be treated with the compound and monitored for growth or death relative to the same cells cultured in the absence of the compound. A cessation of growth or a slowing of the growth rate (i.e., the doubling rate), e.g., by 10% or more, is indicative of tumor cell inhibition.
Alternatively, tumor cell inhibition can be monitored by administering the compound to an animal model of the tumor of interest. Examples of experimental animal tumor models are known in the art and described in the examples herein. A cessation of tumor growth (i.e., no further increase in size) or a reduction in tumor size (i.e., tumor volume) or cell number (e.g., at least a 10% decrease in either) in animals treated with a compound as described herein relative to tumors in control animals not treated with the compound is indicative of tumor growth inhibition.
To monitor the efficacy of tumor treatment in a human, tumor size or tumor cell titer is measured before and after initiation of the treatment, and treatment is considered effective if either the tumor size or titer ceases further growth, or if the tumor is reduced in size or titer, e.g., by at least 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%, that is, the absence of the tumor).
Methods of determining the size or cell titer of a tumor in vivo vary with the type of tumor, and include, for example, various imaging techniques well known to those in the medical imaging or oncology fields (MRI, CAT, PET, etc.), as well as histological techniques and flow cytometry.
For the antitumor method of the invention, a typical effective dose of the compounds given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about mg/kg of body weight of the subject.

VIII. Method of Inhibiting LipoxyõcLenase In another embodiment, the present invention also provides for a method of treating diseased states, in particular inflammation, caused by the 5-lipoxygenase system and/or by the synthesis of the Leukotrienes C4, D4, E4 and F4 as well as Leukotriene B4 in mammals, especially in human subjects. This method comprises administering to a subject an effective amount of ECO-04601. Compound ECO-04601 may be used alone or in combination with other anti-inflammatory compounds to treat or prevent disease states related to inflammation including pulmonary conditions, inflammation, cardiovascular conditions, central nervous system conditions or skin conditions. More specific diseases include gastritis;
erosive esophagitis; inflammatory bowel disease; ethanol-induced hemorrhagic erosions;
hepatic ischemia; ischemic neuronal injury; noxious agent induced damage or necrosis of hepatic, pancreatic, renal, neuronal or myocardial tissue; liver parenchymal damage caused by hepatoxic agents such as CCI4 and D-galactosamine; ischemic renal failure; disease-induced hepatic damage; trauma-or stress-induced cell damage; asthma; multiple sclerosis; ischemic reperfusion;
edema; rheumatoid arthritis; viral encephalitis; bacterial pneumonia;
neurodegeneration; Alzheimer's disease and glycerol-induced renal failure.
For the method of the invention related to the 5-lipoxygenase system and/or the biosynthesis of Leukotrienes, a typical effective unit dose of ECO-04601 given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
The inhibition of lipoxygenase enzymes is monitored using methods well known in the art and as described in the examples herein. A decrease in enzyme activity by at least 10%, relative to the activity in the absence of a compound as described herein is indicative of effective inhibition of lipoxygenase activity.
Farnesyl dibenzodiazepinone compounds useful according to the invention can be used to reduce or prevent inflammation. Among the hallmarks of local acute inflammation are heat, redness, swelling, pain and loss of function. These changes are induced largely by changes in vascular flow and caliber, changes in vascular permeability and leukocyte exudation (Robbins et al., "Pathologic Basis of Disease", 6th Ed., W.B. Saunders Co., Philadelphia, PA). Anti-inflammatory therapy performed using compounds useful according to the invention can be monitored for success by tracking any of these changes. For example, a decrease in swelling (e.g., at least 10% decrease following treatment) or reported pain (e.g., a sustained decrease of 1 point or more on a 1-10 scale reported by the patient, with 10 being the worst pain experienced in association with this disorder prior to treatment, and 0 being no pain) can be used to indicate successful treatment.
Other measurable hallmarks of inflammation inciude leukocyte infiltration and inflammatory cytokine levels. These hallmarks can be monitored by biopsy of the affected tissue. A decrease of 10% or more in leukocyte infiltration in fixed, stained tissue relative to infiltration in similar tissue prior to treatment can be used to indicate successful treatment, as can a decrease of 10% or more in the level of any given inflammatory cytokine, relative to the level before treatment. Those skilled in the art can readily assay for inflammatory cytokine levels in tissue, blood, or other fluid samples. Alternatively, the level of systemic indicators of inflammation such as C
reactive protein levels and erythrocyte sedimentation rate can be monitored.
Each of these has established normal ranges in medicine, and treatment is considered successful if one or more of such indicators goes from outside the normal range to inside the normal range after the initiation of treatment.

IX. Method of Inhibiting Bacterial Growth In another embodiment, the present invention relates to a method for treating bacteriaf infection in a mammalian subject in need thereof, comprising the step of administering to the mammal a therapeutically effective amount of compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
According to another embodiment, the invention provides a method of decreasing bacterial quantity in a biological sample. This method comprises the step of contacting the biological sample with a compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
This method is effective if the number of bacteria decreases by at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
These pharmaceutical compositions effective to treat or prevent a bacterial infection which comprise ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof in an amount sufficient to measurably decrease bacterial quantity, and a pharmaceutically acceptable carrier, are another embodiment of the present invention. The term "measurably decrease bacterial quantity", as used herein means a measurable change in the number of bacteria between a sample containing the inhibitor and a sample not containing the inhibitor.
Agents which increase the susceptibility of bacterial organisms to antibiotics are known. For example, U.S. Pat. No. 5,523,288, U.S. Pat. No. 5,783,561 and U.S.
Pat. No. 6,140,306 describe methods of using bactericidal/permeability-increasing protein (BPI) for increasing antibiotic susceptibility of gram-positive and gram-negative bacteria. Agents that increase the permeability of the outer membrane of bacterial organisms have beeri described by Vaara, M. in Microbiological Reviews (1992) pp. 395-411, and the sensitization of gram-negative bacteria has been described by Tsubery, H., et al, in J. Med. Chem . (2000) pp. 3085-3092.
For the method of the irivention related to treatment of subjects with a bacterial infection, a typical effective unit dose of ECO-04601, a compound described herein or a pharmaceutically acceptable derivative or prodrug thereof given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
Another preferred embodiment of this invention relates to a method, as described above, of treating a bacterial infection in a mammal in need thereof, but : .~,.., :.~,..~,.....~,,...~..... .~. _ .. . .. .-~.__. ___.

further comprising the step of administering to the mammal an agent which increases the susceptibility of bacterial organisms to antibiotics.
According to another preferred embodiment, the invention provides a method, as described above, of decreasing bacterial quantity in a biological sample, but further comprising the step of contacting the biological sample with an agent which increases the susceptibility of bacterial organisms to antibiotics.
Methods of decreasing bacterial quantity are effective if the number of bacteria decreases at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
The pharmaceutical compositions and methods of this invention will be useful generally for controlling bacterial infections in vivo. Examples of bacterial organisms that may be controlled by the compositions and methods of this invention include, but are not limited to the following organisms: Streptococcus pneumoniae, Streptococcus pyrogenes, Enterococcus fecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, E. coli, Serratia marcesens, Staphylococcus aureus, Coagulase negative Staphylococcus, Haemophilus infuenzae, Bacillus anthracis, Mycoplasma pneumoniae, and Staphylococcus epidermidis. The compositions and methods will therefore be useful for controlling, treating or reducing the advancement, severity or effects of nosocomial or non-nosocomial infections. Examples of nosocomial uses include, but are not limited to, urinary tract infections, pneumonia, surgical wound infections, bacteremia and therapy for febrile neutropenic patients. Examples of non-nosocomial uses include but are not limited to urinary tract infections, pneumonia, prostatitis, skin and soft tissue infections and intra-abdominal infections.
In addition to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this invention may also be employed in compositions to treat or prevent the above-identified disorders.
A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitory active metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, IC50 and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant figures and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set in the examples, Tables and Figures are reported as precisely, as possible. Any numerical values may inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

EXAMPLE 1: PREPARATION OF PRODUCTION CULTURE

Unless otherwise noted, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO), (Aldrich). Micromonospora spp. (deposit accession number IDAC 070303-01) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, MI). An inoculum for the production phase was prepared by transferring the surface growth of the Micromonospora spp. from the agar plates to 125-mL
flasks containing 25 mL of sterile medium comprised of 24 g potato dextrin, 3 g beef extract, 5 g Bacto-casitone, 5 g glucose, 5 g yeast extract, and 4 g CaCO3 made up to one liter with distilled water (pH 7.0). The culture was incubated at about 28 C for approximately 60 hours on a rotary shaker set at 250 rpm. Following incubation, 10 mL of culture was transferred to a 2L baffled flask containing 500 mL of sterile production medium containing 20 g/L potato dextrin, 20 g/L glycerol, 10 g/L
Fish meal, 5 g/L Bacto-peptone, 2 g/L CaCO3, and 2 g/L (NH4)2SO4, pH 7Ø
Fermentation broth was prepared by incubating the production culture at 28 C
in a rotary shaker set at 250 rpm for one week.

EXAMPLE 2: ISOLATION

500 mL ethyl acetate was added to 500 mL of fermentation broth prepared as described in Example 1 above. The mixture was agitated for 30 minutes on an orbital shaker at 200 rpm to create an emulsion. The phases were separated by centrifugation and decantation. Between 4 and 5 g of anhydrous MgSO4 was added to the organic phase, which was then filtered and the solvents removed in vacuo.
An ethyl acetate extract from 2 L fermentation was mixed with HP-20 resin (100 mL; Mitsubishi Casei Corp., Tokyo, Japan) in water (300 mL). Ethyl acetate was removed in vacuo, the resin was filtered on a Buchner funnel and the filtrate was discarded. The adsorbed HP-20 resin was then washed successively with 2 x 125 mL of 50% acetonitrile in water, 2x125 mL of 75% acetonitrile in water and 2 x 125 mL of acetonitrile.
Fractions containing the compound of Formula II were evaporated to dryness and 100 mg was digested in the 5 mL of the upper phase of a mixture prepared from chloroform, cyclohexane, methanol, and water in the ratios, by volume, of 5:2:10:5.
The sample was subjected to centrifugal partition chromatography 'using a High Speed Countercurrent (HSCC) system (Kromaton Technologies, Angers, France) fitted with a 200 mL cartridge and prepacked with the upper phase of this two-phase system. The HSCC was run with the lower phase mobile and the compound of Formula II was eluted at approximately one-half column volume. Fractions were collected and the compound of Formula II was detected by TLC of aliquots of the fractions on commercial Kieselgel 60F254 plates. Compound could be visualized by inspection of dried plates under UV light or by spraying the plates with a spray containing vanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently heating the plate. Fractions contained substantially pure compound of Formula II, although highly colored. A buff-colored sample could be obtained by chromatography on HPLC as follows.
6 mg of sample was dissolved in acetonitrile and injected onto a preparative HPLC column (XTerra ODS (10 m), 19x150mm, Waters Co., Milford, MA), with a 9 mL/min flow rate and UV peak detection at 300 nm. The column was eluted with acetonitrile/buffer (20 mM of NH4HCO3) according to the following gradient shown in Table 1.

Table I
Time min Water (%) Acetonitrile (%) Fractions containing the compound of Formula II eluted at approximately 11.0 min and were combined, concentrated and lyophilized to give a yield of 3.8 mg compound.

Alternative Protocol 1 The compound of Formula II was also isolated using the following alternative protocol. At the end of the incubation period, the fermentation broth from the baffled flasks of Example 1 was centrifuged and the supernatant decanted from the pellet containing the bacterial mycelia. 100 mL of 100% MeOH was added to the mycelial pellet and the sample was stirred for 10 minutes and centrifuged for 15 minutes.
The methanolic supernatant was decanted and saved. 100 mL of acetone was then added to the mycelial pellet and stirred for 10 minutes then centrifuged for minutes. The acetonic supernatant was decanted and combined with the methanolic supernatant. Finally, 100 mL of 20% MeOH/H20 was added to the mycelial pellet, stirred for 10 minutes and centrifuged for 15 minutes. The supernatant was combined with the acetonic and methanolic supernatants.
The combined supernatant was added to 400 ml of HP-20 resin in 1000 mL of water and the organics were removed in vacuo. The resulting slurry was filtered on a Buchner funnel and the filtrate was discarded. Adsorbed HP-20 resin was washed successively with 2x500mL of 50% MeOH/H20, 2x500rnL of 75% MeOH/H20 and 2x500mL of MeOH.

The individual washes were collected separately and analyzed by TLC as described above. Those fractions containing the compound of Formula II were evaporated to near dryness and lyophilized. The lyophilizate was dissolved in methanol and injected onto a preparative HPLC column (Xterra ODS (10 m), 19x150mm, Waters Co., Milford, MA) with a flow rate of 9 mlJmin and peak detection at 300 nm.
The column was eluted with acetonitrile/buffer (5 mM of NH4HCO3) according to gradient shown in Table 2.

Table 2 Time min Buffer (%) Acetonitrile (%) Fractions containing the compound of Formula il were combined, concentrated and lyophilized to yield about 33.7 mg of compound.
Alternative Protocol 2 liters of the whole broth from Example 1 are extracted twice with equal volumes of ethyl acetate and the two extracts are combined and concentrated to dryness. The dried extract is weighed, and for every gram of dry extract, 100 mL of MeOH-H20 (2:1 v/v) and 100 mL of hexane is added. The mixture is swirled gently but well to achieve dissolution. The two layers are separated and the aqueous layer is washed with 100 mL of hexane. The two hexane layers are combined and the combined hexane solution is washed with 100 mL methanol:water (2:1, vlv). The two methanol:water layers are combined and treated with 200 mL of EtOAc and mL of water. The layers are separated and the aqueous layer is extracted twice more with 200 mL portions of EtOAc. The EtOAc layers are combined and concentrated. The residue obtained will be suitable for final purification, either by HSCC or by HPLC as described above. This extractiori process achieves a ten-fold purification when compared with the extraction protocol used above.

EXAMPLE 3: ELUCIDATION OF THE STRUCTURE OF COMPOUND OF
FORMULA H.

The structure of the compound of Formula II was derived from spectroscopic data, including mass, UV, and NMR spectroscopy. Mass was determined by electrospray mass spectrometry to be 462.6 (FIGURE 1), UVmax 230nm with a shoulder at 290 nm (FIGURE 2). NMR data were collected dissolved in MeOH-d4 including proton (FIGURE 3), and multidimensional pulse sequences gDQCOSY
(FIGURE 4), gHSQC (FIGURE 5), gHMBC (FIGURE 6), and NOESY (FIGURE 7).
A number of cross peaks in the 2D spectra of ECO-04601 are key in the structural determination. For example, the farnesyl chain is placed on the amide nitrogen by a strong cross peak between the proton signal of the terminal methylene of that chain at 4.52 ppm and the amide carbonyl carbon at 170 ppm in the gHMBC
experiment. This conclusion is confirmed by a cross peak in the NOESY spectrum between the same methylene signals at 4.52 ppm and the aromatic proton signal at 6.25 ppm from one of the two protons of the tetra substituted benzenoid ring.

Based on the mass, UV and NMR spectroscopy data, the structure of the compound was determined to be the structure of Formula II.

EXAMPLE 4: ANTIBACTERIAL ACTIVITY (MINIMAL INHIBITORY
CONCENTRATION DETERMINATION) Minimal Inhibitory Concentration (MIC) is defined as the lowest concentration of drug that inhibits more than 99% of the bacterial population. The MIC determination of ECO-04601 against bacteria strains (Bacillus subtilis - ATCC 23857;
Micrococcus luteus - ATCC 9341) was performed using broth microdilution assay (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;
Approved Standard-Fifth Edition. NCCLS document M7-A5 (ISBN 1-56238-394-9).
NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA.).

Test compound preparation: The test article ECO-04601 is prepared as 100X
stock solutions in DMSO, with concentrations ranging from 3.2 mg/mI to 0.0625 mg/ml (a two-fold dilution series over 10 points). The first dilution (3.2mg/ml) was prepared by resuspending 0.5 mg of each test article in 156.25 pl of DMSO. The stock is then serially diluted by two-fold decrement to obtain the desired concentration range.
Inoculum preparation: From an overnight culture in Mueller Hinton (MH) broth, cell density for each indicator strain (Bacillus subtilis; Micrococcus luteus) was adjusted to 0.5 Mc Farland units in 0.85% saline, then further diluted 1/100 in appropriate assay medium (- 1 X 106 cells/ml).

MIC determination: The 100X ECO-04601 solutions was diluted 50 times in MH
broth and dispensed in a 96 well plate, one test concentration per column of wells, columns in total. The 11 t" column of wells contained MH broth with 1% DMSO, the 12th column of wells contained 100 NI of broth alone. 50 pl of the final cell dilution of each indicator strain was added to each corresponding well of the microplate containing 50 pl of diluted drug or media alone. Assay plates were incubated at 35 C
for 24 hrs.

The results of the MIC for the compound of ECO-04601, shown in Table 3, demonstrate a range of antibacterial effects:

Table 3 Indicator strain MIC (pg/mL) Bacillus subtilis ATCC 23857 12.5 Micrococcus luteus ATCC 9341 6.25 EXAMPLE 5. ANTICANCER ACTIVITY IN VITRO AGAINST HUMAN AND ANIMAL
TUMOR CELL LINES FROM VARIOUS TISSUES

Culture conditions: The cell lines listed in Table 4 were used to characterize the cytotoxicity of ECO-04601 against human and animal tumor cell lines. These cell lines were shown to be free of mycoplasma infection and were maintained in the appropriate media (Table 4) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, under 5% CO2 at 37 C. Cells were passaged two to three times per week. Viability was examined by staining with 0.25%
trypan blue and only flasks where cell viability was >95% were used for this study.

Cell lines amplification and plating: Tumor cells were seeded (1-3 x 103 cells per 100 pL) in 96-well flat bottom microtiter plates and incubated at 37 C and 5% CO2 for 16 hrs before treatment in drug-free medium supplemented with 10% serum.
Evaluation of inhibitory activity on cell proliferation: Cells were incubated for 96 hrs with 6 loglo-fold concentrations of the test substance starting at 10pg/ml (20 pM).
The test substance stock solution (5 mg/mL) was initially diluted at 1/70 fold in medium supplemented with serum. Other concentrations were then obtained from 1/10 fold successive dilutions in the same supplemented medium. Cell survival was evaluated 96 h later by replacing the culture media with 150 pL fresh medium containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH
7.4.
Next, 50 pL of 2.5 mg/mL of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer solution, pH 7.4, was added. After 3-4h of incubation at 37 C, the medium and soluble MTT was removed, and 200 pL of dimethyisulfoxide was added to dissolve the precipitate of reduced MTT
followed by addition of 25 pL glycine buffer (0.1 M glycine plus 0.1 M NaCi, pH 10.5). The absorbance was determined at 570 nm with a microplate reader. Results were expressed as the concentration of drug which inhibits 50% of the cell growth (IC50).
The IC50 values shown in Table 4 demonstrated a pharmacologically relevant cytotoxic activity of ECO-04601 against a variety of tumor types such as leukemias, melanomas, pancreatic and breast carcinomas.

Table 4 Cell lines Type Origin Source Culture medium IC50 x10-6M
K562 Leukemia Human ATCC RPM11640 8.6 m elo eneous P388 Leukemia Mouse ATCC RPMI 1640 10.9 183 Leukemia Human ATCC RPMI '1640 2.7 B16 1710 Melanoma Mouse ATCC RPMI 1640 11.4 SK-MEL 28 Melanoma Human ATCC RPMI 1640 14.0 SK-MEL Melanoma Human ATCC RPMI1640 14.3 28VEGF (expressing VEGF) SK-MEL-1 Melanoma Human ATCC EMEM 1% non- 14.1 essential amino acid 1% Sodium u vate Panc 96 Pancreatic Human ATCC RPMI '1 % Sodium 12.5 carcinoma u vate Panc 10.05 Pancreatic Human ATCC RPMI 1% Sodium 14.2 carcinoma puryvate Insulin MCF-7 Breast Human ATCC RPMI 1640 9.7 adenocarcinoma EXAMPLE 6. ANTICANCER ACTIVITY IN VITRO AGAINST VARIOUS HUMAN
TUMOR CELL LINES FROM THE U.S. NATIONAL CANCER INSTITUTE PANEL
A study measuring the in vitro antitumor activity of ECO-04601 was performed by the National Cancer Institute (National Institutes of Health, Bethesda, Maryland, USA) against panel of human cancer cell lines in order to determine the ECO-04601 concentrations needed to obtain a 50% inhibition of cell proliferation (G150). The operation of this unique screen utilizes 50 different human tumor cell lines, representing leukemia, melanoma and cancers of the lu,ng, colon, brain, ovary, breast, prostate, and kidney.

Culture conditions and platinq:
The human tumor cell lines of the cancer-screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 pL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines (Table 5). After cell inoculation, the microtiter plates were incubated at 37 C, 5% C 2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz).

Evaluation of inhibitory activity on cell proliferation:
ECO-04601 was provided as a lyophilized powder with an estimated purity of 90+%. The compound was stored at -20 C until day of use. ECO-04601 was solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 501ag/mL gentamicin. Additional four, 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus control.
Aliquots of 100 Nl of these different drug dilutions were added to the appropriate microtiter wells already containing 100 Nl of medium, resulting in the required final drug concentrations (8.0 x 10-5 M to 8.0 x i 0-9 M).
Following drug addition, the plates were incubated for an additional 48 h at 37 C, 5% C02, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 pl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4 C. Supernatants were discarded, and the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (100 p 1) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air-dried.

Bound stain was subsequently solubilized with 10 mM trizmaTM base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 pl of 80% TCA (final concentration, 16% TCA).
The growth inhibitory activity of ECO-04601 was measured by NCI utilizing the G150 value, rather than the classical IC50 value. The G150 value emphasizes the correction for the cell count at time zero and, using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], G150 is calculated as [(Ti-Tz)/(C-Tz)] x 100 = -50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.

Result:
ECO-04601 shows a significant antitumor activity against several types of tumor as revealed by the NCI screening. Results of the screen are shown in Table 5, and more detailed results of activity against gliomas are shown in Example 7 (Table 6).

Table 5 Inoculation G150 Origin Density (x10'sM) Cell Line Name Type (number of cells/well) CCRF-CEM Leukemia Human 40,000 1.08 K-562 Leukemia Human 5,000 1.43 RPMI-8226 Leukemia Human 20,000 3.15 A549/ATCC Non-Small Cell Lung Human 7,500 9.10 EKVX Non-Small Cell Lung Human 20,000 0.23 HOP-62 Non-Small Cell Lung Human 10,000 8.29 NCI-H226 Non-Small Cell Lung Human 20,000 2.00 NCI-H23 Non-Small Cell Lung Human 20,000 2.02 NCI-H460 Non-Small Cell Lung Human 7,500 13.60 NCI-H522 Non-Small Cell Lung Human 20,000 3.44 COLO 205 Colon Human 15,000 12.70 HCT-116 Colon Human 5,000 2.92 HCT-15 Colon Human 10,000 9.73 HT29 Colon Human 5,000 20.70 SW-620 Colon Human 10,000 2.72 SF-268 CNS Human 15,000 4.94 SF-295 CNS Human 10,000 12.70 SF-539 CNS Human 15,000 0.0075 SNB-19 CNS Human 15,000 2.90 SNB-75 CNS Human 20,000 7.71 U251 CNS Human 7,500 2.19 LOX IMVI Melanoma Human 7,500 4.53 M14 Melanoma Human 15,000 4.57 SK-MEL-2 Melanoma Human 20,000 25.0 SK-MEL-28 Melanoma Human 10,000 11.6 SK-MEL-5 Melanoma Human 10,000 7.80 UACC-257 Melanoma Human 20,000 2.31 UACC-62 Melanoma Human 10,000 1.55 IGR-OV1 Ovarian Human 10,000 3.11 OVCAR-3 Ovarian Human 10,000 13.50 OVCAR-4 Ovarian Human 15,000 9.67 OVCAR-5 Ovarian Human 20,000 2.81 OVCAR-8 Ovarian Human 10,000 2.65 SK-OV-3 Ovarian Human 20,000 4.00 786-0 Renal Human 10,000 6.99 A498 Renal Human 25,000 22.30 ACHN Renal Human 10,000 3.10 CAKI-1 Renal Human 10,000 15.20 RXF 393 Renal Human 15,000 7.71 SN12C Renal Human 15,000 3.85 UO-31 Renal Human 15,000 19.70 DU-145 Prostate Human 10,000 3.56 MCF7 Breast Human 10,000 10.10 NCI/ADR-RES Breast Human 15,000 18.30 MDA-MB- Breast Human 20,000 2.72 HS 578T Breast Human 20,000 2.76 MDA-MB-435 Breast Human 15,000 15.30 BT-549 Breast Human 20,000 0.11 T-47D Breast Human 20,000 0.77 The results indicate that ECO-04601 was effective against most of the human tumor cell lines that have been assayed in the NCI screening panel suggesting a broad anticancer activity against several types of human cancer.

EXAMPLE 7: IN VITRO ANTIPROLIFERATIVE STUDY AGAINST A PANEL OF
GLIOMA CELL LINES

The anticancer activity of ECO-04601 was evaluated using a panel of glioma cancer cell lines shown in Table 6, and the 50% inhibition of cell proliferation (IC50) was determined.

Culture conditions:
The cell lines listed in Table 6 were shown to be free of mycoplasma infection and were maintained on DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, under 5% COZ at 37 C. Cells were passaged once a week. Prior to use the cells were detached from the culture flask by treating with trypsin for five to ten minutes. The cells were counted with a Neubauer glass slide and viability assessed by 0.25% trypan blue exclusion.
Only flasks with >95% cell viability, were used in the study.

Cell lines amplification andplating:
Cells, 5 x 103 cells per well in 100 pL drug-free medium supplemented with 10% serum, were plated in 96-well flat bottom microtiter plates and incubated at 37 C for 48 hrs before treatment.

Evaluation of inhibito activity on cell proliferation:
Cells (in triplicate wells) were incubated 96 hrs with medium containing different concentrations of ECO-04601, starting at 5.0 ug/ml (10 pM). The compound was used in a solution of 1% DMSO in D-MEM or RPMI media (or other equivalent media). The concentrations of ECO-04601 were as follows: 10 pM (5.0 pg/ml), 1 pM
(0.50 pg/ml), 0.5 pM (0.25 pg/mI), 0.1 pM (0.050 pg/mi), 0.5 pM (0.025 pg/mi), 0.01 PM (0.0050 ug/ml), 0.001 pM (0.00050 Ng/ml). Negative controls were cells treated with vehicle alone (1% DMSO in culture medium). Positive controls were cells treated with 4 to 6 increasing concentrations of cisplatin (CDDP) (data not shown).
The optical density was measured before incubation (time 0) and following 96 hrs of incubation with test compound in order to measure the growth rate of each cell line.
At the end of the cell treatment, cell culture media was replaced with 150 pl of fresh medium containing 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Then 50 NI of 2.5 mg/mI of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide in PBS pH 7.4, were added to each well and the culture plates incubated for 4 hrs at 37 C. The resulting supernatant was removed and formazan crystals were dissolved with 200 NI of DMSO followed by 25 ial of glycine buffer (0.1 M glycine plus 0.1 M NaCI, pH 10.5). The optical density was read in each well using a single wavelength spectrophotometer piate reader at 570 nm.
Results were expressed as the concentration of drug, which inhibits 50% of the cell growth (IC50). Each of the cell lines was tested in at least 3 independent experiments.
Results shown in Table 6 confirmed the activity of ECO-04601 against different brain cancer cell lines including gliosarcoma, which is the most malignant form of type IV glioblastoma multiform. Gliosarcomas are a mixture of glial and endothelial cells and are resistant to any chemotherapy.

Table 6 Cell lines Type Origin Source IC50 (x10-6M) 9L Gliosarcoma Rat ATCC 6.82 2.90 GHD Astrocytoma Human ATCC 6.29 2.98 U 373 Astrocytoma Human ATCC 3.83 1.37 GL26 Glioblastoma Human ATCC 8.93 1.10 C6 Glioblastoma Rat ATCC 4.28 2.82 Oligodendrogliom ATCC
DN a Human 3.26 0.93 Oligodendrogliom ATCC
GHA a Human 1.78 0.84 EXAMPLE 8: EFFECT ON THE ENZYMATIC ACTIVITY OF HUMAN
LIPOXYGENASE (5-LO) 5-Lipoxygenase catalyzes the oxidative metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE), the initial reaction leading to formation of leukotrienes. Eicosanoids derived from arachidonic acid by the action of lipoxygenases or cycloxygenases have been found to be involved in acute and chronic inflammatory diseases (i.e. asthma, multiple sclerosis, rheumatoid arthritis, ischemia, edema) as well in neurodegeneration (Alzheimer disease), aging and various steps of carcinogenesis, including tumor promotion, progression and metastasis.
The aim of this study was to determine whether ECO-04601, is able to block the formation of leukotrienes by inhibiting the enzymatic activity of human 5-LO.
Methods employed are based on Carter et al (1991) J. Pharmacol. Exp. Ther.
256(3):929-937, and Safayhi (2000), Planta Medica 66:110-113.

Experimental Design:
Human peripheral blood mononuclear cells (PMNs) were isolated through a Ficoll-Paque density gradient. PMNs were stimulated by addition A23187 (30 pM
final concentration). Stimulated PMNs were adjusted to a density of 5 x106 cells/mL
in HBBS medium and incubated with the vehicle control (DMSO), ECO-04601 (at final concentrations of 0.1, 0.5, 1, 2.5, 5 and 10 pM) and NDGA as positive control (at final concentrations of 3, 1, 0.3, 0.1 and 0.03 pM) for 15 minutes at 37 C.
Following incubation, samples were neutralized with NaOH and centrifuged.
Leukotriene B4 content was measured in the supernatant using an Enzyme Immunosorbant Assay (EIA) assay.

Results:
Results shown in Figure 8 demonstrated that ECO-04601 inhibited the activity of human 5-LO with an apparent EC50 = 0.93 pM (versus 0.1 pM for the positive control NDGA) and therefore displays anti-inflammatory properties.

EXAMPLE 9: IN VIVO EFFICACY IN A GLIOMA MODEL

The aim of this study was to test whether ECO-04601 administered by i.p.
route prevents or delays tumor growth in C6 glioblastoma cell-bearing mice, and to determine an effective dosage regimen.

Animals:
A total of 60 six-week-old female mice (Mus musculus nude mice), ranging between 18 to 25 g in weight, were observed for 7 days before treatment.
Animal experiments were performed according to ethical guidelines of animal experimentation (Charte du comitd d'ethique du CNRS, ,juillet 2003) and the English guidelines for the welfare of animals in experimental neoplasia (WORKMAN, P., TWENTYMAN, P., BALKWILL, F., et a!. (1998). United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the welfare of animals in experimental neoplasia (Second Edition, July 1997; British Journal of Cancer 77.= 1-10). Any dead or apparently sick mice were promptly removed and replaced with healthy mice. Sick mice were euthanized upon removal from the cage. Animals were maintained in rooms under controlled conditions of temperature (23t2 C), humidity (45 5%), photoperiodicity (12 hrs light / 12 hrs dark) and air exchange.
Animals were housed in polycarbonate cages (5/single cage) that were equipped to provide food and water. Animal bedding consisted of sterile wood shavings that were replaced every other day. Food was provided ad libitum, being placed in the metal lid on the top of the cage. Autoclaved tap water was provided ad libitum. Water bottles were equipped with rubber stoppers and sipper tubes. Water bottles were cleaned, sterilized and replaced once a week. Two different numbers engraved on two earrings identified the animals. Each cage was labelled with a specific code.

Tumor Cell Line:
The C6 cell line was cloned from a rat glial tumor induced by N-nitrosomethyurea (NMU) by Premont et al. (Premont J, Benda P, Jard S., [3H]
norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation. Biochim Biophys Acta. 1975 Feb 13;381(2):368-76.) after series of alternate cuiture and animal passages.
Cells were grown as adherent monolayers at 37 C in a humidified atmosphere (5% C02, 95% air). The culture medium was DMEM supplemented with 2 mM L-glutamine and 10% fetal bovine serum. For experimental use, tumor cells were detached from the culture flask by a 10 min treatment with trypsin-versen. The cells were counted in a hemocytometer and their viability assessed by 0.25%
trypan blue exclusion.

Preparation of the Test Article:
For the test article, the following procedure was followed for reconstitution (performed immediately preceding injection). The vehicle consisted of a mixture of benzyl alcohol (1.5%), ethanol (8.5%), propylene glycol (27%), PEG 400 (27%), dimethylacetamide (6%) and water (30%). The vehicle solution was first vortexed in order to obtain a homogeneous liquid. 0.6 mL of the vortexed vehicle solution was added to each vial containing the test article (ECO-04601). Vials were mixed thoroughly by vortexing for 1 minute and inverted and shaken vigorously. Vials were mixed again prior to injection into each animal.

Animal Inoculation with tumor cells:
Experiment started at day 0 (Do). On Do, mice received a superficial intramuscular injection of C6 tumor cells (5 x 105 cells) in 0.1 mL of DMEM
complete medium into the upper right posterior leg.

Treatment regimen and Results:
In a first series of experiments, treatment started 24 hrs following inoculation of C6 cells. On the day of the treatment, each mouse was slowly injected with pL of test or control articles by i.p. route. For all groups, treatment was performed until the tumor volume of the saline-treated mice (group 1) reached approximately 3 cm3 (around day 16). Mice of group 1 were treated daily with a saline isosmotic solution for 16 days. Mice of group 2 were treated daily with the vehicle solution for 16 days. Mice of group 3 were treated daily with 10 mg/kg of ECO-04601 for 16 days. Mice of group 4 were treated every two days with 30 mg/kg of ECO-04601 and received 8 treatments. Mice of group 5 were treated every three days with 30 mg/kg of ECO-04601 and received 6 treatments. Measurement of tumor volume started as soon as tumors became palpable (>100 mm3; around day 11 post-inoculation) and was evaluated every second day until the end of the treatment using callipers.
As shown in Table 7 and Figure 9, the mean value of the tumor volume of all ECO-04601 treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 7 indicates a statistically significant value, while "ns" signifies not significant.

Table 7 Treatment Treatment Tumor volume % P
regimen (mm) Inhibitio value (mean SEM) n Saline Q1 x 16 3,004.1 - -249.64 Vehicle Q1 x 16 2,162.0 350.0 28.0% >0.05 solution ns ECO-04601 01 x 16 1,220.4 59.4% <0.01 (10 mg/kg) 283.46 *
ECO-04601 Q2 x 8 1,236.9 58.8% <0.01 (30 mg/kg) 233.99 *
ECO-04601 Q3 x 6 1,184.1 60.6% <0.01 (30 mg/kg) 221.45 *

In a second series of experiments, treatment started at day 10 following inoculation of C6 cells when tumors became palpable (around 100 to 200 mm).
Treatment was repeated daily for 5 consecutive days. On the day of the treatment, each mouse was slowly injected with 100 pL of ECO-04601 by i.p. route. Mice of group 1 were treated daily with saline isosmotic solution. Mice of group 2 were treated daily with the vehicle solution. Mice of group 3 were treated daily with 20 mg/kg of ECO-04601. Mice of group 4 were treated daily with 30 mg/kg of ECO-04601. Mice were treated until the tumor volume of the saline-treated control mice (group 1) reached around 4 cm3. Tumor volume was measured every second day until the end of the treatment using callipers. As shown in Table 8 and Figure 10, the mean vaiue of the tumor volume of all ECO-04601 treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 8 indicates a statistically significant value, while "ns" signifies not statistically significant.

Histological analysis of tumor sections showed pronounced morphological changes between ECO-04601-treated tumors and control groups. In tumors treated with ECO-04601 (20 - 30 mg/kg), cell density was decreased and the nuclei of remaining tumor cells appeared larger and pycnotic while no such changes were observed for vehicle-treated mice (Figure 11).

Table 8 Treatment Treatment Tumor volume % P
regimen (mm) lnhibitio value (mean SEM) n Saline Q1 x5 4,363.1 - -614.31 Vehicle solution Q1 x 5 3,205.0 26.5% >0.05 632.37 ns ECO-04601 Q1 x 5 1,721.5 60.5% <0.01 (20 mg/kg) 374.79 *
ECO-04601 Q 1 x 5 1,131.6 74.1% <0.01 (30 mg/kg) 525.21 *

EXAMPLE 10: GENERATION OF VARIANTS OF ECO-04601 ACCORDING TO
THE INVENTION

Variants of the ECO-04601 molecule, for example those identified herein as Formulae III-LIX, can be generated by standard organic chemistry approaches.
General principles of organic chemistry required for making and manipulating the compounds described herein, including functional moieties, reactivity and common protocols are described, for example, in "Advanced Organic Chemistry," 3rd Edition by Jerry March (1985). In addition, it will be appreciated by one of ordinary skill in the art that the synthetic methods described herein may use a variety of protecting groups, whether or not they are explicitly described. A "protecting group" as used herein means a moiety used to block one or more functional moieties such as reactive groups including oxygen, sulfur or nitrogen, so that a reaction can be carried out selectively at another reactive site in a polyfunctional compound. General principles for the use of protective groups, their applicability to specific functional groups and their uses are described for example in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York (1999).

Scheme 1: Epoxide variants The epoxide compounds of the present invention (e.g., compounds according to exemplary Formulae VII-XIV) are made from the compound of Formula II (ECO-04601) by treatment with any of a number of epoxidizing reagents such as perbenzoic acid, monoperphthalic acid or more preferably by m-chloroperbenzoic acid in an inert solvent such as tetrahydrofuran (THF) dichloromethane or 1,2-dichloroethane. It will be appreciated by one of ordinary skill in the art that slightly greater than one molecule equivalent of epoxidizing agent will result in the maximal yield of mono-epoxides, and that the reagent, solvent, concentration and temperature of the reaction will dictate the ratio of specific mono-epoxides formed. It will also be appreciated that the mono-epoxides will be enantiomeric mixtures, and that the di-epoxides and the tri-epoxide can be prepared as diastereomers and that the conditions of the reaction will determine the ratios of the products. One skilled in the art will appreciate that under most conditions of reactions the product will be a mixture of all possible epoxides and that these may be separated by standard methods of chromatography. Exemplary approaches to the generation of mono-, di-and tri-epoxides are provided below.
A) Mono-epoxides of the Formulae Vil, V(ll, and IX by epoxidation of the compound of Formula II:

Formula VII Formula VIII

0 CH3 CH3 H3 H~ JH, H3 N /
N / . / CH3 0 PN-HO H OH HO H OH
N-HO HO

HO H / \ OH Formula IX
HO

To a solution of the compound of Formula II dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexTM LH-20 to isolate a mixture of predominantly the compounds of Formulae VII, VIII and IX, contaminated with some unchanged starting material and some di- and tri- epoxides. The compounds of Formulae VII, VIII and XIX are separated and purified by HPLC using the system described in Example 2 for the purification of the compound of Formulae II. In a typical experiment yields of 15% to 25% are obtained for each of the compounds of Formulae VII, VIII and IX.

B) Synthesis of Compounds of Formulae X, XI, and XII by di-epoxidation of Compound of Formula II:
Formula X Formula XI
0 H3 CH3 CH~ H3 CH3 CH, N 0 CH' O O
N
HO H OH
HO I / \ OH H
H
HO HO

HO i Formula XII
H OH
HO

To a solution of the compound of Formula II dissolved in tetrahydrofuran (THF) is added 2.3 equivalents of ineta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate a mixture of predominantly the compounds of Formulae X, XI and XII, contaminated with traces of unchanged starting material and some mono- and tri- epoxides. The Compounds of Formulae X, XI and XII are separated and purified by HPLC using the system described in Example 2 for the purification of the compound of Formulae II. In a typical experiment, yields of 15% to 20% are obtained for each of the compounds of Formulae X, XI and XII.

C) Synthesis of Compound of Formula XIII by tri-epoxidation of Compound of Formula II:
0 CH3 CHg CHs i ~

H H' H Formula XIII
HO

To a solution of the compound of Formula II, dissolved in tetrahydrofuran (THF), is added 3.5 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate the compound of Formula XIII as a mixture of diasteriomers in a yield of 80+%.

Scheme 2: Synthesis of Compound of Formula III by N-acetylation of Compound of Formula II.
0 CH, CH, CH
N CHs Formula III
HO
; b 0=0 OH

To a solution of Compound of Formula II dissolved in tetrahydrofuran (THF) is added 1.2 equivalents of acetic anhydride and a few drops of triethylamine.
The reaction mixture allowed to stand at room temperature for 1-2 hours and then evaporated to dryness under reduced pressure to obtain the Compound of Formula III in an essentially pure form in an almost quantitative yield.

Scheme 3: Syntheses of Compounds of Formulae IV and V by N-alkylation of Compound of Formula II.

O H, CH, CH3 N~ CHs Formula IV R = benzyl Formula V R= ethyl ~t/ )-Ho HO OH 17 R To a solution of Compound of Formula II dissolved in terachloroethylene is added 1.2 equivalents of the appropriate alkyl bromide (benzyl bromide for the compound of formula IV or ethyl bromide for the Compound of Formula V). The reaction mixture the reaction mixture is heated under reflux for 1-2 hours and then evaporated to dryness under reduced pressure to obtain the Compound of Formula IV or the Compound of Formula V respectively, in an essentially pure form in an almost quantitative yield.

Scheme 4: Syntheses of Compounds of Formulae XL, XLI and XLII by catalytic reduction of Compound of Formula II.

0 CH3 CH3 CH0 CH3 CHs H, qNCH3 CHg Ho NFormula XL HoFormula XLI
H OH H OH
HO HO
O CH3 H3 Ha / ~

HO / / \ OH Formula XLII
H ~
HO

A solution of the Compound of Formula 11 (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where one millimole of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of the Compounds of Formulae XL, XLI and XLII
contaminated by unreacted starting material and minor amounts of over reduced products. The desired products may be separated and purified by HPLC or HSCC
chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of the Compounds of Formulae XL, XLI and XLII.
Scheme 5: Syntheses of Compounds of Formulae XLIII, XLIV and XLV by catalytic reduction of Compound of Formula II.

O CH3 CH3 CH3 O H3 CH, CH3 ~ CHg PNt' ~ CHs Formula XLIV
Ho Formula XLIII HO OH
H
H OH
H
HO

0 CHs H3 CH, i ~
N CHy HO ~ / 1 Formula XLV
H ~ OH
HO

A solution of the Compound of Formula 11 (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where two millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of the Compounds of Formulae XLIII, XLIV and XLV
contaminated by trace amounts unreacted starting material and minor amounts of under and over reduced products. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of the Compounds of Formulae XLIII, XLIV and XLV.

Scheme 6: Syntheses of Compound of Formula XLVI by catalytic reduction of Compound of Formula II.

/

HO ; ~ \ oH Formula XLVI
H ~
HO

A solution of the Compound of Formula II (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where three millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain an essentially pure sample of the Compound of Formula XLVI.

Scheme 7: Syntheses of Compound of Formula VI by peracetylation of Compound of Formula II.

0 H, H, H, a qNt' N / / CH
C \ O~cH Formula VI
H,C 0 O
CH
O

A solution of the Compound of Formula II (100 mg) in acetic anhydride (5 ml) is treated with pyridine (250 uI). The reaction mixture is allowed to stand overnight at room temperature and is then diluted with toluene (100 ml). The toluene solution is washed well with aqueous 5% sodium bicarbonate solutions, then with water and is finally concentrated under reduced pressure to give an essentially pure sample of the Compound of Formula VI in almost quantitative yield.

Scheme 8: Syntheses of Compound of Formula LI by opening the epoxide of Compound of Formula VII.

O H~ H H3 OH OH
N i \ Formula LI
HO
H OH
HO

A solution of the Compound of Formula VII (100 mg) in tetrahydrofuran (50 ml) is treated with 1 N aqueous hydrochloric acid (5 ml). The reaction mixture is stirred overnight at room temperature and is then diluted with toluene (100 ml) and water (200 ml). The toluene layer is separated and the aqueous layer is extracted with a further 100 ml of toluene. The combined toluene layers are washed once more with water (50 ml) and the separated and dried under vacuum to give the vicinal glycol Compound of Formula LI.

Scheme 9: Syntheses of Compounds of Formulae XLVII, XLIX and LI by ozonolysis of Compound of Formula II.

0 H, 0 CH, CH, 0 1-11 N

N o ~ i \ Formula XLIX
i\ Formula XLVII H_ OH
HO OH HO
H ~
HO

/
Formula LI
HO OH
H
HO

A solution of the Compound of Formula II (462 mg) in dry ethyl acetate (200 ml) in an ozonolysis apparatus is cooled to below -20 C. A stream of ozone-containing oxygen is passed into the solution from an ozone generator, which has been precalibrated such that the rate of ozone generation is known. To obtain predominantly the compound of Formula XLVII the passage of ozone is halted after 0.9 millimole have been generated. To obtain predominantly the compound of Formula XLIX the ozone passage is halted after 2 millimoles have been generated and to obtain the compound of Formula LI as the predominant product 3.3 millimoles of ozone are generated.
At the completion of the ozonolysis, the reaction mixture is transferred to an hydrogenation apparatus, 5% palladium on calcium carbonate catalyst (0.2 g) is ,..~ . .~,_ ~_~b~..:~~~... ..~:, .~......,~... .~ .~.._.~..a,.y added to the reaction mixture which is maintained at less than -20 C and is hydrogenated. When hydrogen uptake is complete the hydrogen atmosphere is replaced with nitrogen and the reaction mixture is allowed to come to room temperature, filtered to remove catalyst and the filtrate is concentrated. The crude product may be purified by chromatography using either HPLC or HSCC with the systems as described in Example 2 to give, dependent on the amount of ozone used, Compounds of Formulae XLVII, XLIX and LI.

Scheme 10: Synthesis of Compound of Formulae XLVIII by reduction of the aidehyde of Compound of Formula XLVII.

0 CH, CH3 OH
N
Formula XLVIII
HO
H OH
HO

A solution of the Compound of Formula XLVIII (50 mg) in isopropanol (5 ml) is cooled in an ice-salt bath and sodium borohydride (10 mg) is added and the mixture is stirred for 20 minutes. It is then diluted with water (20 ml) and extracted twice with toluene (10 ml portions) at ambient temperature. The combined toluene extracts are filtered and the filtrate is concentrated to give the Compound of Formula XLVII.

Scheme 11: Syntheses of Compounds of Formulae XIV and XV by epoxidation of the Compound of Formula XLII.

Ho Formula XIV HO Formula XV
OH H OH
H
HO HO

To a solution of Compound of Formula XLII dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate a mixture of predominantly the Compounds of Formulae XIV, and XV, contaminated with some unchanged starting material and some diepoxide. The Compounds of Formulae XIV
and XV are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of the Compound of Formulae II. In a typical experiment yields of 35% to 40% are obtained for each of the Compounds of Formulae XIV and XV.

Scheme 12: Synthesis of Compound of Formulae XIX by epoxidation of the Compound of Formula XL.

CH, CH3 CH, ~ 0 0 HO ; / \ Formula XIX
H OH
HO

To a solution of Compound of Formula XL dissolved in tetrahydrofuran (THF) is added 2.2 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate essentially pure Compound of Formulae XIX in good yield.

Scheme 13: Syntheses of Compounds of Formulae XXVI, XXVII and XXVIII by esterification of the Compound of Formula II.
H3 Ha 0 CH3 CH, H, N CH, / CH3 o N ~\ Formula XXVI Ho H ~ Formula XXVII

HaC HO 0 H
HO Formula XXVIII

N
HO
To a solution of Compound of Formula II dissolved in toluene (9 parts) tetrahydrofuran (1 part), cooled in an ice-bath is added 1.1 equivalents of acetic anhydride and two drops of boron trifluoride etherate. The reaction is maintained cool in an ice bath and stirred at 0 C for 1-2 hours. The reaction mixture is then poured into aqueous 5% sodium bicarbonate solution shaken and the toluene layer is removed. The aqueous layer is re-extracted with toluene and the combined toluene layers are concentrated to a mixture of predominantly the Compounds of Formulae XXVI, XXVII and XXVIII, contaminated with some unchanged starting material and some diacetates. The Compounds of Formulae XXVI, XXVII and XXVIII are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of the Compound of Formulae II. In a typical experiment yields of 25% to 30% are obtained for each of the Compounds of Formulae XXVI, XXVII and XXVIII.

Scheme 14: Syntheses of Compounds of Formulae XXXIII, XXXIV and XXXV by methylation of the Compound of Formula II.

0 CH3 CH3 CH3 O H, CH3 CH, CH3 N ~ / / CH3 Formula )CXXIII Formula XXXIV
o HO
HaC- H OH H OH

~ / / /

Formula XXXV
HO O-CH, H
HO
A solution of the Compound of Formula II (1 g) in tetrahydrofuran 50 (ml) is titrated with exactly one equivalent of sodium methoxide, allowed to stand for minutes at room temperature and then treated with 1.2 equivalents of dimethylsulphate. Heat the mixture under reflux for one hour, cool to room temperature and pour into a mixture of toluene (200 ml) and water (200 ml).
The layers are separated and the aqueous layer is extracted once more with an equal portion of toluene. The combined toluene layers are washed once with 1 N
aqueous acetic acid and then concentrated to s crude product, which is predominantly a mixture of the Compounds of Formulae )cXXIII, XXXIV and XXXV with some unchanged starting material and traces of over-methylated derivatives. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtairi approximately 200 mg of each of the Compounds of Formulae XXXIII, XXXIV and XXXV.

EXAMPLE 11: GENES AND PROTEINS FOR THE PRODUCTION OF
COMPOUNDS OF FORMULA

Micromonospora sp. strain 046-ECO11 is a representative microorganism useful in the production of the compound of the invention. Strain 046-ECO11 has been deposited with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 on March 7, 2003 and was assigned IDAC accession no. 070303-01. The biosynthetic locus for the production of the compound of Formula II
was identified in the genome of Micromonospora sp. strain 046-ECO11 using the genome scanning method described in USSN 10/232,370, CA 2,352,451 and Zazopoulos et. al., Nature Biotechnol., 21, 187-190 (2003).
The biosynthetic locus spans approximately 52,400 base pairs of DNA and encodes 43 proteins. More than 10 kilobases of DNA sequence were analyzed on each side of the locus and these regions were deemed to contain primary genes or genes unrelated to the synthesis of the compound of Formula II. As illustrated in FIGURE 12, the locus is contained within three sequences of contiguous base pairs, namely Contig 1 having the 36,602 contiguous base pairs of SEQ ID NO: 1 and comprising ORFs 1 to 31 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63), Contig 2 having the 5,960 contiguous base pairs of SEQ ID NO: 64 and comprising ORFs 32 to 35 (SEQ ID NOS: 66, 68, 70 and 72), and Contig 3 having the 9,762 base pairs of SEQ ID NO: 73 and comprising ORFs 36 to 43 (SEQ ID NOS: 75, 77, 79, 81, 83, 85, 87 and 89). The order, relative position and orientation of the 43 open reading frames representing the proteins of the biosynthetic locus are illustrated schematically in FIGURE 12. The top line in FIGURE 12 provides a scale in base pairs. The gray bars depict the three DNA contigs (SEQ ID NOS: 1, 64 and 73) that cover the locus. The empty arrows represent the 43 open reading frames of this biosynthetic locus. The black arrows represent the two deposited cosmid clones covering the locus.

The biosynthetic locus will be further understood with reference to the sequence listing which provides contiguous nucleotide sequences and deduced amino acid sequences of the locus from Micromonospora sp. strain 046-ECO11.
The contiguous nucleotide sequences are arranged such that, as found within the biosynthetic locus, Contig 1(SEQ ID NO: 1) is adjacent to the 5' end of Contig (SEQ ID NO: 64), which in turn is adjacent to Contig 3 (SEQ ID NO: 73). The ORFs illustrated in FIGURE 12 and provided in the sequence listing represent open reading frames deduced from the nucleotide sequences of Contigs 1, 2 and 3 (SEQ
ID NOS: 1, 64 and 73). Referring to the Sequence Listing, ORF 1 (SEQ ID NO: 3) is the polynucleotide drawn from residues 2139 to 424 of SEQ ID NO: 1, and SEQ ID
NO: 2 represents that polypeptide deduced from SEQ ID NO: 3. ORF 2(SEQ ID
NO: 5) is the polynucleotide drawn from residues 2890 to 4959 of SEQ ID NO: 1, and SEQ ID NO: 4 represents the polypeptide deduced from SEQ ID NO: 5. ORF 3 (SEQ ID NO: 7) is the polynucleotide drawn from residues 7701 to 5014 of SEQ
ID
NO: 1, and SEQ ID NO: 6 represents the polypeptide deduced from SEQ ID NO: 7.
ORF 4 (SEQ ID NO: 9) is the polynucleotide drawn from residues 8104 to 9192 of SEQ ID NO: 1, and SEQ ID NO: 8 represents the polypeptide deduced from SEQ ID
NO: 9. ORF 5 (SEQ ID NO: 11) is the polynucleotide drawn from residues 9192 to 10256 of SEQ ID NO: 1, and SEQ ID NO: 10 represents the polypeptide deduced from SEQ ID NO: 11. ORF 6(SEQ ID NO: 13) is the polynucleotide drawn from residues 10246 to 11286 of SEQ ID NO: 1, and SEQ ID NO: 12 represents the polypeptide deduced from SEQ ID NO: 13. ORF 7 (SEQ ID NO: 15) is the polynucleotide drawn from residues 11283 to 12392 of SEQ ID NO: 1, and SEQ ID
NO: 14 represents the polypeptide deduced from SEQ iD NO: 15. ORF 8 (SEQ ID
NO: 17) is the polynucleotide drawn from residues 12389 to 13471 of SEQ ID NO:
1, and SEQ ID NO: 16 represents the polypeptide deduced from SEQ ID NO: 17. ORF
9 (SEQ ID NO: 19) is the polynucleotide drawn from residues 13468 to 14523 of SEQ ID NO: 1, and SEQ ID NO: 18 represents the polypeptide deduced from SEQ
ID NO: 19. ORF 10 (SEQ ID NO: 21) is the polynucleotide drawn from residues 14526 to 15701 of SEQ ID NO: 1, and SEQ ID NO: 20 represents the polypeptide deduced from SEQ ID NO: 21. ORF 11 (SEQ ID NO: 23) is the polynucleotide drawn from residues 15770 to 16642 of SEQ ID NO: 1, and SEQ ID NO: 22 represents the polypeptide deduced from SEQ ID NO: 23. ORF 12 (SEQ ID NO: 25) is the polynucleotide drawn from residues 16756 to 17868 of SEQ ID NO: 1, and SEQ ID NO: 24 represents the polypeptide deduced from SEQ ID NO: 25. ORF 13 (SEQ ID NO: 27) is the polynucleotide drawn from residues 17865 to 18527 of SEQ
ID NO: 1, and SEQ ID NO: 26 represents the polypeptide deduced from SEQ ID NO:
27. ORF 14 (SEQ ID NO: 29) is the polynucleotide drawn from residues 18724 to 19119 of SEQ ID NO: 1, and SEQ ID NO: 28 represents the polypeptide deduced from SEQ ID NO: 29. ORF 15 (SEQ ID NO: 31) is the poiynucleotide drawn from residues 19175 to 19639 of SEQ ID NO: 1, and SEQ ID NO: 30 represents the polypeptide deduced from SEQ ID NO: 31. ORF 16 (SEQ ID NO: 33) is the polynucleotide drawn from residues 19636 to 21621 of SEQ ID NO: 1, and SEQ ID
NO: 32 represents the polypeptide deduced from SEQ ID NO: 33. ORF 17 (SEQ ID
NO: 35) is the polynucleotide drawn from residues 21632 to 22021 of SEQ ID NO:
1, and SEQ ID NO: 34 represents the polypeptide deduced from SEQ ID NO: 35. ORF
18 (SEQ ID NO: 37) is the polynucleotide drawn from residues 22658 to 22122 of SEQ ID NO: 1, and SEQ ID NO: 36 represents the polypeptide deduced from SEQ
ID NO: 37. ORF 19 (SEQ ID NO: 39) is the polynucleotide drawn from residues 24665 to 22680 of SEQ ID NO: 1, and SEQ ID NO: 38 represents the polypeptide deduced from SEQ ID NO: 39. ORF 20 (SEQ ID NO: 41) is the polynucleotide drawn from residues 24880 to 26163 of SEQ ID NO: 1, and SEQ ID NO: 40 represents the polypeptide deduced from SEQ ID NO: 41. ORF 21 (SEQ ID NO: 43) is the polynucleotide drawn from residues 26179 to 27003 of SEQ ID NO: 1, and SEQ ID NO: 42 represents the polypeptide deduced from SEQ ID NO: 43. ORF 22 (SEQ ID NO: 45) is the polynucleotide drawn from residues 27035 to 28138 of SEQ
ID NO: 1, and SEQ ID NO: 44 represents the polypeptide deduced from SEQ ID NO:
45. ORF 23 (SEQ ID NO: 47) is the polynucleotide drawn from residues 28164 to 28925 of SEQ ID NO: 1, and SEQ ID -NO: 46 represents the polypeptide deduced from SEQ ID NO: 47. ORF 24 (SEQ ID NO: 49) is the polynucleotide drawn from residues 28922 to 30238 of SEQ ID NO: 1, and SEQ ID NO: 48 represents the polypeptide deduced from SEQ ID NO: 49. ORF 25 (SEQ ID NO: 51) is the polynucleotide drawn from residues 30249 to 31439 of SEQ ID NO: 1, and SEQ ID
NO: 50 represents the polypeptide deduced from SEQ ID NO: 51. ORF 26 (SEQ iD
NO: 53) is the polynucleotide drawn from residues 31439 to 32224 of SEQ ID NO:
1, and SEQ ID NO: 52 represents the polypeptide deduced from SEQ ID NO: 53. ORF

27 (SEQ ID NO: 55) is the polynucleotide drawn from residues 32257 to 32931 of SEQ ID NO: 1, and SEQ ID NO: 54 represents the polypeptide deduced from SEQ
ID NO: 55. ORF 28 (SEQ ID NO: 57) is the polynucleotide drawn from residues 32943 to 33644 of SEQ ID NO: 1; and SEQ ID NO: 56 represents the polypeptide deduced from SEQ ID NO: 57. ORF 29 (SEQ ID NO: 59) is the polynucleotide drawn from residues 34377 to 33637 of SEQ ID NO: 1, and SEQ ID NO: 58 represents the polypeptide deduced from SEQ ID NO: 59. ORF 30 (SEQ ID NO: 61) is the polynucleotide drawn from residues 34572 to 34907 of SEQ ID NO: 1, and SEQ ID NO: 60 represents the polypeptide deduced from SEQ ID NO: 61. ORF 31 (SEQ ID NO: 63) is the polynucleotide drawn from residues 34904 to 36583 of SEQ
ID NO: 1, and SEQ ID NO: 62 represents the polypeptide deduced from SEQ ID NO:
63. ORF 32 (SEQ ID NO: 66) is the polynucleotide drawn from residues 23 to of SEQ ID NO: 64, and SEQ ID NO: 65 represents the polypeptide deduced from SEQ ID NO: 66. ORF 33 (SEQ ID NO: 68) is the polynucleotide drawn from residues 1702 to 2973 of SEQ ID NO: 64, and SEQ ID NO: 67 represents the polypeptide deduced from SEQ ID NO: 68. ORF 34 (SEQ ID NO: 70) is the polynucleotide drawn from residues 3248 to 4270 of SEQ ID NO: 64, and SEQ ID
NO: 69 represents the polypeptide deduced from SEQ ID NO: 70. ORF 35 (SEQ ID
NO: 72) is the polynucleotide drawn from residues 4452 to 5933 of SEQ ID NO:
64, and SEQ ID NO: 71 represents the polypeptide deduced from SEQ ID NO: 72. ORF
36 (SEQ ID NO: 75) is the polynucleotide drawn from residues 30 to 398 of SEQ
ID
NO: 73, and SEQ ID NO: 74 represents the polypeptide deduced from SEQ ID NO:
75. ORF 37 (SEQ ID NO: 77) is the polynucleotide drawn from residues 395 to of SEQ ID NO: 73, and SEQ ID NO: 76 represents the polypeptide deduced from SEQ ID NO: 77. ORF 38 (SEQ ID NO: 79) is the polynucleotide drawn from residues 3388 to 1397 of SEQ ID NO: 73, and SEQ ID NO: 78 represents the polypeptide deduced from SEQ ID NO: 79. ORF 39 (SEO ID NO: 81) is the polynucleotide drawn from residues 3565 to 5286 of SEQ ID NO: 73, and SEQ ID NO: 80 represents the polypeptide deduced from SEQ ID NO: 81. ORF 40 (SEQ ID NO: 83) is the polynucleotide drawn from residues 5283 to 7073 of SEQ ID NO: 73, and SEQ ID
NO: 82 represents the polypeptide deduced from SEQ ID NO: 83. ORF 41 (SEQ ID
NO: 85) is the polynucleotide drawn from residues 7108 to 8631 of SEQ ID NO:
73, and SEQ ID NO: 84 represents the polypeptide deduced from SEQ ID NO: 85. ORF

42 (SEQ ID NO: 87) is the polynucleotide drawn from residues 9371 to 8673 of SEQ
ID NO: 73, and SEQ ID NO: 86 represents the polypeptide deduced from SEQ ID
NO: 87. ORF 43 (SEQ ID NO: 89) is the polynucleotide drawn from residues 9762 to 9364 of SEQ ID NO: 73, and SEQ ID NO: 88 represents the polypeptide deduced from SEQ ID NO: 89.
Some open reading frames provided in the Sequence Listing, namely ORF 2 (SEQ ID NO: 5), ORF 5 (SEQ ID NO: 11), ORF 12 (SEQ ID NO: 25), ORF 13 (SEQ
ID NO: 27), ORF 15 (SEQ ID NO: 31), ORF 17 (SEQ ID NO: 35), ORF 19 (SEQ ID
NO: 39), ORF 20 (SEQ ID NO: 41), ORF 22 (SEQ ID NO: 45), ORF 24 (SEQ ID NO:
49), ORF 26 (SEQ ID NO: 53) and ORF 27 (SEQ ID NO: 55) initiate with non-standard initiation codons (eg. GTG - Valine, or CTG - Leucine) rather than standard initiation codon ATG methionine. AII ORFs are listed with the appropriate M, V or L amino acids at the amino-terminal position to indicate the specificity of the first codon of the ORF. It is expected, however, that in all cases the biosynthesized protein will contain a methionine residue, and more specifically a formylmethionine residue, at the amino terminal position, in keeping with the widely accepted principle that protein synthesis in bacteria initiate with methionine (formylmethionine) even when the encoding gene specifies a non-standard initiation codon (e.g. Stryer BioChemistry 3rd edition, 1998, W.H. Freeman and Co., New York, pp. 752-754).
ORF 32 (SEQ ID NO: 65) is incomplete and contains a truncation of 10 to 20 amino acids from its carboxy terminus. This is due to incomplete sequence information between Contigs 2 and 3(SEQ ID NOS: 64 and 73, respectively).
Deposits of E. coli DH10B vectors, each harbouring a cosmid clone (designated in FIGURE 12 as 046KM and 046KQ respectively) of a partial biosynthetic locus for the compound of Formula II from Micromonospora sp.
strain 046-ECO11 and together spanning the full biosynthetic locus for production of the compound of Formula II have been deposited with the International Depositary Authority of Canada, Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 on February 25, 2003. The cosmid clone designated 046KM was assigned deposit accession numbers IDAC 250203-06, and the cosmid clone designated 046KQ was assigned deposit accession numbers IDAC
250203-07. Cosmid 046KM covers residue 1 to residue 32,250 of Contig 1 (SEQ ID
NO: 1). Cosmid 046KQ covers residue 21,700 of Contig 1 (SEQ ID NO: 1) to residue 9,762 of Contig 3 (SEQ ID NO: 73). The sequence of the polynucleotides comprised in the deposited strains, as well as the amino acid sequence of any polypeptide encoded thereby are controlling in the event of any conflict with any description of sequences herein.
The deposit of the deposited strains has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
The deposited strains are provided merely as convenience to those skilled in the art and are not an admission that a deposit is required for enablement. A license may be required to make, use or sell the deposited strains, and compounds derived therefrom, and no such license is hereby granted.
In order to identify the function of the proteins coded by the genes forming the biosynthetic locus for the production of the compound of Formula li the gene products of ORFs 1 to 43, namely SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 were compared, using the BLASTP
version 2.2.10 algorithm with the default parameters, to sequences in the National Center for Biotechnology Information (NCBI) nonredundant protein database and the DECIPHER database of microbial genes, pathways and natural products (Ecopia BioSciences Inc. St.-Laurent, QC, Canada).
The accession numbers of the top GenBankT " hits of this BLAST analysis are presented in Table 14 along with the corresponding E values. The E value relates the expected number of chance alignments with an alignment score at least equal to the observed alignment score. An E value of 0.00 indicates a perfect homolog.
The E values are calculated as described in Altschul et al. J. Mol. Biol., 215, (1990). The E value assists in the determination of whether two sequences display sufficient similarity to justify an inference of homology.

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Table 14 ~
ORF Famil # aa GenBank homology Probabilit % Identity % Similarity Proposed function of GenBank match 1 ABCC 571 NP_736627.1, 590aa 1.00E-107 222/496 (44.76%) 278/496 (56.05%) BC
transporter Corynebacterium efficiens NP_600638.1, 510aa 5.00E-80 184/500 (36.8%) 260/500 (52%) BC transporter Corynebacterium efficiens NP600638.1, 510aa 3.OOE-12 58/195 (29.74%) 84/195 (43.08%) BC transporter Corynebacterium efficiens 2 RECH 689 CAC93719.1, 923aa 3.00E-17 57/158 (36.08%) 87/158 (55.06%) regulator[Lechevalieria aerocolonigenes]
BAC55205.1, 943aa 3.OOE-12 51/170 (30%) 81/170 (47.65%) transcriptional activator [Streptomyces sp.
NP_631154.1, 932aa 3.OOE-07 29/63 (46.03%) 40/63 (63.49%) re u(ator. Stre tom ces coelicolor A3(2) 3 REGD 895 CAC93719.1, 923aa 3.OOE-20 92/330 (27.88%) 142/330 (43.03%) regulator [Lechevalieria aerocolonigenes]
BAC55205.1, 943aa 1.00E-15 80/277 (28.88%) 101/277 (36.46%) ctivator [Streptomyces sp. TP-A0274]
NP_733725.1, 908aa 3.00E-12 95/339 (28.02%) 140/339 (41.3%) regulator Stre tom ces coelicolor A3(2)) 4 IDSA 362 NP_601376.2, 371aa 2.OOE-80 158/321 (49.22%) 208/321 (64.8%) 3GPP synthase [Corynebacterium glutamicum NP_738677.1, 366aa 3.OOE-79 158/330 (47.88%) 204/330 (61.82%) olyprenyl synthase,Corynebacterium efficiens W
NP_216689.1, 352aa 2.00E-78 153/331 46_22% 203/331 (61.33%) idsA2 M
cobacterium tuberculosis H37Rv]
MVKA 354 BAB07790.1, 345aa 2.00E-71 150/326 (46.01%) 193/326 (59.2%) mevalonate kinase [Streptomyces sp. CL190] OD o BAB07817.1, 334aa 5.OOE-66 145/324 (44.75%) 185/324 (57.1%) mevalonate kinase [Kitasatospora griseola] o NP_720650.1, 332aa 3.00E-36 95/327 (29.05%) 157/327 (48.01%) mevalonate kinase [Streptococcus mutans o 6 DMDA 346 BAB07791.1, 350aa 2.OOE-88 177/305 (58.03%) 199/305 (65.25%) diphosphomevalonate decarboxylase [Streptomyces sp.
BAB07818.1, 300aa 2.OOE-69 145/275 (52.73%) 168/275 (61.09%) mevalonate diPH
decaroboxylase [Kitasatospora griseola]
NP785307.1, 325aa 3.OOE-44 105/307 (34.2%) 141/307 45.93%) di hos homevalonate decarboxylase [Lactobacillus plantarum 7 MVKP 369 BAB07792.1, 374aa 4.OOE-93 183/365 (50.14%) 220/365 (60.27%) phosphomevalonate kinase [Streptomyces sp. CL190]
BAB07819.1, 360aa 6.00E-77 171/358 (47.77%) 202/358 (56.42%) phosphomevalonate kinase [Kitasatospora griseola]
AAG02442.1, 368aa 2.OOE-31 102/354 28.81% 149/354 42.09% phosphomevalonate kinase Enterococcus faecalis 8 IPPI 360 Q9KWF6, 364aa 1.00E-128 238/361 (65.93%) 269/361 (74.52%) Isopentenyl-diphosphate delta-isomerase Q9KWG2, 363aa 1.00E-128 230/349 (65.9%) 270/349 (77.36%) Isopentenyl-diphosphate delta-isomerase NP_814639.1, 347aa 5.OOE-73 154/348 (44.25%212/348 60.92%) isopentenyl di hos hate isomerase Enterococcus faecalis 9 HMGA 351 BAA70975.1, 353aa 1.00E-165 284/348 (81.61%) 317/348 (91.09%) 3-hydroxy-3-methylglutaryl coenzyme A reductase [Streptomyces sp.]
3-hydroxy-3-methylglutaryl coenzyme A reductase [Kitasatospora BAA74565.1, 353aa 1.00E-160 282/347 (81.27%) 310/347 (89.34%) riseola]

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BAA74566.1, 353aa 1.00E-155 277/347 79.83% 299/347 86.17% 3 hydroxy 3 methylglutaryl coenzyme A reductase [Streptomyces sp.] y KASH 391 BAB07795.1, 389aa 1.00E-148 260/386 (67.36%) 3001386 (77.72%) 3-hydroxy-3-methylglutaryl CoA synthase [Streptomyces sp. CL190]
BAB07822.1, 346aa 1.00E-136 239/343 (69.68%) 268/343 (78.13%) HMG-CoA synthase [Kitasatospora griseolal CAD24420.1, 388aa 6.OOE-79 166/385 (43.12%) 210/385 (54.55%) HMG-CoA synthase [Paracoccus zeaxanthinifaciens]
11 IPTN 290 NP631248.1, 295aa 5.OOE-22 79/282 (28.01%) 124/282 (43.97%) hypothetical protein [Streptomyces coelicolor A3(2)]
AAN65239.1, 324aa 5.OOE-06 70/278 (25.18%) 112/278 40.29%) cloQ Stre tom ces roseochromogenes subsp. oscitans 12 SPKG 370 AAM78435.1, 344aa 5.OOE-48 112/208 (53.85%) 131/208 (62.98%) two-component sensor [Streptomyces coelicolor A3(2)]
NP_630507.1, 382aa 5.OOE-48 112/208 (53.85%) 131/208 (62.98%) sensor kinase [Streptomyces coelicolor A3(2)]
ZP 00058991.1, 407aa 9.OOE-34 88/198 (44.44%) 114/198 (57.58%) Signal transduction histidine kinase [Thermobifida fusca 13 RREB 220 NP_630508.1, 224aa 3.OOE-79 148/220 (67.27%) 179/220 (81.36%) regulatory protein [Streptomyces coelicolor A3(2)]
ZP_00058992.1, 221aa 4.OOE-67 129/218 (59.17%) 163/218 (74.77%) Response regulator [Thermobifida fusca]
NP 625364.1, 221aa 6.OOE-66 134/222 60.36% 164/222 3.87% response regulator Stre tom ces coelicolor A3(2)]

14 UNES 131 No hit - - - -UNEZ 154 NP649459.2, 628aa 7.60E-02 21/55 (38.18%) 33/55 (60%) CG1090-PB
[Drosophila melanogaster]
NP_730819.1, 473aa 7.60E-02 21/55 (38.18%) 33/55 (60%) CG1090-PA [Drosophila melanogaster]
AAM1 1079.1, 428aa 7.60E-02 21/55 38.18% 33/55 60% GH23040p [Drosophila melano aster co 16 OXDS 661 NP_242948.1, 500aa 1.00E-52 129/433 (29.79%) 197/433 (45.5%) unknown conserved protein [Bacillus halodurans] ~ O1 ZP_00091617.1, 480aa 3.OOE-32 123/426 (28.87%) 175/426 (41.08%) Putative multicopper oxidases [Azotobacter vinelandii]
NP_252457.1, 463aa 1.00E-31 115/408 (28.19%) 170/408 (41.67%) metallo-oxidoreductase Pseudomonas aeruginosa PA01 17 UNFD 129 NP_437360.1, 127aa 7.00E-33 73/121 (60.33%) 87/121 (71.9%) bleomycin resistance protein family [Sinorhizobium meliloti]
AA091879.1, 123aa 1.OOE-31 68/117 (58.12%) 86/117 (73.5%) unknown [uncultured bacterium]
NP_103287.1, 131aa 1.00E-23 59/122 48.36% 76/122 (62.3%) unknown rotein Mesorhizobium loti Anthranilate/para-aminobenzoate synthase [Pseudomonas 19 CSMB 661 ZP_00137697.1, 769aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%) eruginosa phenazine biosynthesis protein PhzE [Pseudomonas aeruginosa NP_250594.1, 627aa 1.OOE-166 319/622 (51.29%) 408/622 (65.59%) PA01]
ZP_00137701.1, 687aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%) Anthraniiate/
ara-aminobenzoate synthas [Pseudomonas aeruginosa AAKD 427 P41403, 421aa 1.00E-64 161/420 (38.33%) 214/420 (50.95%) spartokinase (Aspartate kinase) ZP_00057166.1, 445aa 2.OOE-64 154/415 (37.11%) 218/415 (52.53%) Aspartokinases [Thermobifida fusca]

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~
AAD49567.1, 421aa 6.00E-64 152/412 (36.89%) 216/412 (52.43% aspartokinase subunit A[Amycolatopsis mediterranei]
~0 21 ALDB 274 NP275722.1, 266aa 2.00E-53 104/231 (45.02%) 147/231 (63.64%) conserved protein [Methanothermobacterthermautotrophicus] () NP_614692.1, 270aa 2.OOE-52 104/240 (43.33%) 146/240 (60.83%) Fructose-1,6-bisphosphate aldolase [Methanopyrus kandieri AV19] ~
fructose-bisphosphate aldolase [Methanosarcina acetivorans str.
NP_615406.1, 267aa 2.OOE-50 99/231 (42.86%) 141/231 61.04% 2A
22 UNFC 367 NP_275723.1, 378aa 4.OOE-46 116/308 (37.66%) 171/308 (55.52%) conserved protein [Methanothermobacter thermautotrophicus]
NP_614691.1, 402aa 2.00E-45 115/295 (38.98%) 163/295 (55.25%) alternative 3-dehydroquinate synthase [Methanopyrus kandleri NP248244.1, 361aa 2.OOE-43 103/255 (40.39%) 150/255 (58.82%) conserved hypothetical protein Methanococcus'annaschii 23 HYDK 253 NP_577771.1, 247aa 4.00E-14 55/178 (30.9%) 87/178 (48.88%) metal-dependent hydrolase [Pyrococcus furiosus DSM 3638]
NP142108.1, 247aa 1.00E-12 50/151 (33.11%) 78/151 (51.66%) hypothetical protein PH0093 [Pyrococcus horikoshiij NP_125791.1, 248aa 1.00E-11 42/151 (27.81%) 76/151 50.33% hypothetical protein [Pyrococcus ab ssi 24 ADSA 438 NP_070499.1, 433aa 2.OOE-41 122/347 (35.16%) 171/347 (49.28%) coenzyme F390 synthetase [Archaeoglobus fulgidus NP_618724.1, 434aa 5.OOE-41 119/345 (34.49%) 171/345 (49.57%) coenzyme F390 synthetase [Methanosarcina acetivorans NP_632700.1, 437aa 7.OOE-41 121/345 (35.07%) 171/345 (49.57%) Coenzyme F390 synthetase [Methanosarcina mazei Goel] 25 HOXV 396 ZP_00027430.1, 442aa 8.00E-76 152/358 (42.46%) 211/358 (58.94%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum]

NP_627457.1, 420aa 1.OOE-71 161/420 (38.33%) 216/420 (51.43%) salicylate hydroxylase [Streptomyces coelicolor A3(2)]
ZP_00033877.1, 403aa 2.OOE-68 146/395 (36.96%) 200/395 (50.63%) 2- ol renyl 6 metho henol h dro lase Burkholderia fun orum 26 SDRA 261 NP_391080.1, 261aa 6.OOE-58 119/261 (45.59%) 149/261 (57.09%) 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase [Bacillus subtilisj ~ O1 ZP_00059512.1, 260aa 1.00E-55 116/259 (44.79%) 144/259 (55.6%) Dehydrogenase [Thermobifida fusca]
AAG31126.1, 257aa 9.OOE-55 117/257 (45.53%) 144/257 (56.03%) MxcC Stigmatella aurantiaca]
27 DHBS 224 051790, 207aa 7.,00E-60 110/198 (55.56%) 142/198 (71.72%) isochorismatase 051518, 207aa 1.00E-58 110/198 (55.56%) 140/198 (70.71%) isochorismatase NP_391077.1, 312aa 2.OOE-58 106/203 52.22% 139/203 (68.47%) isochorismatase [Bacillus subtilis 28 SDRA 233 NP_103491.1, 242aa 9.00E-21 74/230 (32.17%) 112/230 (48.7%) acyl-carrier protein reductase [Mesorhizobium loti]
AAL14912.1, 245aa 1.OOE-15 65/229 (28.38%) 100/229 (43.67%) short-chain dehydrogenase [Rhizobium leguminosarum bv. trifolii]
NP_902480.1, 235aa 7.00E-15 67/229 (29.26%) 100/229 43.67% oxidoreductase [Chromobacterium violaceum 29 UNIQ 246 S18541, 281aa 4.50E-02 43/146 (29_45%) 63/146 (43.15%) hypothetical protein 3- Streptomyces coelicolor NP629228.1, 281aa 5.90E-02 43/146 (29.45%) 63/146 (43.15%) h othetical protein [Streptomyces coelicolor A3(2)]
30 UNFE 111 ZP_00058149.1, 130aa 1.00E-10 35/97 (36.08%) 47/97 (48.45%) membrane protein [Thermobifida fusca]
NP_737701.1, 120aa 1.00E-09 37/111 (33.33%) 51/111 (45.95%) hypothetical protein [Corynebacterium efficiens W
O
O
Cn y NP_827629.1, 118aa 7.OOE-09 35/105 33.33% 51/105 48.57% hypothetical protein [Streptomyces avermitilis MA 4680] 0 31 EFFT 559 ZP_00058148.1, 537aa 2.OOE-67 165/517 (31.91%) 253/517 (48.94%) Predicted symporter [Thermobifida fusca] -~-[
NP626090.1, 544aa 4.OOE-66 162/521 (31.09%) 257/521 (49.33%) transport protein [Streptomyces coelicolor A3(2)]
NP_827630.1, 549aa 7.OOE-63 160/523 (30.59%) 256/523 48.95% sodium-dependent s m orter Stre tom ces avermitilis 2,4-dihydroxybenzoate monooxygenase [Sphingobium 32 HOYH 532 AAM96655.1, 544aa 2.OOE-92 206/526(39.16%) 279/526 (53.04%) hlorophenolicum]
ZP_00029353.1, 543aa 1.00E-73 188/539 (34.88%) 263/539 (48.79%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum]
NP 769326.1, 569aa 5e-62 173/519 (33.33%) 251/519 48.36% b1r2686 Brad rhizobium'a onicum db' 33 DAHP 423 T03226, 391aa 1.00E-111 207/383 (54.05%) 259/383 (67.62%) hypothetical protein - Streptomyces hygroscopicus ZP_00137693.1, 405aa 3.00E-87 172/385 (44.68%) 233/385 (60.52%) DAHP synthase [Pseudomonas aeruginosa UCBPP-PA14]
NP250592.1, 405aa 1.00E-86 169/380 (44.47%) 232/380 (61.05%) henazine bios nthesis protein PhzC Pseudomonas aeruginosa 34 REGG 340 BAC53615.1, 346aa 1.OOE-67 142/307 (46.25%) 192/307 (62.54%) regulator protein [Streptomyces kasugaensis]
S44506, 424aa 3.OOE-66 141/305 (46.23%) 182/305 (59.67%) regulator protein -Streptomyces glaucescens AAK81822.1, 348aa 1.00E-65 141/323 (43.65%) 192/323 (59.44%) transcriptional re ulator Stre tom ces lavendulae rn 35 UNFJ 493 ZP_00073237.1, 678aa 7.OOE-35 124/454 (27.31%) 197/454 (43.39%) RTXtoxins [Trichodesmium erythraeum IMS101]
NP484716.1, 433aa 3.OOE-05 109/470 (23.19%) 172/470 (36.6%) similar to vanadium chloroperoxidase [Nostoc sp.
ZP00067005.1, 667aa 7.40E-02 37/139. 26_62% 52/139 (37.41%) h thetical protein [Microbulbifer degradans 2-40 36 RECI 112 NP627088.1, 125aa 3.OOE-17 48/100 (48%) 59/100 (59%) hypothetical protein . [Streptomyces coelicolor A3(2)] O1 NP846017.1, 109aa 7.00E-15 40/101 (39.6%) 60/101 (59_41%) hypothetical protein [Bacillus anthracis str. Ames]
NP_241272.1, 174aa 9.OOE-15 39/106 (36.79%) 62/106 (58.49%) unknown conserved protein [Bacillus halodurans]
37 UNIQ 325 NP422203.1, 187aa 1.00E-03 24/61 (39.34%) 36/61 (59.02%) hypothetical protein [Caulobacter crescentus CB15 38 OXAH 663 ZP_00058724.1, 659aa O.00E+00 370/647 (57.19%) 435/647 (67.23%) Acyl-CoA dehydrogenases [Thermobifida fusca]
AAB97825.1, 433aa 5.OOE-93 203/446 (45.52%) 251/446 (56_28%) acyl-CoA oxidase [Myxococcus xanthus]
AAF14635.1, 694aa 5.OOE-85 211/565 (37.35%) 292/565 (51.68%) 1 ac I-CoA
oxidase [Petroselinum cris um 39 ABCA 537 T14162, 574aa 9.OOE-62 189/509 (37%) 240/509 (47%) ABC transport protein - Mycobacterium smegmatis NP_624808.1 4.OOE-60 184/540 (35%) 251/540 (46%) BC transporter [Streptomyces coelicolor A3(2)]
NP822745.1 8.OOE-32 124/392 31 % 168/392 (42%) BC transportert Stre tom ces avermitilis MA-4680]
40 ABCA 596 T14180, 1122aa 1.0OE-i07 236/594 (39.73%) 300/594 (50.51%) exiT
protein - Mycobacterium smegmatis AAC82548.1, 589aa 1.00E-107 234/583 (40.14%) 295/583 (50.6%) unknown [Mycobacterium smegmatis]
NP_624810.1, 601aa 3.OOE-97 222/593 (37.44%) 283/593 (47.72%) ABC-trans orter Stre tom ces coelicolor A3(2)]

W

cn methyitransferases [Bacillus cereus 41 UNIQ 507 NP_831570.1, 676aa 8.OOE-07 62/262 (23.66%) 116/262 (44.27%) NP655735.1, 676aa 2.OOE-06 61/262 (23.28%) 116/262 (44.27%) ubiE/COQ5 methyltransferase family [Bacillus anthracis NP_844290.1, 681aa 2.OOE-06 61/262 23.28% 116/262 44.27% hypothetical protein [Bacillus anthracis str. Ames]
42 232 NP830809.1, 208aa 8.OOE-08 46/210 (21.9%) 74/210 (35.24%) Transporter, LysE family [Bacillus cereus ]
NP844737.1, 210aa 2.OOE-07 46/210 (21.9%) 74/210 (35.24%) homoserine/threonine efflux protein[Bacillus anthracis NP655752.1, 208aa 1.00E-06 47/210 (22.38%) 75/210 (35.71%) L sE, LysE type translocator [Bacillus anthracis 43 132 NP_827272.1, 127aa 4.OOE-09 38/107 (35.51%) 52/107 (48.6%) hypothetical protein [Streptomyces avermitilis MA-4680]
NP_246491.1, 112aa 5.90E-02 21/94 22.34% 44/94 46.81% unknown Pasteurelia multocida O
N
iP
iP
O
t N
~ O
p N ~
t O
rn N

The ORFs encoding proteins involved in the biosynthesis of compounds of Formula II are assigned a putative function and grouped together in families based on sequence similarity to known proteins. To correlate structure and function, the protein families are given a four-letter designation used throughout the description and figures as indicated in Table 15. The meaning of the four letter designations is as follows: AAKD designates an amino acid kinase; ABCA and ABCC designate ABC transporters; ADSA designates an amide synthetase; ALDB designates an aldolase function; CSMB designates a chorismate transaminase; DAHP designates a 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate synthase activity;
DHBS designates a 2,3-dihydro-2,3-dihydroxybenzoate synthase activity; DMDA
designates a diphosphomevalonate decarboxylase; EFFT designates an efflux protein; HMGA designates a 3-hydroxy-3-methylglutaryl-CoA reductase; HOXV
designates a monooxygenase activity; HOYH designates a hydroxylase/
decarboxylase activity; HYDK designates a hydrolase activity; IDSA designates an isopentenyl diphosphate synthase; IPPI designates an isopentenyl diphosphate isomerase; IPTN designates an isoprenyltransferase; KASH designates 3-hydroxy-methylglutaryl-CoA synthase; MVKA designates a mevalonate kinase; MVPK
designates a phosphomevalonate kinase; OXAH designates an acylCoA oxidase;
OXDS designates an oxidoreductase; RECH, RECI, REGD, REGG and RREB
designate regulators; SDRA designates a dehydrogenase/ketoreductase, SPKG
designates a sensory protein kinase; UNES, UNEZ, UNFA, UNFC, UNFD, UNFE, UNFJ and UNIQ designate proteins of unknown function.

Table 15 FAMILY FUNCTION
AAKD amino acid kinase; strong homology to primary aspartate kinases, converting L-aspartate to 4-phospho-L-aspartate ABCA ABC transporter ABCC ABC transporter ADSA adenylating amide synthetase ALDB aldolase; similarity to fructose-1,6-biphosphate aldolase that generates D-giyceraldehyde-3Ph, precursor of D-erythrose-4Ph involved in the shikimate pathway CSMB chorismate transaminase, similarity to anthranilate synthase DAHP DAHP synthase, class II; involved in formation of aminoDAHP from PEP and erythrose-4-phosphate DHBS 2,3-dihydro-2,3-dihydroxybenzoate synthase (isochorismatase) DMDA diphosphomevalonate decarboxylase (mevalonate pyrophosphate decarboxylase) EFFT efflux protein HMGA HMG-CoA reductase; converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate plus CoA in isoprenoid biosynthesis HOXV FAD monooxygenase; shows homology to a variety of monooxygenases including salicylate hydroxylases, zeaxanthin epoxidases HOYH hydroxylase/decarboxylase; FAD-dependent rnonooxygenase HYDK hydrolase IDSA isoprenyl diphosphate synthase, catalyzes the addition of 2 molecules of isopentenyl pyrophosphate to dimethylallyl pyrophosphate to generate GGPP
IPPI isopentenyl diphosphate isomerase, catalyzes the isomerization of IPP to produce dimethylallyl diphosphate IPTN isoprenyltransferase; catalyzes covalent N-terminal attachment of isoprenyl units to amide groups of nitrogen-containing heterocycle rings KASH HMG-CoA synthase; condenses acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA
MVKA mevalonate kinase; converts mevalonate to 5-phosphomevalonate in the mevalonate pathway of isoprenoid biosynthesis MVKP phosphomevalonate kinase; converts 5-phosphomevalonate to 5-diphosphomevalonate in the mevalonate pathway of isoprenoid biosynyhesis OXAH acyl CoA oxidase OXDS oxidoreductase RECH regulator RECI regulator; similarity to PadR transcriptional regulators involved in repression of phenolic acid metabolism REGD transcriptional regulator; relatively large regulators with an N-terminal ATP-binding domain containing Walker A and B motifs and a C-terminal LuxR type DNA-binding domain REGG regulator RREB response regulator; similar to response regulators that are known to bind DNA
and act as transcriptional activators SDRA dehydrogenase/ketoreductase, NAD-dependent SPKG sensory protein kinase, two component system UNES unknown function UNEZ unknown function UNFA unknown function UNFC unknown function UNFD unknown function UNFE putative membrane protein UNFJ unknown function UNIQ unknown function Biosynthesis of the compound of Formula II involves the action of various enzymes that synthesize the three building blocks of the compound, namely the farnesyl-diphosphate component (FIGURE 13), the 3-hyd roxy-anth ran i late-ad enylate component (FIGURE 14a) and the 2-amino-6-hydroxy-benzoquinone component (FIGURE 14b) that are subsequently condensed to form the final compound (FIGURE 15).
The farnesyl-diphosphate biosynthesis involves the concerted action of seven enzymes (FIGURE 13). ORF 10 (KASH) (SEQ ID NO: 20) encodes a hydroxymethylglutaryl-CoA synthase that catalyzes an aldol addition of acetyl-CoA
onto acetoacyl-CoA to yield 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This product is subsequently reduced through the action of ORF 9 (HMGA) (SEQ ID NO:
18) to form mevalonic acid (MVA). ORF 5 (MVKA) (SEQ ID NO: 10) phosphorylates mevalonate to 5'-phosphomevalonate using ATP as the phosphate donor. The next step in the farnesyi-diphosphate biosynthesis is the phosphorylation reaction of_ the 5'-phosphomevalonate to 5'-pyrophosphomevalonate (DPMVA) that is catalyzed by ORF 7 (MVKP) (SEQ ID NO: 14). Subsequent decarboxylation of 5'-pyrophosphomevalonate catalyzed by ORF 6 (DMDA) (SEQ ID NO: 12) yields isopentenyl diphosphate (IPP) which is then converted to dimethylallyldiphosphate (DMADP) through the action of ORF 8(IPPI) (SEQ ID NO: 16) that has isomerase enzymatic activity. The final step in the biosynthesis of farnesy!-diphosphate is the condensation of one molecule of dimethylallyldiphosphate with two molecules of isopentenyl diphosphate catalyzed by the isoprenyl diphosphate synthase ORF 4 (IDSA) (SEQ ID NO: 8). The described pathway involved in synthesis of farnesyl-diphosphate is entirely consistent with related mevalonate pathways described in other actinomycete species (Takagi et al., J. Bacteriol. 182, 4153-4157, (2000)).
Biosynthesis of the 3-hydroxy-anthranilate component involves the use of precursors derived from the shikimate pathway (FIGURE 14a). Chorismic acid is transaminated through the action of ORF 19 (CSMB) (SEQ ID NO: 38) to form aminodeoxyisochorismic acid. This enzyme resembles anthranilate synthases and is likely to catalyze specifically the transfer of the amino group using glutamine as the amino donor. The next step involves isochorismatase activity and is mediated by ORF 27 (DHBS) (SEQ ID NO: 54). This reaction consists in the removal of the pyruvate side chain from aminodeoxyisochorismic acid to form 6-amino-5-hydroxy-cyclohexa-1,3-dienecarboxylic acid. This compound is subsequently oxidized through the action of ORF 26 (SDRA) (SEQ ID NO: 52) yielding 3-hydroxy-anthranilic acid. ORF 24 (ADSA) (SEQ ID NO: 48) catalyzes the activation of 3-hydroxy-anthranilic acid through adenylation generating the 3-hydroxy-anthranilate-adenylate component (FIGURE 14a).
Biosynthesis of the 2-amino-6-hydroxy-benzoquinone component of the compound of Formula II, requires components derived from the aminoshikimate pathway. FIGURE 14b depicts the series of enzymatic reactions involved in the biosynthesis of this constituent. ORF 21 (ALDB) (SEQ ID NO: 42) resembles aldolases involved in the generation of precursors of D-erythrose-4-phosphate which is part of the aminoshikimate pathway used for the generation of 2-amino-6-hydroxy-[1,4]-benzoquinone. ORF 33 (DAHP) (SEQ ID NO: 67) catalyzes the initial step in the aminoshikimate pathway that corresponds to the formation of 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (amino DAHP) from phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E-4Ph). Subsequent reactions leading to 3-amino-5-hydroxy-benzoic acid are catalyzed by enzymes provided by primary metabolism biosynthetic pathways present in Micromonospora sp. strain 046-ECO11. ORF 25 (HOXV) (SEQ ID NO: 50) hydroxylates 3-amino-5-hydroxy-benzoic acid at position 2, generating 3-amino-2,5-dihydroxy-benzoic acid.
This intermediate is further modified by ORF 32 (HOYH) (SEQ iD NO: 65) that catalyzes a decarboxylative oxidation reaction yielding 6-amino-benzene-1,2,4-triol.
A final oxidation reaction is performed by ORF 16 (OXDS) (SEQ ID NO: 32) yielding 2-amino-6-hydroxy-[1,4]-benzoquinone (FIGURE 14b).
Assembly of the three components resulting in the compound of Formula li is catalyzed by ORFs 24 and 11 (FIGURE 15). ORF 24 (ADSA) (SEQ ID NO: 48) catalyzes the condensation of the adenylated 3-hydroxy-anthranilate with the 2-amino-6-hydroxy-[1,4]-benzoquinone component. A spontaneous condensation between the free amino group of the 3-hydroxy-anthranilate and one of the carbonyl groups present on the 2-amino-6-hydroxy-[1,4]-benzoquinone component occurs yielding a dibenzodiazepinone intermediate. This compound is further modified through transfer of the farnesyl group of the farnesyl-diphosphate intermediate onto the nitrogen of the amide of the dibenzodiazepinone catalyzed by ORF 11 (IPTN) (SEQ ID NO: 22) and resulting in the formation of the compound of Formula II

(FIGURE 15).

Additional ORFs, namely ORF 2 (RECH) (SEQ ID NO: 4), ORF 3 (REGD) (SEQ ID NO: 6), ORF 12 (SPKG) (SEQ ID NO: 24), ORF 13 (RREB) (SEQ ID NO:
26), ORF 34 (REGG) (SEQ ID NO: 69) and ORF 36 (RECI) (SEQ ID NO: 74) are involved in the regulation of the biosynthetic locus encoding the compound of Formula H. Other ORFs, namely ORF 1 (ABCC) (SEQ ID NO: 2), ORF 31 (EFFT) (SEQ ID NO: 62), ORFs 39 and 40 (ABCA) (SEQ ID NOS: 80 and 82, respectively) and ORF 42 (SEQ ID NO: 86) are involved in transport. Other ORFs involved in the biosynthesis of the compound of Formula Il include ORF 20 (AAKD) (SEQ ID NO:
40), ORF 23 (HYDK) (SEQ ID NO: 46), ORF 38 (OXAH) (SEQ ID NO: 78) as well as ORFs 14, 15, 17, 18, 22, 29, 30, 35, 37, 41 and 43 (SEQ ID NOS: 28, 30, 34, 34, 44, 58, 60, 71, 76, 84 and 88, respectively) of unknown function.

TABLE 16: PREFERRED MEDIA COMPOSITION FOR PRODUCTION OF ECO-Component QB MA KH RM JA FA Ex 1 pH* 7.2 7.5 7 6.85 7.3 7.0 7.0 Glucose 12 10 10 10 Sucrose 100 Lactose Cane molasses 15 Corn starch 30 Soluble starch 10 25 Potato dextrin 20 40 20 Corn steep solid Corn steep liquor 5 15 Dried yeast 2 Yeast extract 5 Malt extract 35 PharmamediaTM 10 15 Glycerol 20 NZ-Amine 5 10 Soybean powder 15 Soybean flour Meat extract Bacto-peptone 5 MgSO4.7H2O 1 MgCI2. 6H20 CaCO3 4 1 2 2 2 NaCI 5 (NH4)2 SO4 2 2 K2 SO4 0.25 MnCIZ.4H2O
MgCI2.6HZ0 10 FeCIZ.4Hz0 ZnCIZ
Na2HPO4 3 Thiamine Casamino acid 0.1 Proflo oil 4 Trace element 2 solution *3 ml/L
Fish meal 10 Unless otherwise indicated all the ingredients are in gm/L.
"3 Trace elements solution contains: ZnCl2 40 mg; Fe CI3 6H20 (200 mg); CuC12 2H20 (10 mg);
MnC12.4H20; Na2B407.10H20 (10mg); (NH4) s M07024 .4HZ0 (10 mg) per litre.
*5 The pH is to adjusted as marked prior to the addition of CaCO3.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

SEQUENCE LISTING
<110> ECOPIA BIOSCIENCES INC.
Bachmann, Brian 0.
McAlpine, James B.
Zazopoulos, Emmanuel Farnet, Chris M.
Piraee, Mahmood <120> FARNESYL DIBENZODIAZEPINONE, PROCESSES FOR ITS PRODUCTION AND ITS USE
AS A PHARMACEUTICAL

<130> 3005-7PCT
<150> USSN 60/441,126 <151> 2003-01-21 <150> USSN 60/492,997 <151> 2003-08-07 <150> USSN 60/518,286 <151> 2003-11-10 <160> 89 <170> PatentIn version 3.0 <210> 1 <211> 36602 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 1 ccggtgcacc gggttctcca ggatcgccgt cgcgcccacc ggccccgaca ggtagacgac 60 gttcagggac ttgccgcgcc cttcgtagtt ggcccgcacc acctgcgcgt cgccgatccg 120 gcccctggtc tccagcgtgc ggttctccca cacctgccat ccgacgaagg tcaggaacag 180 cgcggtgaac agggacgtga cgagcagcca gaggccagct gtcagcacgg tcgccccctc 240 gccccgtagc aggccgagga cgacctcctc gtagcgcgag gggcggccga cggggccggt 300 gcccgctccg tcgacagcca tcccgccgct ccttcgccga ctgccccgga catccacggt 360 agccagcgag tccagtccgg tgaggaaggg gtggcgagaa gtcgatatga ctgagaggca 420 tatttatgac tcccagtcat atcgctcgga agtgaccgaa cgacctgacg ccgccggggc 480 tgtgagcggc agcgtgggcc aggccgcgag gtcctggagc atctgccggt cgtgggtggc 540 gacgaccacc gccgcccggg tcgtcagcag ggcggcggtg aggtcgtcga ccagcggcgc 600 cgacaggtgg ttcgtcggtt cgtcgaggat cagcaggtcg ggacgttcgg ccaggcgcag 660 cgccaggttc agccgccgtt gctgtccgtg cgacatccgg ccgacggggg tacgccgggc 720 ctcggcgtcg agcaggttcg tcgcgctcag cggcagggcc gtgccggagc cgacgcgccc 780 gctggagcgg agccggccca cgtgctgctc gtacaggtcg tgcgcgagca gcgccggcgg 840 ccagtcgggc acctcctgac cgaggtacgc gacgcgcgcg ccggacaggt gccggacctc 900 Z

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Gly Asn Asp Glu Gln Val Leu Arg Glu Ile Leu Gly Val Asp Val His Arg Glu Leu Ile Asp Phe Ala Gly Gly Ala Gly Gly Asn Pro His Leu Val Ala Glu Leu Ala Arg Gly Leu Ala Glu Glu Gly Leu Ile Arg Glu Thr Asn Gly Arg Ala Glu Leu Val Ser Arg Arg Ile Pro Arg Arg Val Leu Ser Phe Val Met Arg Arg Leu Asn Asp Val Ser Ala Gly Cys Gln Gln Phe Leu Lys Val Ala Ala Ala Leu Gly Arg Ser Phe Met Leu Glu Asp Val Ser Arg Met Leu Gly Arg Ser Ser Ala Ala Leu Leu Pro Pro Val Asp Glu Ala Ile Ala Ser Gly Phe Val Val Ala Ala Glu His Gln Leu Ala Phe Gln Ser Asp Phe Leu Leu Arg Gly I1e Ile Glu Ser Ile Pro Gly Pro Ala Arg Asp Ala Leu Arg Arg Glu Ala Met Ser Leu Ser Gly Arg Arg Arg Pro Ala Ala Asp Gln Asn Arg Arg Leu Asp Ala Ala Pro Thr Ala Pro Val Ser Ala Thr Gly Glu Asp Ala Thr Gly Ser Cys Ser Arg Ala His Arg Leu Ile Met Asn Gly Asn Ala Lys Ala Gly Ile Arg Val Ala Glu Ala Val Leu Ala Gly Pro Ala Ala Ser Leu Ala Ala Arg Arg Asp Ala Glu Ala Cys Leu Val Leu Ala Asp Leu Leu Leu Gly Gly Glu Gly Gly Gly Pro Met Thr Glu Ala Ile Leu Arg Glu Arg Asp Ala Glu Ser Gly Asp Ala Ala Leu Ala Met Ala Leu Thr Ala Arg Ser Thr Gly Leu Trp Ser Ala Gly Lys Leu Ala Glu Gly Leu Lys Leu Gly Arg Ala Ala Val Arg Ala Gly Ala Glu Ala Glu Pro Val Trp Arg Leu His Ala Gln Leu Ala Leu Ala Gly Lys Leu Ala Asn Leu Arg Clu Phe Asp Glu Ala Glu Ala Leu I1e Asn Glu Ala Glu Ala Gly Leu Arg Gly Leu Pro Ala Pro Ile Trp Thr Ala Ala Thr Ala Val Met Arg Ser Arg Leu Leu Leu Gln Ala Gly Arg Ile Gly Glu Ala Arg Arg Glu Ala Ala Leu Ala Thr Thr Ala Val Glu Gly Asp Ala Val Pro Met Leu Arg Pro Leu Ala Tyr Ala Val Leu Ser Thr Ala Ser Phe Tyr Met Gly Asp Leu Pro Ala Ala Ile Glu Tyr Leu Arg Arg Gly Gln Arg Asp Ala Asp Arg His Val Val Leu Asp Ser Val Gln Tyr Ser Trp Ala Glu Val Leu Ile Thr Val Lys Gln Glu Gly Pro Arg Ala Ala Ala Gln Leu Leu Ala Gly Lys His His Arg Leu Pro Thr Gln Arg Arg Leu Tyr Val Glu Val Pro Ser Ala Ala Ala Phe Leu Val Leu Leu Ala Arg Asp Val Asp Asp Arg Asp Leu Glu Arg Arg Val Leu Asp Thr Val Asn Gly Leu Ala Ala Asp Asn Pro Arg Ile Gln Val Val Ser Leu Thr Ala Met His Ala His Ala Leu Ala Asn Ser Ala Pro Ala Ala Leu Ala Leu Ile Ile Val Gln Ser Arg Asp Pro Ile Ser Val Ala Leu Ala Thr Glu Glu Leu Ala Lys Leu Tyr Ala Ala Gln Ala Gln Ala Gly Gly Arg Pro Ala Thr Pro Ala Arg Ala Glu Glu Ala Ala Thr Pro Pro Ala Ser Cys Trp Ser Thr Leu Ser Asp Met Glu Gln Arg Ile Ala Tyr Leu Val Ser Val Gly Leu Thr Asn Arg Gln Ile Ala Lys Gin Val His Leu Ser Ala His Thr Val Asn Tyr His Leu Arg Lys Ile Tyr Arg Lys Leu Gly Phe Asn Thr Arg Ala Glu Leu Ala His Ala Ala Ala Thr Tyr Ser Gly Arg Ala Ala Ile Tyr Ser Met Ser Gly Asp Gln Asp Trp Gly Ala Gly Ser Met Thr Gly Lys Ala Ser O'VLT bPababaaba pqaqabP-eaa boqaPPbbPb aaSaabbqab abbqbbaqoq SbaaaPbbba 089T paqb,2abqba q-eaqpaqaba bbqaaabaab baa~abab~~ Pvbabbqaba bo-eaoaba2a OZ9T bql2oabaa-ea qaob12aqbaq bb-eaaqebb'e 000012LOa25b abaabaqabb b0-2eOqb5oe 09ST o-eboqaoqba baaba-evbaq aa-ebqbaa-eb aebbqba2bo boaabbqabq aaqbbqaoqq 005T aaboaboa ba bl2baabqbbs baqbaPqaqo abaaba bpab aeaaa blaob aopaaeab-eL-OfIfIZ obbbaboqab qab-2aaobao baobbbobao abb~-ebbeab ~-eaqbba-eoq -ebqabqbel2b 08~T babbbqbaqa LDqb2abqbbo qapbaqa ajb bqba-eoaboa -ebbabo-ebbb ob-eabbbbba OZ~T bb-eoqao2qb -eba qi?Bobaa baoobqoa-eb bbbbqva-eqo qqaaqoaba a i2a bpo:~abqb 09ZT babaeqooba qaqoabbabi abq-eboabqb babaebbbbb -eb6qba aba a vaaeoobbqa OOZZ bobbobblebb baqbobobbLo bbbbajr:?bba bbbbabbpa o jobqa bqqbb Doolbbobqp O:PZZ bqbbobbopb aboabbopbb qojpboobab Daa bqopbbo bobqoa bbba bPPbbobbpb 080T o~-eoq7ebqqb abbi2boobb-e bo*ebaqqb-eb abooqoa-epb aboqa-eL-pbb boaboqaba b 0Z0Z ojab-eaz)oba eabjajbabb qbqbba~~-eb az)bb-ebbabD bbbabbbab-4 bbabbobbba 096 -2bbbqab2vb qaobbb,ebbo bb;abePPbb babbojbbqb qz>bbboaeaa lbbaa aboa p 006 bqababbqpb abbqapa baa bo25qbbaaq bsbaabapbo boppba babq aa qpbabbvb 0IV8 aapbqpba aa bbabbabbbv bbbbabbajo b;abqaqpba abbqa bqbbq aqbqbabbeb 08L babz)-ebqbab baaabqobaq abaqbabaob baaabbaaba qoqqbbabb,e ba aboqbaba OZL q-4,2obboab5 i2ebobavpbb ba~-eb~e-eq2 bqaa.baaipab abbbaa aqqb qoa~-ebboa2 099 aabo-ebb-ebfi bbaa,ebobab -ebqbbaabab ao,2qaababb aba-ebbq-Ibb aaba~~~b~a 009 a-ebz)abbabb aaobabba-eb abbbaa~~~~ ab-eb:j-ebabb Pb-4bovba-e:j qoabaebaba 0t'5 a aboaabbba aaqqpaaqb-e baa2aq-pabb ababqobqao jqa*ebob-eb-e aq;qoaba-qa 08t, Ppaq-eabsba oba abaqbaq boq-qabbbaj Pobaqpbabb 2bapbb:jbba z)baaoqabqa 0Z:v oabbabbz)qb aq-ebaobbbq abqeebpbaq qqbaebb-ebb q-ebpobbbqq 09~ -eabbaboa b-q qbb2e6qqaq -4b,eabi2oabq abbaababpo qbqebq-e-eb~ q-ebaqbabqe 00~ oqbqqqqbpb qab:[bababb aaoa~~~~bo bba aa:jbqbb ~;,2pbbabbb a qbbopi?iaoiR
0TIZ b-ebbboq:~,ab q-42bbblebe2 baoba-4abbb obobabaqoe 'eboaboqbbq oopobaaq2p 08Z pbbobba abq bb;bbba baq qopbqqpbqa ftbaba apab qboeboqbbb ba qaoqpbpb OZZ ababqaa,4b5 Pabpbapbae Pbbbbbbabb bb-Ipaa bpoa aqbabbaabb bbbboaboob 09 bbqbbbboaq pppbaeobba basbpoabab bboappbpbb bbbaabbavb bbap2apbqp <00t>
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gcccaggcgg ggggacggcc ggcgacgccg gcccgcgccg aggaggccgc caccccgccg 1800 gcgagctgct ggtcgaccct gtccgacatg gagcagcgga tcgcctacct ggtgagcgtg 1860 ggtctgacga accggcagat cgccaagcag gtccacctgt ccgcgcacac cgtcaactac 1920 cacctgcgga agatctaccg gaaactgggt ttcaacaccc gggccgagct ggcgcacgcc 1980 gcggccacgt actccggccg ggcggcgatc tactccatga gcggcgacca ggactggggc 2040 gccggatcca tgaccggcaa ggccagctga 2070 <210> 6 <211> 895 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 6 Met Val Ile Met Asn Arg Met Ala Gly Arg Gly Gln Glu Leu Ser Ser Leu Gly Glu Leu Leu Asp Ala Thr Met Arg Gly Ser Gly Gly Cys Val Val Val Asp Gly Pro Phe Gly Ile Gly Lys Thr His Leu Leu Lys Val Thr Gly Leu Glu Ala Ala Ala Arg Gly Leu Thr Val Val Ala Gly Arg Ala Ser Val Thr Asp Gln Pro Val Pro Val His Leu Leu Val Asn Phe Leu Arg His Ala Met Pro Gly Glu Ala Ala Val Glu Gln Leu Ala Leu Pro Gly Ala Asn Pro Phe Trp Leu Ile Asp Arg Val Gly Asp Leu Val Glu Val Ala Ala Arg Arg Arg Pro Leu Val Val Ala Leu Asp Asp Ala Gln Arg Ile Asp Asp Val Ser Ala Leu Ala Leu Arg Gly Leu Val Pro Arg Leu Ala Ser Ser Pro Val Leu Trp Leu Leu Ala Arg Arg Pro Val Ala Ala Gly Ser Ile Ala Gln His Ala Val Asp Trp Leu Ala Glu His Val Ala Val Arg Val Arg Leu Arg Glu Pro Gly Glu G'u Ala Val Ala Asp Leu Cys Ala Gly Ile Leu Gly Ala Arg Pro Asp Ala Ser Val Leu Arg Trp Ala Ala Arg Cys Gly Gly Asn Pro Lys Val Met Glu Ile Val Phe Ser Ala Phe Ile Lys Ala Gly Gln Met Ile Ile Val Asp Gly Ala Ala Ser Val Val Ser Asp Glu Leu Pro Asp Gly Val Leu Ala Ala Val Arg Gly Leu Leu Glu Glu Leu Pro Pro Pro Leu Arg Arg Leu Leu Ala Ala Gly Gly Arg Leu Gly His Thr Phe Pro Val Asp Arg Val Thr Gly Leu Leu Asp Gly Ser Ala Ala Asp Val Ser Ala Ala Ile Asp Glu Ala Val Arg Val Gly Leu Ile Arg Arg Asp Gly Ala Glu Leu Thr Phe Ala His Pro Val Leu Gly Glu Ala Leu Arg His Ala Ala Tyr Pro Glu Pro Glu Arg Ala Glu Pro Gly Ser Ala Pro Ala Pro Ala Ala Gly Asp Pro Val Arg Arg Gly Arg Pro Asp Pro Arg Pro Gly Thr Pro His Ser Pro Ala Gly Val Arg Val Thr Arg Ser Ala Pro Asp Ala Ala Thr Pro Ala Ala Thr Ala Gly Pro Arg Ser Gly Arg Cys Gly Cys Asp Asp Val Ala Ala Ala Ala Val Ser His Leu Glu Asn Gly Ser Ala Glu Ala Pro Arg Ala Leu Ala Arg Ala Leu Arg Leu Leu Ala Gly Ala Gly Arg Ala Ala Glu Ala Gly Arg Leu Ala Glu Val Met Leu Arg Arg Asp Leu Ala Ala Asp Val Glu Ala Gln Leu Val Leu Glu Leu Gly His Gly Met Arg Ala Ala Gly Ser His Arg Leu Ala Ala Gly Phe Leu Arg Arg Thr Gln Ala Arg His Asp Val Cys Glu Leu Asp Arg Ala Lys Leu Asp Arg Ala Leu Ala Asp Thr Thr Lys His Leu Gly Cly Ala Ser Ser Ala Glu Leu Glu Pro Arg His Gln Ser Pro Gly Cys Ala Pro Gly Arg Arg Pro Leu Trp Thr Trp Leu Val Arg Ala Leu Gly Ala Ala Asp Gln Leu Asp Glu Ala Gln Ala Val Leu Asp Thr Val Arg Pro Leu Ala Gln Glu Pro Ser His Thr Gly Ser Glu Ser Leu Trp Arg Gly His Arg Ala Glu Leu Leu Ala Ala Ala Gly Arg Leu Asp Glu Ala Arg Ala Glu Ala Glu Ala Ala Leu Arg Ala Ala Asp His Ser Arg Pro Gly Asp Cys Val Pro Ala Arg Leu Val Leu Ala His Leu Gly Val His His Gly Asp Leu Ala Thr Ala Ser Asp Gln Leu Arg Ala Ala Glu Arg Leu Ala Ser Ala Asp Asp Ser Ala Arg Met Asp Trp Ala Leu Ala Arg Phe His Ala Ala Ser Gly Arg Pro Ala Met Met Val Gln Thr Leu Ile Asn Val Ala Gly Gln Val Ala Pro Asp Pro Leu Leu Phe Thr Glu Ala Pro Ala Ala Ala Ala Thr Leu Val Arg Gln Ala Arg Arg Ala Gly Leu Asp Ala Glu Ala Glu Arg Ala Val Glu Val Ala Arg Arg Val Ala Arg Gly Asn Pro Phe Val Gln Ser Leu Ala Ala Ala Ala Glu His Ala Ala Gly Leu Leu Arg Asp Asp Pro Ala Ala Leu Leu Arg Ala Ala Asp Leu His Arg Leu Ala Gly Arg Thr Leu Ala Ala Ala Gly Ala Val Glu Asp Ala Ala Arg Ser Thr Arg Asp Arg Ala Glu Ala Thr Arg Leu Leu Glu Ala Ala Thr Asp Gly Tyr Arg Glu Cys Gly Ala Arg Arg Asp Leu Glu Arg Val Glu Ala Glu Leu Arg Gly Leu Pro Ala His Asn Val Arg Pro Leu Val Pro Asp Arg Pro Arg Ser Gly Trp Glu Ser Leu Thr Ser Ala Glu Leu Arg Val Val Arg Ala Ile Val Asp Gly Met Thr Asn Arg Glu Ala Ala Ser Ser Leu Phe Leu Ser Pro His Thr Val Asp Ser His Leu Arg Arg Val Phe Ser Lys Leu Asp Ile Asn Ser Arg Val Glu Leu Thr Arg Cys Phe Ile Ala His Glu Ala Val Arg Pro Ala Leu Ala Thr Thr Arg Gln Pro Ala Ser Ala Gly o~
OTYLT 6ba ebbaabb abeabbqa bq obpboabbEa apaa bbabab bqoqabaqbp bboqabboop 089T apaqbpaaab pbbpababbq a baa2ba2;5 aapol2bbzab lbbabb-eaba bb2bo-ebaja OZ9T beaq-ebaobb a bobbbqaba bbba aqbb;a bbqaaLobbqb qaboabbabb aabbaaabab 095T obqa bbboaa oqbi2aa pabb aaaabpbbqa bpbaabaalo alaabqbbab bbqaopobpp 005T bapaaPaPbb a ba:jabobbb aapbbqobe-e oababaapbb qa52bobqbq boebapaaba Ot, 17T aabbpabavb boababqaaq qabba abbob bqaaboapaa b-eabbaabaa bbbabqebbb 08~T apa-ebbbqo7e ebaqabqbaq abpababbV8 aqbaL-bbabb a boqoopbab oabaalobqS
OZ~T bqbbpbba ba ;aabaabbaa bbebaabaab bbabbbbabb bbaabbqabq aababqobab 09ZT 4ba aabbqap abpbapaaba 65-ebaabaaq pbboppbpbb qao2aaoqbq baabaabPab 00ZT babbqbapba -ebabqbbbab lbbaabbbaq ababaabbbb obbapbabaa baaobapaab 0:~TT babaPbbaab 0500qo5abo paqbabopqb abbaaboaoa ola-ea aaaba pbbbaaabbo 080T baaqPbaaob bobbbabobb a aqbboaapb obbbabbabb aapa bbooba baoqpbbaoo OZOT bpboabqbab vbbaaei2bba aapqbobaab apaabaqqab abbebpbbaq abqbbooapa 096 aa boqqaapb qab-ebbabab bavbabopba vqpbqa bbba qbbbabZbba bbpbapba qp 006 baboaboaqb qbapboaba o bboqabbopb bqobqaabbb opbqbbboop baqbaooqqq 0:~ 8 baPaPaabba qabbaabba b baabbabaqa bqaababbab qabaaaaaba a bjabpbbvb 08L bqabqabbbo baqqbaabaa baqoaqbqbb apbaaobqab Pbapbaaqbq bbqbboqbab OZL babbbbapbb qbaqpaqpbq -ebpaobbaab 62paqpaqqb ababpaqqaq baqpbebbq2 099 bqbbppbaaa Peabbabbab qabaaobbab bb;obabIaa qbaaaaabap bbaabbaaab 009 abba qaaq-ea bbaababqbq aapbaabbqb babbpbbpba bbboabpbob abqabbapqb 0v5 bbapqbbabo qbapabpbaa bbqabbqavb aqbaa bapab Paqabaqpbo q6bbaabaab 08fi oqbbaabbao baaabbqabq abbqa qabqb baabaqaaqb abb;aababa abqbaqa bbb 0Ztl ababqaoabb ~aaabab~a~ ba~ba~ba~~ ababeaaaba ~ba~bb~aaa baqbbqbaqa 09~ baaobabba a bababbabaq bbpbaqbbqa jsbabbaqbb boapboqpbq obbqaqqbaa 00~ appoababbb 00540005aq abpabvbajb babbabppba bbaaabqpba bapaababqa 0tlZ aqqoppaqbo baaabqbbaa bpaqPbbaPa qbabpQabbb abbbaabbqb 08T bqbpopbqab bbobaaabba bbabbpbbqa abba o-eaqbb Pebq3b4ao7P aaaPbPPabb 0ZT oq-eabbaqqb aabbbaVbaq baqbaqbobq abbbbbaaqp bbbbabqeoa poaba2baqa 09 bqappbbbbb qqeaqa oqb~ ~p-ebbpabbb ababbbbabb qpabaqsPbq 2aqv3qb5qP
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ctggacgagg cacgcgccga ggcggaggcg gcgctgcgag ccgccgacca ctcccggccg 1800 ggcgactgcg tgccggcgcg cctggtcctg gcccacctcg gcgtgcacca cggtgacctc 1860 gccacggcca gcgaccagtt gcgggcggcc gagcggctgg cctccgccga cgactcggcg 1920 cggatggact gggcgctggc ccggttccac gctgccagcg gccgtccggc gatgatggtg 1980 cagacgctga tcaacgtcgc cggacaggtc gcacccgatc cgctgctgtt caccgaggcg 2040 ccggccgctg cggcgacgct cgtacgccag gcccgccggg cggggctcga cgcggaggcc 2100 gagcgcgccg tggaggtcgc ccggcgcgtc gcccgcggca acccgttcgt ccagtcgctg 2160 gcggcggcgg cggaacacgc cgcgggtctc ctgcgcgacg atccggcggc gctgctgcgg 2220 gccgcggatc tgcaccggct cgccggccgt acgctcgcgg cggccggcgc ggtggaggac 2280 gcggcccgca gcacccggga ccgggccgag gccacccgtc tgctcgaggc cgcgacggac 2340 ggctaccggg agtgcggcgc gcgacgcgac ctggagcgcg tggaggccga gctgcgtggc 2400 ctgccggctc acaacgtccg cccgctggtc cccgaccggc cccggtcggg gtgggagagc 2460 ctgaccagcg cggagctgcg ggtcgtgcgg gccatcgtgg acgggatgac caaccgcgag 2520 gcggcgagtt cgctgttcct gtccccgcac accgtcgaca gtcacctgcg gcgcgtcttc 2580 tccaagctcg acatcaacag ccgggtggaa ctgacccgct gcttcatcgc gcacgaggcg 2640 gtccggccgg cgctggccac cacacgccag ccggcgtccg ccggctga 2688 <210> 8 <211> 362 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 8 Met Thr Val Gly Tyr Leu Gly Thr Val Thr Asp Ser Ala Pro Val Asp Ala Ala Leu Arg Asp Phe Phe Ala Glu Arg Arg Ala Glu Ala Arg Glu Leu Gly Asp Asp Phe Ala Ala Leu Val Ala Glu Leu Glu Ser Tyr Val Leu Arg Gly Gly Lys Arg Ile Arg Pro Ala Phe Ala Trp Leu Gly Trp Ile Gly Ala Gly Gly Asp Pro Glu Asp Pro Val Ala Thr Ala Val Leu Asn Ala Cys Ala Gly Phe Glu Leu Leu His Ala Ser Gly Leu Ile His Asp Asp Ile Ile Asp Ala Ser Gln Thr Arg Arg Gly His Pro Ala Ala His Val Ala Tyr Ala Glu Arg His Arg Ala Arg Arg Phe Ser Gly Asp Pro Gly Thr Phe Gly Thr Gly Thr Ala Ile Leu Ile Gly Asp Leu Val Leu Ile Trp Ala Asp Val Leu Val Arg Ala Ser Gly Leu Pro Ala Asp Ala His Val Arg Val Ser Pro Val Trp Ser Ala Val Arg Ser Glu Val Met Tyr Gly Gln Leu Leu Asp Leu Ile Ser Gln Val Ser Arg Ser Glu Asp Val Asp Ala Ala Leu Arg Ile Asn Gln Tyr Lys Thr Ala Ser Tyr Thr Val Glu Arg Pro Leu Gln Phe Gly Ala Ala Ile Ala Gly Ala Asp Asp Asp Leu Phe Ala Ala Tyr Arg Ala Phe Gly Ala Asp Val Gly Ile Ala Phe Gln Leu Arg Asp Asp Leu Leu Gly Val Phe Gly Asp Pro Val Val Thr Gly Lys Pro Ser Gly Asp Asp Leu Arg Glu Gly Lys Arg Thr Val Leu Leu Ala Thr Ala Leu Lys Arg Ala Asp Glu Arg Asp Pro Asp Ala Ala Ala Tyr Leu Arg Ala Lys Val Gly Thr Asp Leu Ala Asp Glu Glu Ile Ala Arg Ile Arg Ala Ile Phe Arg Asp Val Gly Ala Val Glu Glu I1e Glu Arg Gln Ile Ser Gln Arg Thr Asp Arg Ala Leu Ala Ala Leu Glu Ala Ser Ser Ala Thr Ala Pro Ala Lys His Gln Leu Ala Asp Met Ala Ile Lys Ala Thr Gln Arg Ala Gln <210> 9 <211> 1089 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 9 atgaccgtcg gatatctcgg gacggtcacc gactcggcgc ccgtcgacgc cgcgctgcgc 60 gacttcttcg ccgagcgccg cgccgaggca cgcgagctcg gcgacgactt cgcggccctg 120 gtcgccgagc tggagagcta cgtcctgcgg ggcggcaagc gcatccggcc cgccttcgcc 180 tggctgggct ggatcggcgc cggcggcgac ccggaggacc cggtggcgac cgcggtgctg 240 aacgcctgcg ccgggttcga gctgctgcac gcgtccggcc tcatccacga cgacatcatc 300 gacgcgtcgc agacccgccg cggccatccc gccgcgcacg tcgcgtacgc cgaacggcat 360 cgggcgcggc gcttctccgg tgacccggga acgttcggca ccggcaccgc catcctgatc 420 ggagacctcg tcctgatctg ggccgacgtc ctggtccgcg cctccggcct gccggccgac 480 gcgcacgtgc gggtctcgcc ggtgtggtcg gcggtgcgct ccgaggtcat gtacggccag 540 ctgctcgatc tgatcagcca ggtgagccgg agcgaggacg tcgacgcggc gctgcgcatc 600 aaccagtaca agaccgcgtc gtacacggtg gagcggccac tgcagttcgg cgcggcgatc 660 gccggcgcgg acgacgacct cttcgcggcc taccgcgcct tcggcgccga cgtgggtatt 720 gccttccagc tgcgcgacga cctgctcggc gtgttcggcg acccggtggt gacgggcaag 780 ccgtccggcg acgacctgcg ggagggcaag cggacggtcc tgctcgccac ggcgctcaag 840 cgcgccgacg aacgggaccc ggacgcggcg gcctacctgc gggcgaaggt cggcacggac 900 ctcgcggacg aggagatcgc ccgcatccgc gccatcttcc gcgacgtcgg cgcggtcgag 960 gagatcgagc ggcagatctc gcagcgcacc gaccgggcgc tggccgcgct ggaggcgagc 1020 agcgccaccg cccccgcgaa gcatcagctc gccgacatgg cgatcaaggc cacccagcgg 1080 gcccagtga 1089 <210> 10 <211> 354 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 10 Met Ser Thr Glu Pro Val Thr Val Val Ala Arg Gly Val Leu Asp Gly Arg Gly Asp Gly Pro Gly Arg Leu Gly Thr Gly Arg Ala His Gly Lys Ala Ile Leu Leu Gly Glu His Ala Val Val Tyr Gly Ala Pro Ala Leu Ala Val Pro Val Pro Gln Leu Thr Ala Val Ala Lys Ala Arg Arg Ala Gly Gly Asp Gly Gly Asp Glu Val Ser Phe Ala Ile Ala Gly Leu Glu Ser Pro Glu Val Thr Ser Leu Pro Thr Asp Gly Leu Gln His Leu Val Thr Glu Phe Arg G1n Arg Ala Ala Val Thr Glu Pro Met Arg Val Asp Val Leu Val Asp Cys Ala Ile Pro Gln Gly Arg Gly Leu Gly Ser Ser Ala Ala Cys Ala Arg Ala Ala Val Leu Ala Leu Ala Asp Ala Phe Asp Arg Arg Leu Asp Ala Ala Thr Val Phe Asp Leu Val Gln Thr Ser Glu Asn Val Ala His Gly Arg Ala Ser Gly Ile Asp Ala Leu Ala Thr Gly Ala Thr Ala Pro Leu Ile Phe Arg Asn Gly Val Gly Arg Glu Leu Pro Val Ala Met Ala Gly Ala Ala Arg Ala Ala Arg Gly Ser Gly Pro Ala Gly Phe Asp Ala Val Leu Val Ile Ala Asp Ser Gly Val Ser Gly Ser Thr Arg Asp Ala Val Glu Leu Leu Arg Gly Ala Phe Glu Arg Ser Pro Arg Thr Arg Asp Glu Phe Val Ser Arg Val Thr Ser Leu Thr Glu Ala Ala Ala His Asp Leu Leu Gln Gly Arg Val Ala Asp Phe Gly Ala Arg Leu Thr Glu Asn His Arg Leu Leu Arg Glu Val Gly Ile Ser Thr Glu Arg Ile Asp Arg Met Val Asp Ala Ala Leu Ala Ala Gly Ser Pro Gly Ala Lys Ile Ser Gly Gly Gly Leu Gly Gly Cys Met Ile Ala Leu Ala Arg Asp Arg Gln Glu Ser Ala Ala Val Val Arg Ser Val Gln Gln Ala Gly Ala Val Arg Thr Trp Thr Val Pro Met Gly Arg Phe Thr Gly His Asp Asp <210> 11 <211> 1065 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 11 atgtccacgg aaccggtgac cgtcgtcgcc cgcggcgttc tcgacggccg gggtgacggg 60 ccgggccgcc tcggcaccgg ccgcgcccac ggcaaggcca tcctgctggg cgaacacgcc 120 gtcgtgtacg gcgctccggc gctcgccgtc ccggtgccgc aactgaccgc cgtggccaag 180 gcgcggcggg ccggcggcga cggcggcgac gaggtctcct tcgccatcgc cgggctggag 240 agcccggagg tgacgtcgct tccgaccgac ggcctgcaac atctggtgac ggagttccgg 300 cagcgggccg ccgtcaccga gccgatgcgc gtcgacgtgc tcgtggactg cgccatcccg 360 cagggccggg ggctcgggtc gagcgccgcc tgcgcccgcg ccgcggtgct ggccctcgcg 420 gacgcgttcg accgccgcct cgacgccgcc acggtgttcg atctggtgca gacctcggag 480 aacgtggcgc acggccgggc cagcggcatc gacgccctgg ccaccggtgc gaccgcgccg 540 ctgatcttcc gcaacggcgt gggccgggaa ctgccggtcg ccatggcggg cgccgcgcgt 600 gccgcgcgag ggtcgggccc ggccggcttc gacgcggtgc tcgtcatcgc cgacagcggc 660 gtcagcggca gcacccggga cgcggtggag ctgctgcggg gtgccttcga gcgctccccg 720 cgcacgcgcg acgagttcgt cagccgggtg accagcctga ccgaggcggc ggcgcacgac 780 ctgctccagg gccgggtcgc cgacttcggc gcgcggctga ccgagaacca ccggctgttg 840 cgcgaggtcg gcatcagcac cgaacggatc gaccggatgg tcgacgccgc gctcgcggcg 900 ggcagcccgg gcgccaagat cagcggcggt ggcctgggcg gctgcatgat cgcactggcc 960 cgggaccgcc aggaatccgc ggcggtggtg cggagcgtcc agcaggccgg cgccgtccgc 1020 acctggaccg tcccgatggg gaggttcacc ggccatgacg actga 1065 <210> 12 <211> 346 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 12 Met Thr Thr Asp His Arg Ala Glu Pro Ser Glu Pro Ala Leu Asp Arg Pro Ala Thr Ala Val Ala His Pro Asn Ile Ala Leu Ile Lys Tyr Trp Gly Lys Arg Asp Glu Gln Leu Met Ile Pro Tyr Ala Asp Ser Leu Ser Met Thr Leu Asp Val Phe Pro Thr Thr Thr Thr Val Arg Ile Asp Ser Gly Ala Ala Ala Asp Glu Val Val Leu Asp Gly Ser Pro Ala Asp Gly Glu Arg Arg Gln Arg Val Val Thr Phe Leu Asp Leu Val Arg Lys Leu Ala Gly Arg Thr Glu Arg Ala Cys Val Asp Thr Arg Asn Ser Val Pro Thr Gly Ala Gly Leu Ala Ser Ser Ala Ser Gly Phe Ala Ala Leu Ala Leu Ala Gly Ala Ala Ala Tyr Gly Leu Asp Leu Asp Thr Thr Ala Leu Ser Arg Leu Ala Arg Arg Gly Ser Val Ser Ala Ser Arg Ser Val Phe Gly Gly Phe Ala Met Cys His Ala Gly Pro Gly Ala Gly Thr Ala Ala Asp Leu Gly Ser Tyr Ala Glu Pro Val Pro Val Ala Pro Leu Asp Val Ala Leu Val Ile Ala Ile Val Asp Ala Gly Pro Lys Ala Val Ser Ser Arg Glu Gly Met Arg Arg Thr Val Arg Thr Ser Pro Leu Tyr Gln Ser Trp Val Ala Ser Gly Arg Ala Asp Leu Ala Glu Met Arg Ala Ala Leu Leu Gln Gly Asp Leu Asp Ala Val Gly Glu Ile Ala Glu Arg Asn Ala Leu Gly Met His Ala Thr Met Leu Ala Ala Arg Pro Ala Val Arg Tyr Leu Ala Pro Val Thr Val Ala Val Leu Asp Ser Val Leu Arg Leu Arg Ala Asp Gly Val Ser Ala Tyr Ala Thr Met Asp Ala Gly Pro Asn Val Lys Val Leu Cys Arg Arg Ala Asp Ala Asp Arg Val Ala Asp Thr Leu Arg Asp Ala Ala Pro Ser Cys Ala Val Val Val Ala Gly Pro Gly Pro Ala Ala Arg Pro Asp Pro Gly Ser Arg Pro <210> 13 <211> 1041 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 13 atgacgactg accaccgggc ggagccgtcc gagccggcgc tcgaccggcc cgcgaccgcc 60 gtggcccatc cgaacatcgc gctgatcaag tactggggca agcgcgacga gcagctgatg 120 atcccgtacg ccgacagcct gtcgatgacg ctcgacgtct tcccgaccac caccaccgtc 180 cggatcgaca gcggcgcggc ggccgacgag gtcgtcctcg acggctcgcc cgccgacggc 240 gaacggcgac agcgcgtcgt caccttcctg gacctggtac gcaagctggc cgggcgcacg 300 gaacgggcct gcgtcgacac ccgcaactcc gtgcccaccg gcgccggcct ggcgtcctcg 360 gcgagcggat tcgccgccct cgccctcgcc ggcgccgccg cgtacggcct cgacctggac 420 accaccgcgc tgtcccgcct ggcccggcgg ggatccgtgt cggcctcccg gtcggtcttc 480 ggcggcttcg cgatgtgcca cgcaggcccc ggcgccggga ccgccgcgga cctcggctcc 540 tacgccgagc cggtgcccgt cgcgcccctc gacgtcgcgc tggtgatcgc gatcgtcgac 600 gccgggccga aggcggtgtc gagccgcgag gggatgcggc gaaccgtccg gacctccccg 660 ctctatcagt cgtgggtcgc ctccggccgc gccgacctgg ccgagatgcg ggccgcgctg 720 ctccagggag acctggacgc ggtcggcgag atcgccgaac gcaacgccct cggcatgcac 780 gccaccatgc tggccgcccg gccggcggtg cgctacctgg cgccggtcac tgtcgccgtg 840 ctcgacagcg tgctgcgcct gcgcgccgac ggcgtctccg cctacgccac gatggacgcg 900 ggaccgaacg tcaaggtgct ctgccgccgc gcggacgccg accgggtcgc cgacaccctg 960 cgcgacgccg cgccgagctg cgccgtggtc gtcgccggac cggggccggc ggcccggccg 1020 gacccgggca gccggccgtg a 1041 <210> 14 <211> 369 <212> PRT
<213> Micromonospora sp. strain 046-ECO1.1 <400> 14 Val Thr Gly Pro Gly Ala Val Arg Arg His Ala Pro Gly Lys Leu Phe Val Ala Gly Glu Tyr Ala Val Leu Glu Pro Gly His Pro Ala Leu Leu Val Ala Val Asp Arg Gly Val Asp Val Thr Val Ser Gly Ala Asp Ala His Leu Val Val Asp Ser Asp Leu Cys Pro Glu Gln Ala Cys Leu Arg Trp Gln Asp Gly Arg Leu Val Gly Ala Gly Asp Gly Gln Pro Ala Pro Asp Ala Leu Gly Ala Val Val Ser Ala Ile Glu Val Val Gly Glu Leu Leu Thr Gly Arg Gly Leu Arg Pro Leu Pro Met Arg Val Ala Ile Thr Ser Arg Leu His Arg Asp Gly Thr Lys Phe Gly Leu Gly Ser Ser Gly Ala Val Thr Val Ala Thr Val Thr Ala Val Ala Ala Tyr His Gly Val Glu Leu Ser Leu Glu Ser Arg Phe Arg Leu Ala Met Leu Ala Thr Val Arg Asp Gly Ala Asp Ala Ser Gly Gly Asp Leu Ala Ala Ser Val Trp Gly Gly Trp Ile Ala Tyr Gln Ala Pro Asp Arg Ala Ala Val Arg Glu Met Ala Arg Arg Arg Gly Val Glu Glu Thr Met Arg Ala Pro Trp Pro Gly Leu Arg Val Arg Arg Leu Pro Pro Pro Arg Gly Leu Ala Leu Glu Val Gly Trp Thr Gly Glu Pro Ala Ser Ser Ser Ser Leu Thr Gly Arg 8~
006 aqPbbbaqaB baaqbbPbaP baPbbqabPb aabqq3b4b3 Pabbaaabbb abbaaqbbPa 0IV8 apabqabqap Pbbpaapbap babbbbabPb bqababapba qpaabaaeqb abqbqb;bpb 08L be aabPq bao bi?aa-eaqqab 12bbqbbabab baaabeabbb babb-qbbaaa qaabaabbqa OZL bbabbbaa,2b 4qba-4abPab Pab12babbaa b-ebabbaaeb bqabbbqbb-e bbqababaqa 099 abb-4babaae aa2aabqabb abbaaqbbba b-4aabbbaab b-jaaababab ab;Pba215pb 009 bPba;babba babbabbaba bb-4 LDb-ebaba b-jbaabbaba baaPbaaaba bbeaa-e;aab Ot'S a~~bb~abba bbbbqa:[bab Pbabaabb-la qob~bbabba a~aaba~baa babba-eb;ba 08IV b-qbbaebabb qabqpbabb-:~ abbaaq:jbba ba-4PPbaqab a:[bqabPbbj bbbbaPaaPq 0Ztl babaabbqb-e abaaebqbba paabaqbpap bqbbabbbba bpbaqbbba; aabbaqqbPP
09~ bapabbapba baapabqabb aabPaapaqp babbqbbbab qpaaabqaba aababqabbb 00~ -eba-ebbaaPb -q3aa.a72Pbab ba:lbb-qbb-eb a~~babba~a qbb~baabab baqa3ab325 0:~Z aaababbaab Pabbbapbab bbababbaqb aqabbaabba PbbPabbqbb abqaabqbab 08T bpabpbbaaa bqaqaa-ebaa qapbaqbqqb aqaapaaaba Pbaababbaa qaqbaapaqb 0ZZ apbbqbpbbb bPaPbaqbba bbqbbqabqa babbaaapaa bbbaabpbbq abqbbabapq 09 bpbqbbaaba qbaqqbqabp Pabbbaabab apaabaabab qbaababbba aabbaapbqb ST <00:~>
TT00H-9V0 uzPa4s =ds PaodsououiozazN <~TZ>
KNQ <ZZZ>
OTTT <TTZ>
ST <0ZZ>
bzy 59~ 09~ SS~
PTV zaS ~TO usV aus uT0 STH TPA uT0 2TI ozd qaN oad na7 T~A -I~TO
OS~ Sf7~ 0IV~
PTV PTV PTK dzZ uT0 nT0 bsV na2 baV PTV sus bzV Jus PTV PTK zus SEE 0~~ 5Z~
PTV dsV naZ na2 PTK aTI ATO s.F0 dsv ~TO ~TO r, TO PTV ~TO aaS oad 0Z~ STE OTE SO~
s,~2 eTV PTV -,~TO ~TO TPn zus nT0 PTV PTv dsv sz~0 na7 PTV aus na'i bzV ozd aus aud aTI -erTO naZ bzV TPA nT0 dsV dsV na7 nT0 PTv naZ

IPA szH bxy PTy baV baV TpA uT0 STH na2 na2 nTD uT0 dsV dsK ATO

baK nT0 na2 PTV dSX aTI PTV aus bzV TPA SA0 nT0 uT0 zag bay zaS
SSZ 0SZ 5tlZ
aus aud aaS das bav PTV oaa aaS ATO baV das bav aaS PTV PTV narI
ZO-90-600Z O6~996Z0 Fi'J

ttcacccccc ggctgacggc gctgtgcgac gccgccgaga ccgtcggcgg cgcggccaaa 960 ccgtccggcg ccggtggcgg ggactgcggc atcgcgttgc tggacgccac cgccgcgacg 1020 cggaccgcgc ggctgcgcga gcagtgggcc gccgccgggg tgctccccat gccgatccag 1080 gtccatcaga cgaacgggag cgcgcgatga 1110 <210> 16 <211> 360 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 16 Met Ile Ala Asn Arg Lys Asp Asp His Val Arg Leu Ala Ala Glu Gln Gln Gly Arg Leu Gly Gly His His Glu Phe Asp Asp Val Ser Phe Val His His Ala Leu Ala Gly Ile Asp Arg Ser Asp Val Ser Leu Ala Thr Ser Phe Gly Gly Ile Asp Trp Pro Val Pro Leu Cys Ile Asn Ala Met Thr Gly Gly Ser Thr Lys Thr Gly Leu Ile Asn Arg Asp Leu Ala Ile Ala Ala Arg Glu Thr Gly Val Pro Ile Ala Thr Gly Ser Met Ser Ala Tyr Phe Ala Asp Glu Ser Val Ala Glu Ser Phe Ser Val Met Arg Arg Glu Asn Pro Asp Gly Phe Ile Met Ala Asn Val Asn Ala Thr Ala Ser Val Glu Arg Ala Arg Arg Ala Val Asp Leu Met Arg Ala Asp Ala Leu Gln Ile His Leu Asn Thr Ile Gln Glu Thr Val Met Pro Glu Gly Asp Arg Ser Phe Ala Ala Trp Gly Pro Arg Ile Glu Gln Ile Val Ala Gly Val Gly Val Pro Val Ile Val Lys Glu Val Gly Phe Gly Leu Ser Arg Glu Thr Leu Leu Arg Leu Arg Asp Met Gly Val Arg Val Ala Asp Val Ala Gly Arg Gly Gly Thr Asn Phe Ala Arg Ile Glu Asn Asp Arg Arg Asp Ala Ala Asp Tyr Ser Phe Leu Asp Gly Trp Gly Gln Ser Thr Pro Ala Cys Leu Leu Asp Ala Gln Gly Val Asp Leu Pro Val Leu Ala Ser Gly Gly Ile Arg Asn Pro Leu Asp Val Val Arg Gly Leu Ala Leu Gly Ala Gly Ala Ala Gly Val Ser Gly Leu Phe Leu Arg Thr Leu Leu Asp Gly Gly Val Pro Ala Leu Leu Ser Leu Leu Ser Thr Trp Leu Asp Gln Ile Glu Ala Leu Met Thr Ala Leu Gly Ala Arg Thr Pro Ala Asp Leu Thr Arg Cys Asp Leu Leu Ile Gln Gly Arg Leu Ser Ala Phe Cys Ala Ala Arg Gly Ile Asp Thr His Arg Leu Ala Thr Arg Ser Gly Ala Thr His Glu Met Ile Gly Gly Ile Arg <210> 17 <211> 1083 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 17 atgatcgcca accgcaagga cgaccacgtc cggctcgccg ccgagcagca gggccggctc 60 ggcggtcacc acgagttcga cgacgtgtcc ttcgtgcacc acgccctggc cggcatcgac 120 cggtccgacg tctcgctggc cacgtcgttc ggcggcatcg actggccggt gccgctgtgc 180 atcaacgcga tgaccggcgg cagcaccaag accggcctga tcaaccggga cctggcgatc 240 gcggcccggg agaccggcgt accgatcgcc accgggtcga tgagcgccta cttcgccgac 300 gagtcggtgg ccgagagttt cagcgtgatg cgccgggaga accccgacgg gttcatcatg 360 gccaacgtca acgccaccgc ctccgtcgaa cgggcccggc gggctgtcga cctgatgcgg 420 gccgacgcgc tgcagatcca cctgaacacc atccaggaga cggtgatgcc ggagggggac 480 cggtcgttcg ccgcctgggg gccgcggatc gaacagatcg tcgccggcgt cggtgtgccg 540 gtgatcgtca aggaggtcgg cttcgggctc agccgcgaaa cgctgctgcg gctgcgggac 600 atgggcgtcc gggtggccga cgtcgccggc cgcggcggca cgaacttcgc gcgcatcgag 660 aacgaccggc gggacgccgc cgactactcc ttcctcgacg ggtggggaca gtcgacaccc 720 gcctgcctgc tggacgccca gggcgtggac ctgcccgtgc tggcctccgg cggcatccgc 780 aacccgctcg acgtggtccg cgggctggcg ctcggcgccg gcgcggccgg ggtgtccgga 840 ctgttcctgc gcacgctcct ggacggcggc gtgccggcgc tgctgtcgct gctgtccacc 900 tggctcgacc agatcgaagc cctgatgacc gccctgggcg cgcggacccc ggccgacctg 960 acccgctgcg acctgctgat ccagggtcgg ctgagcgcgt tctgcgcggc ccggggcatc 1020 gacacccacc gcctcgccac ccgttccggc gccacccacg agatgatcgg aggcattcga 1080 tga 1083 <210> 18 <211> 351 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 18 Met Asn Asp Ala Ile Ala Gly Val Pro Met Lys Trp Val Gly Pro Val Arg Ile Ser Gly Asn Val Ala Gln Ile Glu Thr Glu Val Pro Leu Ala Thr Tyr Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala Lys Ile Ser Arg Met Val Glu Ala Gly Ile Val Ala Thr Leu Val Asp Glu Arg Met Thr Arg Ser Val Phe Val Arg Ala Lys Asp Ala Gln Thr Ala Tyr Leu Ala Ser Leu Glu Val Asp Ala Arg Phe Asp Glu Leu Arg Asp Ile Val Arg Thr Cys Gly Arg Phe Val Glu Leu Ile Gly Phe His His Glu Ile Thr Ala Asn Leu Leu Phe Leu Arg Phe Ser Phe Thr Thr Gly Asp Ala Ser Gly His Asn Met Ala Thr Leu Ala Ala Asp Ala Leu Leu Lys His Ile Leu Asp Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile Ser Gly Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile Leu Gly Arg Gly Lys Asn Val Val Thr Glu Leu Val Val Pro Arg Glu Ile Val His Asp Ser Leu His Thr Thr Ala Ala Ala Ile Ala G1n Leu Asn Val His Lys Asn Met Ile Gly Thr Leu Leu Ala Gly Gly Ile Arg Ser Ala Asn Ala His Tyr Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala Thr Gly Gln Asp Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Thr Val Ala Glu Asp Arg Asp Gly Asp Leu Tyr Phe Ser Cys Thr Leu Pro Asn Leu Ile Val Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val Glu Glu Asn Leu Glu Arg Leu Gly Cys Arg Ala Ser Arg Asp Pro Gly Glu Asn Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys Gly Glu Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met Arg Ala His Val Arg Leu Glu Arg Pro Thr Glu Thr Thr Lys Ile Gly Ala <210> 19 <211> 1056 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 19 atgaacgacg cgatcgccgg tgtgcccatg aaatgggtag gtcccgtgcg gatctcggga 60 aacgtggcgc agatcgagac ggaggttccg ctcgccacgt acgagtcgcc gctctggccg 120 tccgtcggcc ggggcgcgaa gatctcccgg atggtcgagg cgggcatcgt cgccacgctc 180 gtcgacgagc gcatgacccg ctcggtgttc gtgcgcgcca aggacgcgca gaccgcctac 240 ctggcctcgc ttgaggtcga cgcgcggttc gacgaactgc gtgacatcgt gcgcacctgc 300 ggcaggttcg tcgagctgat cgggttccac cacgagatca ccgcgaacct gctgttcctg 360 cggttcagtt tcaccaccgg cgacgcgtcc gggcacaaca tggcgacgct ggccgccgac 420 gcgctgctga agcacatcct ggacaccatt ccgggcatct cgtacggctc gatctcgggc 480 aactactgca ccgacaagaa ggccaccgcg ataaacggca ttctcggccg gggcaagaac 540 gtggtcaccg agctggtcgt gccgcgggag atcgtccacg acagcctgca cacgacggcg 600 gcggcgatcg cccagctgaa cgtgcacaag aacatgatcg gcacgttgct cgccggcggt 660 atccgctcgg ccaacgccca ctacgcgaac atgctgctcg ggttctacct ggccacgggt 720 caggacgccg cgaacatcgt cgagggctcc cagggcgtga cggtcgccga ggaccgcgac 780 ggcgacctct acttctcctg cacgctgccc aacctgatcg tgggcaccgt cggcaacggc 840 aaggggctcg gcttcgtcga ggagaacctg gagcggctcg gctgccgcgc ctcgcgtgat 900 ccgggcgaga acgcccggcg gctcgcggtc atcgcggccg cgacggtgct ctgcggcgag 960 ctgtccctgc tcgccgcgca gaccaacccg ggcgagctga tgcgggcgca cgtccggctc 1020 gaacgcccga ccgagaccac gaagatcgga gcctga 1056 <210> 20 <211> 391 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 20 Met Ala Glu Arg Pro Ala Val Gly Ile His Asp Leu Ser Ala Ala Thr Ala His His Val Leu Thr His Glu Thr Leu Ala Ala Ser Asn Gly Ala Asp Val Ala Lys Tyr His Arg Gly Ile Gly Leu Arg Ala Met Ser Val Pro Ala Pro Asp Glu Asp Ile Val Thr Met Ala Ala Ala Ala Ala Ala Pro Val Val Ala Arg His Gly Thr Asp Arg Ile Arg Thr Val Val Phe Ala Thr Glu Ser Ser Val Asp Gln Ala Lys Ala Ala Gly Ile His Val His Ser Leu Leu Gly Leu Pro Ser Ala Thr Arg Val Val Glu Leu Lys Gln Ala Cys Tyr Gly Gly Thr Ala Gly Leu Gln Phe Ala Ile Gly Leu Val His Arg Asp Pro Ser Gln Gln Val Leu Val Ile Ala Ser Asp Val Ser Lys Tyr Ala Leu Gly Glu Pro Gly Glu Ala Thr Gln Gly Ala Ala Ala Val Ala Met Leu Val Gly Ala Asp Pro Ala Leu Val Arg Val Glu Asp Pro Ser Gly Met Phe Thr Ala Asp Val Met Asp Phe Trp Arg Pro Asn Tyr Arg Thr Thr Ala Leu Val Asp Gly His Glu Ser Ile Ser Ala Tyr Leu Gln Ala Leu Glu Gly Ser Trp Lys Asp Tyr Thr Glu Arg Gly Gly Arg Thr Leu Asp Clu Phe Gly Ala Phe Cys Tyr His Gln Pro Phe Pro Arg Met Ala Asp Lys Ala His Arg His Leu Leu Asn Tyr Cys Gly Arg Asp Val Asp Asp Ala Leu Val Ala Gly Ala Ile Gly His Thr Thr Ala Tyr Asn Ala Glu Ile Gly Asn Ser Tyr Thr Ala Ser Met Tyr Leu Gly Leu Ala Ala Leu Leu Asp Thr Ala Asp Asp Leu Thr Gly Arg Thr Val Gly Phe Leu Ser Tyr Gly Ser Gly Ser Val Ala G1u Phe Phe Ala Gly Thr Val Val Pro Gly Tyr Arg Ala His Thr Arg Pro Asp Gln His -Vtl 06Z <TTZ>
ZZ <OTZ>
9LTT bPqPbo boob-2bo-eqo jbobobePoP oqbboa qoqo O'vTT bbba obbqob booP;boobb ba oPooebqb bi2bboobobb ooql2qa Pbob bobbopbo:~b 080T booo~~oobo PobPboPabq qb-ebbbooPj boPboboPqo PboqPb*ebbe obbobbooeb OZOT o~-ebobba bo boopobPa oe bo3oebobo2 oeobaboboo Pqbbbaoobq boqb-4opD5b 096 ooboqq3qq.0 i2bo3baq5ob Pobbooqbbb oi?qobeo;oo ;qobboqboo ebboobbooe 006 b-qooPboPbo obaoPoeboq objo-eobbob oqobbboqo~ P-:~bqPbo-qbo bboPo72-:~3bP
0fI8 oPPobbo-4Pb PbaoboPPoe jboboo-eooP oPobbboaPo abbb5oo.65-a bbaoboboPb 08L aPboqboPbo bo5b5obqa -2 qoPeoqobqo oPa bbooPob obbePoPboo bbqPbbPboo 0ZL a;qboobeoo -2ooPq35qoq qbobobboqq b2bopb5-4oD opabo-4bbob bobobPbooP
099 oeqo72bbPPb bqbo0obbb-e bbqobobbPo bqooP-4aobo o;oqPoo;b-2 bo-eobbbapb 009 oqbbjooobo oP3oPoboa2 -qoppbz)obbo bbqo:~IoPbb q72oqbaPboo booP a q q b'l 0t'5 obbbo~booo 12bbPbo;bob oeqbbqobob booopbbobo bboqboqobq Poobo-4bbob 08IV boboobobbb PoooPbobb-e bobba oobPb qbbbqobobo P;bppboqbq boPbobPaa b OZlv oqPbqbbqoo qbbpobpobo qbooopbqbo opobqbbqoa bboqpoa boq qbpobqopbb 09E bobbopqbbo bbapqabqa a bbPDbPL:>bqo bpboqbbqbb b3oopoobbo qoaooqoobb OOE oqobqoooqo Pooqbopapq Pbbboa bbob bppbobbpoo Pboqbboqbo qbPbboPoob OlvZ oqqbqboqbo opbbooqpbb ooPbooPobb opoobooa ba qbbqbboaba booboa baa b 08T qobqobbqPb opbqboqpop bbPboPbboo oobooobqbo bpbqpbobbb obqobbboqp 0ZT obbqba opa D PqbPPa obbq bopboobobb oppa bpbobo obbqoa oebp bopopopbqo 09 bqbopoqvob obba pba boo booqbqoopb opooqpobbo qbooba oopb iebpba obbqp ZZ <00'v>
TTODH-9f,0 uz~-Tqs -ds PaodsouoIUo-TozW <EZ Z>
VNQ <ZTZ>
9LTT <ZTZ>
TZ <0TZ>

baV ozd nT~ a,~Z TPn bav s,~2 08~ SLE OLE
STH ~TD aa8 naZ ~TD PTV naz baV aAZ oad ~TD aus zus TPA nTE) oad S9E 09E SS~
PZV 03d zrs dsV ~-,TD ~TE) dsV ZPn ozd aud pTV s?H nTf) STH na2 nTE) OS~ SIV~ OlVE
baV xrs zus PTV z,~Z dsV aTi nTE) uTE) bzV bzV dsV aTI PTV PTV baV
SEE 0~E SZE
ZO-90-600Z O6~996Z0 Fi'J

<212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 22 Val Ala Glu Leu Tyr Ser Thr Ile Clu Glu Ser Ala Arg Gln Leu Asp Val Pro Cys Ser Arg Asp Arg Val Trp Pro Ile Leu Ser Ala Tyr Gly Asp Ala Phe Ala His Pro Glu Ala Val Val Ala Phe Arg Val Ala Thr Ala Leu Arg His Ala Gly Glu Leu Asp Cys Arg Phe Arg Thr His Pro Asp Asp Arg Asp Pro Tyr Ala Ser Ala Leu Ala Arg Gly Leu Thr Pro Arg Thr Asp His Pro Val Gly Ala Leu Leu Ser Glu Val His Arg Arg Cys Pro Val Glu Ser His Gly Ile Asp Phe Gly Val Val Gly Gly Phe Lys Lys Ile Tyr Ala Ala Phe Ala Pro Asp Glu Leu Gln Val Ala Thr Ser Leu Ala Gly Ile Pro Ala Met Pro Arg Ser Leu Ala Ala Asn Ala Asp Phe Phe Thr Arg His Gly Leu Asp Asp Arg Val Gly Val Leu Gly Phe Asp Tyr Pro Ala Arg Thr Val Asn Val Tyr Phe Asn Asp Val Pro Arg Glu Cys Phe Glu Pro Glu Thr Ile Arg Ser Thr Leu Arg Arg Thr Gly Met Ala Glu Pro Ser Glu Gln Met Leu Arg Leu Gly Thr Gly Ala Phe Gly Leu Tyr Val Thr Leu Gly Trp Asp Ser Pro Glu Ile Glu Arg Ile Cys Tyr Ala Ala Ala Thr Thr Asp Leu Thr Thr Leu Pro Val Pro Val Glu Pro Glu Ile Glu Lys Phe Val Lys Ser Val Pro Tyr Gly Gly Gly Asp Arg Lys Phe Val Tyr Gly Val Ala Leu Thr Pro Lys Gly Glu Tyr Tyr Lys Leu Glu Ser His Tyr Lys Trp Lys Pro Gly Ala Val Asn Phe Ile <210> 23 <211> 873 <212> DNA
<213> Micromonospora sp. strain 046-EC01l <400> 23 gtggccgagc tctactcgac catcgaggaa tcggcccggc aactggacgt gccgtgttcg 60 cgcgaccggg tctggcccat cctgtccgcg tacggcgacg cgttcgccca tcccgaggcg 120 gtggtcgcct tccgggtggc gaccgcgctg cgtcacgcgg gcgagctgga ctgccggttc 180 cggacgcatc cggacgaccg ggacccgtac gcctcggcgc tcgcccgggg cctcaccccg 240 cgcacggacc accccgtcgg cgcgctgctc tccgaggtcc accggcgctg cccggtggag 300 agccacggca tcgacttcgg ggtggtcggc ggcttcaaga agatctacgc ggccttcgcc 360 ccggacgagc tgcaggtggc cacgtcgctc gccggcattc cggcgatgcc ccgcagcctc 420 gccgcgaacg ccgacttctt cacccggcac ggcctcgacg accgggtcgg cgtgctggga 480 ttcgactacc cggcccggac cgtgaacgtc tacttcaacg acgtgccgcg tgagtgcttc 540 gagccggaga ccatccggtc gacgctgcgc cggaccggga tggccgagcc gagcgagcag 600 atgctccggc tcggcaccgg ggcgttcggg ctctacgtca cgctgggctg ggactccccg 660 gagatcgagc ggatctgcta cgccgcggcg accacggacc tgaccacgct tccggtaccc 720 gtggaaccgg agatcgagaa gttcgtgaaa agcgttccgt acggcggcgg ggaccggaag 780 ttcgtctacg gcgtggcgct gacccccaag ggggagtact acaaactcga gtcgcactac 840 aaatggaagc cgggcgcggt gaacttcatt tga 873 <210> 24 <211> 370 <212> PRT
<213> Micromonospora sp. strain 046-EC01l <400> 24 Val Trp Ala Arg Val Lys Asn Trp Val Val Ala Leu Ala Val Ala Ala Val Leu Met Ile Ser Ala Leu Ala Gly Asp His Pro Ala Pro Glu Gly Leu Gly Leu Leu Gly Phe Ala Leu Val Ala Ala Ser Gly Leu Ala Leu Ala Ala Ser Arg Arg Ala Pro Ile Ala Val Leu Val Ala Thr Gly Leu Cys Val Val Gly Tyr Asn Ala Ile Gly Phe Gly Val Pro Ala Ile Ala Tyr Leu Phe Ala Val Tyr Ala Ala Val Arg Ala Gly His Arg Leu Val Thr Leu Gly Ala Ser Ala Ala Leu Leu Val Val Leu Pro Leu Ala Ile Met Val Ser Pro Ala Asp Gly Ala Leu Lys Glu Ala Leu Ala Gln Ser Arg Gly Val Leu Glu Leu Ala Trp Leu Ile Ala Ala Ala Ala Ala Gly Glu Ala Leu Arg Gln Ala Glu Arg Arg Ala Asp Glu Ala Glu Arg Thr 145 150 1.55 160 Arg Glu G1u Thr Ala Arg Leu Arg Ala Thr Gln Glu Arg Leu His Ile Ala Arg Glu Leu His Asp Ser Leu Thr His Gln Ile Ser Ile Ile Lys Val Gln Ala Glu Val Ala Val His Leu Ala Arg Lys Arg Gly Glu Gln Val Pro Glu Ser Leu Leu Ala Ile Gln Glu Ala Gly Arg Ala Ala Thr Arg Glu Leu Arg Ala Thr Leu Glu Thr Leu Arg Asp Leu Thr Lys Ser Pro Ser His Gly Leu Asp His Leu Pro G1u Leu Leu Ala Gly Ala Glu Lys Ile Gly Leu Ala Thr Thr Leu Thr Ile Glu Gly Asp Gln Arg Asp Val Pro Glu Ala Val Gly Arg Thr Ala Tyr Arg Ile Val Gin Glu Ser Leu Thr Asn Thr Ala Arg His Ala Ser Ala Ala Ala Ala Ala Val Arg Ile Asp Tyr Arg Pro Asp Ala Leu Ser Ile Arg Ile Asp Asp Asp G1y Thr Ala Arg Pro G1y Ala Ala Pro Val Pro Gly Val Gly Leu Leu Gly Met His Glu Arg Val Leu Ala Leu Gly Gly Arg Leu Arg Ala Glu Pro Arg Thr Gly Gly Gly Phe Thr Val Gln Ala Glu Leu Pro Val Val Arg Va1 Pro <210> 25 <211> 1113 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 25 gtgtgggccc gggtgaagaa ctgggtcgtc gcgttggctg tggcggcggt gctgatgatc 60 agcgcgctgg ccggtgacca tcctgccccc gagggcctcg gtctgctcgg cttcgcgctg 120 gtggcggcga gcggcctggc gctggccgcc agtcgtcggg ccccgatcgc cgtgctggtc 180 gccaccgggc tgtgcgtggt gggctacaac gcgatcggct tcggggtgcc cgccatcgcg 240 tacctgttcg cggtctacgc ggcggtccgg gccgggcacc ggctcgtcac gctcggggcg 300 agcgccgccc tgctcgtcgt cctgccgctg gcgatcatgg tctcgcccgc ggacggcgcc 360 ctcaaggagg cgctcgcgca gtcgcggggc gtgctggaac tggcctggct gatcgccgcg 420 gcggcggccg gtgaggcgct gcggcaggcc gaacggcgag cggacgaggc ggaacggacc 480 cgcgaggaga ccgcccggct gcgcgccacc caggagcggc tgcacatcgc acgggagctg 540 cacgactcgc tcacccacca gatctcgatc atcaaggtgc aggcggaggt ggcggtccac 600 ctggcccgca agcggggcga gcaggtgccg gagtcgctgc tggcgatcca ggaggccggc 660 cgggcggcga ctcgcgagct gcgcgcgacc ctggagacgc tgcgtgacct gaccaagtcc 720 ccgtcgcacg ggctcgacca cctcccggag ctgctggccg gggccgagaa gatcggcctg 780 gccaccacgc tgaccatcga gggcgaccag cgggacgtgc cggaggcggt gggccgcacc 840 gcgtaccgga tcgtgcagga gtcgctcacc aacaccgccc ggcacgcctc cgccgcggcc 900 gccgcggtcc ggatcgacta ccgcccggac gcgctgagca tccggatcga cgacgacggg 960 acggcccggc cgggcgccgc cccggtgccc ggcgtcgggc tgctggggat gcacgagcgc 1020 gtcctcgcgc tgggcggccg gctgcgggcg gaaccccgca ccggcggagg cttcaccgtc 1080 caggccgaac tcccggtggt gcgcgtccca tga 1113 <210> 26 <211> 220 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 26 Met Ile Arg Ile Met Leu Leu Asp Asp Gln Pro Leu Leu Arg Ser Gly Phe Arg Ala Leu Leu Asp Ala Glu Asp Asp Ile Glu Val Val Ala Glu Gly Gly Asn Gly Arg Glu Gly Leu Ala Leu Ala Arg Gln His Leu Pro Asp Leu Ala Leu Ile Asp Ile Gln Met Pro Val Met Asp Gly Val Glu Thr Thr Arg Gln Ile Val Ala Asp Pro Ala Leu Ala Gly Val Arg Val Val Ile Leu Thr Asn Tyr Gly Leu Asp Glu Tyr Val Phe His Ala Leu Arg Ala Gly Ala Thr Gly Phe Leu Val Lys Asp Ile Glu Pro Asp Asp Leu Leu His Ala Val Arg Val Ala Ala Arg Gly Asp Ala Leu Leu Ala Pro Ser Ile Thr Arg Met Leu Ile Asn Arg Tyr Val Ser Glu Pro Leu Cys Ala Asp Val Thr Pro Gly Met Glu Glu Leu Thr Asn Arg Glu Arg Glu Ala Val Ala Leu Ala Ala Arg Gly Leu Ser Asn Asp Glu Ile Ala Asp Arg Met Val Ile Ser Pro Leu Thr Ala Lys Thr His Val Asn Arg Ala Met Thr Lys Leu Gln Ala Arg Asp Arg Ala Gln Leu Val Val Phe Ala Tyr Glu Ser Gly Leu Val Ser Pro Gly Asn Arg <210> 27 <211> 663 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 27 atgatcagga tcatgctgct cgacgaccag ccgctgctgc gcagcgggtt ccgcgcgctc 60 ctcgacgccg aggacgacat cgaggtggtg gccgagggcg ggaacggccg ggagggcctg 120 gcgctggccc ggcagcacct gcccgatctc gccctgatcg acatccagat gccggtcatg 180 gacggcgtcg agacgacccg gcagatcgtc gcggatccgg cgctggccgg ggtacgcgtc 240 gtcatcctca ccaactacgg cctcgacgag tacgtcttcc acgcgctgcg cgccggcgcc 300 accggcttcc tggtcaagga catcgagccg gacgacctgc tgcacgccgt gcgggtcgcc 360 gcgcgcggtg acgcgctgct cgcgccgtcg atcacccgga tgctgatcaa caggtacgtg 420 tcggagccgc tctgcgcgga cgtcacgccc ggcatggagg agctgaccaa ccgggaacgc 480 gaggcggtcg ccctggccgc ccggggcctg tccaacgacg agatcgccga t:cgcatggtg 540 atcagcccgc tgaccgcgaa gacccacgtc aaccgcgcca tgaccaagct gcaggcccgc 600 gaccgcgccc agctggtggt gttcgcctac gagtccggcc tggtgtcacc cggcaatcgc 660 tga 663 <210> 28 <211> 131 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 28 Met Phe I1e Arg Arg Leu Leu Thr Ala Ala Ala Ala Gly Val Leu Gly Gly Leu Ala Leu Val Ala Pro Ala Ala Ala Gln Val Thr Ala Ala Asp Gly Asp Gly Gly Ser Gly Arg Ala Gly Ser Val Leu Ala Leu Ala Leu Ala Leu Leu Gly Leu Val Leu Gly Gly Trp Ala Leu Arg Ser Ala Gly Arg Gly Gly Gly Arg Gly Asn Ala Ile Ala Ala Leu Val Leu Ala Val Ala Gly Leu Ile Ala Gly Val Val Ala Leu Ala Gly Ser Asp Gly Gly Val Gly Ser Gly Asn Gly Arg Gly Gly Ala Ile Val Ala Val Val Leu Ala Leu Ile Gly Ile Ala Val Gly Gly Leu Ala Phe Thr Arg Ser Arg Arg Ala Ala <210> 29 <211> 396 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 29 atgttcatcc gtcgtttgct caccgccgcc gcagccggcg tcctcggtgg gctcgcactc 60 gtcgcaccgg cggccgcgca ggtgacggcc gccgacggtg acggtggttc cggccgcgcc 120 ggatccgtgc tggcgctcgc gctcgcgttg ctcggcctcg tcctgggcgg gtgggcgttg 180 cgctccgcgg ggcgcggcgg cggtcgtggc aacgcgatcg ccgcgctggt gctcgcggtg 240 gccggcctga tcgccggcgt ggtcgccctg gccggctccg acggtggtgt cggcagcggc 300 aacggccgtg gtggcgccat cgtggccgtc gtgctggcgc tgatcgggat cgccgtcggc 360 ggcctggcat tcacccgctc ccggcgcgcc gcctga 396 <210> 30 <211> 154 <212> PRT
<213> Micromonospora sp. strain 046-ECOll <400> 30 Met Arg Lys Val Phe Ala Gly Leu Ala Ala Phe Leu Leu Leu Val Leu Val Val Gln Phe Phe Leu Ala Ala Ser Gly Ala Phe Ser Asn Glu Ala Asn Glu Glu Ala Phe Arg Pro His Arg Ile Leu Gly Leu Gly Ser Ile Leu Val Ala Val Val Leu Thr Val Ala Ala Ala Val Met Arg Met Pro Gly Arg Ile Ile Gly Leu Ser Gly Leu VVal Ala Gly Leu Gly Ile Leu Gln Ala Leu Ile Ala Val Ile Ala Lys Ala Phe Gly Asp Ser Ala Gly Asp Ser Ala Val Gly Arg Tyr Val Phe Gly Leu His Ala Val Asn Gly Leu Val Met Val Ala Val Ala Arg Val Ile Leu Arg Ser Val Arg Ala Ala Pro Asp Thr Thr Thr Thr Pro Gly Val Asp Thr Thr Val Thr Gly Pro Ala Ala Asp Ser Ala Arg Thr Ala Ser <210> 31 <211> 465 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 31 atgcgcaaag tgttcgccgg actggcagcg ttcctgctgc tcgtgctcgt ggtgcagttc 60 ttcctggccg ccagcggcgc gttcagcaac gaggccaacg aggaggcgtt ccgccctcac 120 cggatcctgg gcctggggag catcctcgtc gccgtggtgc tgacggtggc c:gccgcggtg 180 atgcggatgc ccggccggat catcggcctg tccggcctgg tcgccgggct gggcatcctg 240 caggccctga tcgcggtcat cgccaaggcg ttcggcgact cggccggtga ctcggccgtc 300 ggccggtacg tgttcggcct gcacgcggtc aacggactgg tgatggtggc cgtcgcccgc 360 gtcatcctgc gcagcgtccg ggcggcgccg gacacgacca ccacgcccgg cgtggacacg 420 acggtcaccg gtccggcggc cgactcggcg cgaacggcgt catga 465 <210> 32 <211> 661 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 32 Met Ser Thr Leu Gln Trp Ile Leu Val Asp His Val Val Ala Leu Leu Gly Val Ala Thr Trp Phe Ala Thr Gly Val Thr Ala Ala Leu Gly Arg His Arg Ile Ala Leu Ala Leu Leu Gly Ala Ala Val Leu Val Thr Val Ala Arg Leu Gly Thr Val Ala Leu Leu Ala Asp Arg Gly Trp Trp Phe Val Gln Glu Lys Val Leu Leu Gly Leu Pro Met Leu Gly Ala Ala Gly Leu Val Ala Val Leu Leu Ala Gly Pro Arg Leu Leu Ala Ala Arg Gln Ser Pro Ala Ala Asp Leu Pro Ala Gly Ala Leu Val Ala Val Leu Thr Ala Gly Phe Ala Ala Leu Ala Gly Leu Val Val Thr Phe Thr Ala Gly Tyr Pro Leu Thr Trp Ser Thr Ala Leu Ile Ala Val Ala Leu Val Cys Ala Ala Ala Leu Leu Thr Ala Arg Val Val Gly Arg Pro Ala Ala Pro Ala Ala Glu Ala Gly Ser Pro Glu His Thr Pro Ala Ala Ala Gly Pro Thr Ala Leu Ser Arg Arg Arg Phe Leu Gly Val Ala Gly Gly Val Val Ala Ala Gly Ala Gly Ala Thr Gly Val Gly Leu Leu Phe Arg Asp Pro Glu Ala Met Val Thr Gly Gly Gly Pro Gly His Ala Gly Gly Ala Arg Pro Lys Val Ser Val Ala Asp Leu Arg Gly Pro Gly Ala Pro Ala Ala Gly Gly Thr Ala Arg Arg His Val Leu Thr Ala Arg Thr Gly Thr Val Thr Ile Pro Ser Gly Arg Pro Ile Asp Ala Trp Ser Tyr Glu Gly Arg Leu Pro Gly Pro Ala Ile Thr Ala Thr Glu Gly Asp Leu Ile Glu Val Thr Leu Arg Asn Ala Asp Ile Glu Asp Gly Val Thr Val His Trp His Gly Tyr Asp Val Pro Cys Gly Glu Asp Gly Ala Pro Gly Ala Thr Gln His Ala Val Gln Pro Gly Gly Glu Phe Val Tyr Arg Phe Gln Ala Asp Gln Val Gly Thr Tyr Trp Tyr His Thr His Gln Ala Ser His Pro Ala Val Arg Lys Gly Leu Tyr Gly Thr Leu Val Val Thr Pro Arg Glu Asp Arg Pro Glu Ala Glu Arg Gly Leu Asp Leu Thr Leu Pro Val His Thr Phe Asp Asp Val Thr Ile Leu Gly Asp Gln Glu Gly Arg Ala Val His Asp Val Arg Pro Gly Gln Pro Val Arg Leu Arg Leu Ile Asn Thr Asp Ser Asn Pro His Trp Phe Ala Val Val Gly Ser Pro Phe Arg Val Val Ala Val Asp Gly Arg Asp Leu Asn Gln Pro Gly Glu Val Arg Glu Val Gly Leu Arg Leu Pro Ala Gly Gly Arg Tyr Asp Leu Thr Leu Ala Met Pro Asp Ala Lys Val Thr Leu Leu Leu Asp Asn Asp Ser Asp Gln Gly Val Leu Leu Arg Pro Pro Gly Val Gly Gly Gly Asp Arg Pro Leu Pro Asp Thr Ala Asp Trp Pro Glu Phe Asp Leu Leu Gly Tyr Gly Glu Pro Ala Pro Val Pro Phe Asp Ala Asp Asp Ala Asp Arg His Phe Thr Ile Val Leu Asp Arg Ala Leu Ala Met Val Asp Gly Lys Pro Ala Tyr Ala Gln Thr Val Asp Gly Arg Ala His Pro Ser Val Pro Asp Gln Leu Val Arg Glu Gly Asp Val Val Arg Phe Thr Val Val Asn Arg Ser Leu Glu 5,65 570 575 Thr His Pro Trp His Leu His Gly His Pro Val Leu Ile Leu Ser Arg Asp Gly Arg Pro Tyr Ser Gly Ser Pro Leu Trp Met Asp Thr Phe Asp Val Arg Pro Gly Glu Val Trp Glu Val Ala Phe Arg Ala Asp Asn Pro Gly Val Trp Met Asn His Cys His Asn Leu Pro His Gln Glu Gln Gly Met Met Leu Arg Leu Val Tyr Asp Gly Val Thr Thr Pro Phe Ala Ser Thr Ser His Ala His <210> 33 <211> 1986 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 33 atgagcacgc tccaatggat cctcgtggac cacgtcgtgg cgctgctcgg tgtcgcgacg 60 tggttcgcaa cgggtgtcac ggcagctctc ggccgccacc ggatcgcgtt ggcgctcctc 120 ggcgccgcgg tgctggtgac agtcgcccgc ctgggcaccg tggcgctgct ggccgaccgc 180 ggctggtggt tcgtccagga gaaggttctg ctggggctgc cgatgctcgg cgccgcgggg 240 ctcgtcgcgg tgctcctggc cggcccgcgc ctgctcgcgg cccggcagtc accggcggcg 300 t,S
TTOOH-9V0 ut~~~s ds -eaodsououzozozW <~TZ>
s'dd <ZTZ>
6ZT <TTZ>
IV ~ <OTZ>

986T eb-40po 086T 23bo20352b 01205-GoOba-a aoooba -eoa -e oqbqbbo-ebo -e;o;bo;abb ob:~obqpbqL-OZ6T obbb-2ob72bb -eoo-eoboob? oo~~~~~~~~ 3-eo3-epbq-eb bqoqbqbbb~ ~q-e-2o-ebba b 098T bbooqqba bb ~bb~bbb~b~ bb-eb-25bbaa b5obqboi2b~ ~~~oi2oeb5q -e5bqb131500 008T obeabboDqo eqboobboob boebDboooq bqDoqpbqob qbbooqpoob ba eobqoovo 0:~LT bbqbooopoo opppboqoob pbboovpoqb bqbba poqqa bobqboqbD2 bbbbbpbbbo 089T DqboqobpoD VbDaDaqboD qDDDIPaPDb obDqbbopbD IbODPb2DDD baP;bDbDoo 0Z9T j55q-GO3bbj DDDbbb33~2b D4O3q53;BD OPDIIDPDDb aDP5005DPb 09ST opboDbOPb3 jqboobqboo oba5b33bQb obbopqobbb -jobq33pboq 4bPb0oDbbI
OoST opbooboopa Pbboobqa bo ooboapbqbb qbbobboqbo bbbooboDDb z)b:~Dbqooqb OV:K Dbbbi2a 3pbo oqa eboevop boqobqa bqo bopoqbb2po oboebboa 6q Pa obbqooap 08~T bqoovbopqb boDbbpbboo boaobqa abo aqobbbDqbb 2bobopqbbp bDbbboobpo 0Z~T o-22a ;a oeb3 b=bba pboq booB5155qb obooqqooob a qobboqboq booboqqbb;
09ZT opobDooppo a qoebDovop poqpbqoqbo bqapbobqbb a obeoobboo oa booqbavb 00ZT o~~o~ba ob~ ba pbbb-ebbp oa pbobbaqo 3;ebapoqbo -2bo2b3j;ba popobqbboo Ot,TT bqobDpb~~o -abbqa bbbDb Dbpbba bppb boDbboovbb -2boba boobo Pbqboqboqo 080T bapbbboeqb qabbbppeDb Dblboa bDoo Dpobaqbobb paovoa ovoE Dopq6b~3-eq 0Z0T bovbbbbqbb e~~2.6bobbs oD;qbboos; a jboqjb2bo bbobbooabe abqbj5ob32o 096 bpa bDeooba bbbDobobDb 5o2bbeba bB 35qbosbqbD pbo2q5bbo2 abbqopobqb 006 o~~~~b~bba 2bbpb~qi23P baa b~e-eobo oqobo,2bqb5 vboqpbqoov bobbbpboop 0IV8 boboo~~q-eo obboobbboo 35qoo5a Dbb b2bD-2:~abpb bqoa bapboq pba o;bo~ebb 08L DoqbDa qqvb o-ea qb~o-eob bbopbbooob DopoqDbqbo i2ooboebDbo bbopobbobb OZL bDbba bboal obabboa a Db bDbob~~o-eb ba5bqb3ojD qbbppooa ob Doa bobbqbb 099 ooBa2opb6D a oobbobbpb boDpoqbbq2 bDbbebbooo 2boboo;ja q objoobboqb 009 0550OVa 353 bboobobbbo bbobaqbbqb Pbbbbboobb qbobbaqooq 4bbooboO53 0lvs ooqbqobobfi ovooa bbbao bbobba bboa bopopobvbb ooa oqa bboa bbpbbaboa b 08V b3o=6=5a oa pbopbboq bbqbbboba b oopoqobqab a ba oboa bob qojba qooob OZIV oqboobaI-eb qa boba ovab ebbqba pbqo booopqbbbo oboa poqqba pbqbbabbqD
09~ obboobbqob 3bDoba jqa b ba ob~o-ebqo bqbboboqbb qoba babboa bboobqoo2b ZO-90-600Z O6~996Z0 Fi'J

<400> 34 Met Thr Ala Asp Leu His Gly Leu Ala Ser Val Arg Tyr Ile Val Asp Asp Val Ser Ala Ala Ile Glu Phe Tyr Thr Thr His Leu Gly Phe Thr Val Ser Thr Ala Phe Pro Pro Ala Phe Ala Asp Val Val Arg Gly Pro Leu Arg Leu Leu Leu Ser Gly Pro Thr Ser Ser Gly Ala Arg Val Thr Pro Ala Asp Ala Ala Gly Cys Gly Arg Asn Arg Ile His Leu Ile Val Asp Asp Leu Asp Ala Glu Arg Glu Arg Leu Glu Arg Ala Gly Val Thr Leu Arg Ser Asp Val Val Ala Gly Pro Gly Gly Arg Gln Phe Leu Ile Ala Asp Pro Ala Gly Asn Leu Val Glu Val Phe Glu Pro Ala Ala Arg Gly <210> 35 <211> 390 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 35 atgaccgcag acctgcacgg cctggccagc gtccgctaca tcgtcgacga cgtgtcggcg 60 gcgatcgagt tctacaccac ccacctgggt ttcacggtgt cgaccgcgtt cccgccggcc 120 ttcgccgacg tggtgcgcgg gccgctgcgg ctcctgctgt ccgggccgac cagctcgggc 180 gcccgggtca ccccggcgga cgcggccggg tgcgggcgca accgcatcca cctgatcgtc 240 gacgatctcg acgccgaacg ggagcggctg gagcgcgccg gggtgacgtt gcgcagcgac 300 gtcgtggccg ggccgggcgg ccgtcagttc ctgatcgccg acccggcggg caacctggtc 360 gaggtgttcg agccggcagc ccgcggctga 390 <210> 36 <211> 178 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 36 Met Leu Thr Ala Val Val Ala Ser Pro His Ser Pro Glu Asn Thr Ser Arg His Pro Thr Gly Gly Asp Ala Val Asp Glu Ala Thr Pro Arg Thr Pro Val Ala Ala Arg Pro Thr Trp Ser Pro Ala Thr Ala Pro Val Trp Leu Val Gly Val Leu Ala Thr Leu Ala Gly Ala Val Ala Ala Glu Ala Phe Thr Leu Ala Ala Arg Gly Phe Gly Val Pro Met Glu Ala Ala Gly Val Trp Glu Glu Gln Ala Gln Ala Ile Pro Val Gly Ala Ile Ala Arg Ser Val Val Leu Trp Ser Ile Gly Gly Ile Val Leu Ala Val Val Val Ala Arg Arg Ala Arg Arg Pro Val Arg Ala Phe Val Ala Gly Thr Val Ala Phe Thr Val Leu Ser Leu Ala Ala Pro Ala Phe Ala Arg Asp Thr Pro Val Ser Thr Gln Leu Val Leu Ala Gly Thr His Val Ile Ala Gly Ala Val Ile Ile Ser Ile Leu Ala Ala Arg Leu Ala Ala Pro Thr Pro Pro Arg <210> 37 <211> 537 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 37 atgttgactg ccgtcgtggc gtccccgcat tctcccgaga acacatcgag gcacccgacc 60 ggaggcgacg ccgtggatga ggccactccc cgaactcccg tcgcggcacg gcccacctgg 120 tcgccggcca ccgctccggt gtggctggtc ggcgtgctgg ccaccctcgc cggggccgtg 180 gccgcggagg cgttcacgct cgccgcccgg ggcttcggcg taccgatgga ggcggccggc 240 gtctgggagg agcaggcgca ggcgatcccg gtgggggcca tcgcccgcag cgtcgtgctc 300 tggtcgatcg gcggaatcgt cctggcggtg gtcgtggcgc ggcgggcccg gcggcccgtg 360 cgtgccttcg tggccggcac cgtcgcgttc accgtgctgt ccctcgccgc gcccgccttc 420 gcccgggaca ccccggtgtc gacgcagctc gtcctcgccg gcacccacgt gatcgccggc 480 gccgtgatca tctccatcct ggccgcgcgg ctcgccgcgc ccaccccgcc ccggtaa 537 <210> 38 <211> 661 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 38 Met Asp Gly Thr Glu Ser Asn Val Thr Gly Phe Pro Asp Leu Leu Ser Gly Leu Gly Gly Asp Gly Arg Ala Phe Ala Leu Leu His Arg Pro Gly Ala Ala Gly Cys Ala Tyr Val Glu Val Leu Thr Gly Glu Val Cys Asp Val Asp Thr Leu Gly Glu Leu Pro Leu Pro Thr Glu Pro Ala Thr Gly Ala Arg His Asp Leu Leu Val Ala Val Pro Tyr Arg Gln Val Thr Glu Arg Gly Phe Asp Cys His Asp Asp Gly Ala Pro Leu Leu Ala Met Arg Val His Glu Gln Phe Gly Leu Asp Arg Gly Gln Ala Leu Ala Gly Leu Pro Glu Arg Gly Val Pro Val Thr Asp Ala Asp Phe Asp Leu Ser Asp Glu Asp Tyr Ala Ala Ile Val Lys Arg Val Val Gly Asp Glu Ile Gly Leu Gly Ala Gly Ser Asn Phe Val Ile Arg Arg Thr Phe Thr Ala Arg Leu Ala Asp Tyr Ser Ile Ala Thr Glu Leu Ala Leu Phe Arg Arg Leu Leu Thr Gly Glu Leu Gly Ser Tyr Trp Thr Phe Leu Phe His Ser Gly Ala Gly Thr Phe Ile Gly Ala Ser Pro Glu Arg His Val Ser Met Ile Asp Gly Thr Val Ser Met Asn Pro Ile Ser Gly Thr Tyr Arg His Pro Pro Asn Gly Pro Ala Val Ser Gly Leu Leu Glu Phe Leu Asn Asp Pro Lys Glu Ala Asn Glu Leu Tyr Met Val Val Asp Glu Glu Leu Lys Met Met Ala Arg Met Cys Ala Ser Gly Gly Gln Val His Gly Pro Phe Leu Lys Glu Met Ala Arg Val Thr His Ser Glu Tyr Ile Leu Thr Gly Arg Ser Asp Leu Asp Val Arg Asp Val Leu Arg Glu Thr Leu Leu Ala Pro Thr Val Thr Gly Ser Pro Ile Glu Asn Ala Phe Arg Val Ile Thr Arg His Glu Thr Thr Gly Arg Gly Tyr Tyr Gly Gly Val Leu Ala Leu Met Gly Arg Asp Ser Ala Gly Ser Arg Thr Leu Asp Ser Ala Ile Met Ile Arg Thr Ala Glu Ile Asp Asp Ala Gly Thr Leu Arg Leu Gly Val Gly Ala Thr Leu Val Arg Asp Ser Lys Pro Glu Ser Glu Val Ala Glu Thr Arg Ala Lys Ala Gly Ala Met Arg Ala Ala Leu Gly Leu Gly Val Asp Pro Asp Gly Pro Asp Gly Gly Arg Thr Thr Ala Ala Arg Ala Arg Ser Ser Leu Ala Thr Asp Pro Arg Val Arg Arg Ala Leu Arg Glu Arg Asn Thr Thr Leu Ser Arg Phe Trp Leu Asp Gly Ala Glu Arg Arg Thr Pro Asn Pro Ala Leu Thr Gly Arg Arg Val Leu Val Val Asp Asn Glu Asp Thr Phe Met Ala Met Leu Asp His Gln Leu Arg Ala Leu Gly Leu Arg Ser Ser Ile Ala Arg Phe Asp Ser Arg Leu Arg Pro Asp Gly His Asp Leu Val Val Val Gly Pro Gly Pro Gly Asp Pro Gly Asp Leu Thr Asp Pro Arg Met Arg Thr Leu Arg Gly Leu Thr Arg Asp Leu Leu Ala Gly Thr Val Pro Phe Leu Ser Ile Cys Leu Gly His Gln Val Leu Ala Ala Glu Leu Gly Phe Pro Leu Ala Arg Arg Ala Val Pro Asn Gln Gly Val Gln Lys Arg Ile Asp Leu Phe Gly Arg Pro Glu Leu Val Gly Phe Tyr Asn Thr Tyr Thr Ala Arg Ser Ala His Asp Val Val Ala Gly Gly Arg Arg Gly Pro Ile Glu Ile Ser Arg Ser Pro Asp Ser Gly Asp Va1 His Ala Leu Arg Gly Pro Gly Phe Arg Ser Val Gln Phe His Leu Glu Ser Val Leu Thr Gln His Gly Pro Arg Ile Leu Gly Asp Leu Leu Val Ser Leu Leu Ala Asp Gly Thr Ala Ala Ala Ala Ala Glu Ala Ala Gly Arg Arg Gly Asn Arg Pro 0'vL't ooPoeqoopo bbqboqopeb Toabboobbo ~~b~oo2bo~ ~bbob~~b~o 089T bqbqbbbeoo ~-eooobqbbo bobobbooob oqonoooqqb bbbqoPeboo Booboqobqb OZ92 bLOOLOObbb qaDbqDqLDD qbqDQqabDD b4bb~P12b53 3b343bq3DL bObODD2DqD
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TT,009-9t0 uTPzqs ds Pzodsououzoza zN <~ZZ>
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ZO-90-600Z O6~996Z0 FiJ

gcccgctccg cgcacgacgt ggtggccggt ggccggcggg gcccgatcga gatcagccgc 1800 agcccggaca gcggggacgt gcacgcgctg cgcggcccgg gattccgttc cgtccagttc 1860 cacctggagt ccgtcctcac ccagcacggc ccacggatcc tgggcgacct gctggtctcc 1920 ctgctcgccg acggcacggc cgccgccgcg gccgaggcgg cgggccggcg cgggaaccgc 1980 ccgtga 1986 <210> 40 <211> 427 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 40 Val Lys Thr Thr Val Asp Val Leu Val Gln Lys Tyr Gly Gly Thr Ser Leu Gln Thr Leu Asp Arg Val Arg His Ala Ala Leu Arg Ile Ala Glu Ala Arg Arg His Gly Ser Ala Val Thr Val Val Val Ser Ala Arg Gly Ser Arg Thr Asp Asp Leu Leu Arg Leu Ala Ala Asp Val Gly Ala Ala Gly Pro Ser Arg Glu Leu Asp Gln Leu Leu Ala Val Gly Glu Ser Glu Ser Ala Ala Leu Met Ala Leu Ala Leu Thr Gly Leu Gly Val Pro Ala Val Ser Leu Thr Gly His Gln Ala Glu Ile His Thr Thr Asp Arg His Gly Asp Ala Leu Ile Ser Arg Ile Gly Ala Ala Arg Val Gl.u Ala Ala Leu Gly Arg Gly Glu Val Ala Val Val Thr Gly Phe Gln Gly Ile Asp Arg Ala Gly Asp Val Ala Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala Leu Ala Ala Arg Leu Arg Ala Ser Ala Cys Glu Ile Tyr Thr Asp Val Asp Gly Val Phe Ser Ala Asp Pro Arg Ile Leu Pro Ala Ala Arg Cys Leu Pro Trp Val Glu Pro Gly Val Met Ala Glu Met Ala Phe Ala Gly Ala Arg Val Leu His Thr Arg Cys Ile Glu Leu Ala Ala Met Glu Gly Val Glu Val Arg Val Arg Asn Ala Ser Ser Gln Ala Pro Gly Thr Ile Val Val Asp Arg Pro Asp Asp Arg Pro Leu Glu Thr Arg Arg Ala Val Val Ala Val Thr His Asp Thr Asp Val Val Arg Val Leu Val His Cys Arg Asp Gly Arg Arg Asp Met Ala Pro Asp Val Phe Glu Va1 Leu Ala Ala His Gly Ala Val Ala Asp Leu Val Ala Arg Ser Gly Pro Tyr Glu Ser Glu Phe Arg Met Gly Phe Thr Ile Arg Arg Ser Gln Ala Glu Ala Val Arg Thr Ala Leu His Asp Leu Thr Ala Ser Phe Asp Gly Gly Val His Phe Asp Glu Asn Val Gly Lys Val Ser Val Val Gly Met Gly Leu Leu Ser Arg Pro Glu His Thr Ala Arg Leu Met Ala Ala Leu Ala Ala Ala Gly I1e Ser Thr Ser Trp Ile Ser Thr Ser Gln Met Arg Leu Ser Val Ile Val Ser Arg Asp Arg Thr Val Asp Ala Val Glu Ala Leu His Arg Ala Phe Arg Leu Asp Arg Ser Glu Pro Ala Asp Ala Thr Ser Leu Thr Ser Arg Arg Ser Ala Thr Ala <210> 41 <211> 1284 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 41 gtgaagacga ctgtggacgt gctggtccag aaatacgggg gcacctcgct gcagaccctc 60 gaccgcgttc ggcacgccgc gctgcggatc gccgaggcgc ggcggcacgg ctccgccgtg 120 acagtggtcg tgtcggcgcg cggcagccgg accgacgacc tgctgcggct ggcggccgac 1.80 gtcggcgccg cgggtccgtc ccgggaactc gaccagttgc tcgcagtcgg cgagtccgag 240 tcggcggcgc tgatggcgct ggcgttgacc gggctgggag tgccggccgt ctcgctgacc 300 gggcaccagg cggagatcca caccaccgac cggcacggcg acgcgctgat ctcgcggatc 360 ggggcggcgc gggtggaagc ggcgctgggc cgtggcgagg tcgccg~~ggt caccggattc 420 cagggcatcg accgggccgg tgacgtcgcc acgctggggc gcggcggctc cgacacgaca 480 gcggtggcgc tcgcggcccg gctccgcgcg tcggcgtgcg agatctacac cgacgtggac 540 ggcgtcttca gcgccgaccc ccgcatcctt ccggcggcgc gttgcctgcc gtgggtggag 600 cccggcgtca tggcggagat ggcgttcgcc ggcgcgcggg tcctgcacac ccgatgcatc 660 gagctggccg ccatggaagg ggtcgaagtg cgcgtgcgca acgcgtcgtc gcaggcgccc 720 ggaacgatag tcgtggaccg gcccgacgac cggccgctgg agacccggcg ggccgtggtg 780 gcggtcaccc acgacaccga tgtcgtccgc gtgctggtgc actgccgcga cggccgccgg 840 gacatggcac ccgacgtgtt cgaggtgctg gccgcccatg gggcggtggc ggacctggtg 900 gcccggtccg ggccctacga gagcgagttc cggatggggt tcaccatccg ccgcagccag 960 gccgaagcgg tgcggaccgc gctgcacgac ctcaccgcgt ccttcgacgg cggggtccac 1020 ttcgacgaga acgtcggcaa ggtgtccgtg gtcggcatgg gcctgctcag ccgccccgag 1080 cacacggccc ggctgatggc ggcgctggcc gcggcgggga tctcgacgag ctggatctcc 1140 acctcccaga tgcggctgtc ggtgatcgtg tcgcgggacc gcaccgtcga cgccgtcgaa 1200 gccctgcacc gcgcgttccg cctggaccgg tccgagccgg cggacgccac gtccctgacc 1260 tcccgccgtt ccgccaccgc ctga 1284 <210> 42 <211> 274 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 42 Val Ala Val Leu Asn Ala Ser Phe Ala Arg Gly Leu Arg Leu Arg Arg Leu Phe Arg Arg Gly Asp Gly Arg Leu Leu Val Val Pro Leu Asp His Ser Val Thr Asp Gly Pro Leu Arg Arg Gly Asp Leu Asn Ser Leu Leu Gly Glu Leu Ala Gly Thr Gly Val Asp Ala Val Val Leu His Lys Gly Ser Leu Arg His Val Asp His Gly Trp Phe Gly Asp Met Ser Leu Ile Val His Leu Ser Val Ser Thr Arg His Ala Pro Asp Pro Asp Ala Lys Tyr Leu Val Ala His Val Glu Glu Ala Leu Arg Leu Giy Ala Asp Ala Val Ser Val His Val Asn Leu Gly Ser Pro Gln Glu Ala Arg Gln Ile Ala Asp Leu Ala Ala Val Ala Gly Glu Cys Asp Arg Trp Asn Val Pro Leu Leu Ala Met Val Tyr Ala Arg Gly Pro Gin Ile Thr Asp Ser Arg Ala Pro Glu Leu Val Ala His Ala Ala Thr Leu Ala Ala Asp Leu G1y TZODa-9T70 ur2a4s =ds 7ezodsououiozozW <~TZ>
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Pro Gly Ala Arg Glu Arg Leu Leu Ala Ala Leu Arg Glu Cys Gly Leu Ala Arg Ala Val Val Cys Ala Gly Gly Thr Ile Asp Leu Asp Arg Leu Ser Arg Gln Leu Val Thr Gly Gly His Val Glu Thr Asp Ala Asp Asn Asp Ala Val Ala Ala Ala Cys Ala Gly Thr Asp Gly Arg Leu Val Pro Phe Phe Phe Ala Asn Pro His Arg Pro Ala Glu Ala Tyr Arg Ala Arg Ala Ala Glu Phe Arg Gly Leu Glu Ile Ser Pro Ala Val His Gly Val Ala Leu Thr Asp Pro Arg Val Ala Asp Leu Val Ala Val Ala Ala Glu Phe Asp His Pro Val Tyr Val Val Cys Leu Asp Arg Pro Gly Ala Gly Val Ala Asp Leu Val Gly Leu Ser Arg Arg Phe Pro Gln Val Ser Phe Val Leu Gly His Ser Gly Val Gly Asn Ile Asp Leu Tyr Ala Leu Thr Leu Ile Gln Asp Glu Pro Asn Ile Ser Leu Glu Thr Ser Gly Gly Tyr Thr Cys Val Ala Glu Ala Ala Leu Arg Arg Leu Gly Asp Asp Arg Val Val Phe Gly Ser Glu Tyr Pro Leu Gln His Pro Ala Val Glu Leu Ala Lys Phe Gln Ala Leu Arg Leu Pro Pro Glu Arg Trp Arg Arg Ile Ala Trp Asp Asn Ala His Arg Leu Leu Gly Glu Glu Lys Arg <210> 47 <211> 762 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 47 atgaccgcgc agccggtgct ggacttccac gtacgcctgg cgccccggcc cggggcgcgg 60 gagcggctgc tcgccgcgct gcgcgagtgc gggctggcgc gggcggtggt gtgcgcgggc 120 ggcaccatcg acctggaccg gctgtcccgc cagctcgtca ccggcggcca cgtcgagacc 180 gacgccgaca acgacgcggt ggcggcggcc tgcgccggca ccgacggccg gctggtgccg 240 ttcttcttcg ccaacccgca ccggccggcc gaggcgtacc gggcccgcgc cgccgagttc 300 cgcggcctgg agatctcacc cgccgtccac ggcgtcgccc tgaccgaccc gcgggtcgcc 360 gacctcgtgg ccgtggcggc ggagttcgac catccggtgt acgtggtctg cctggaccga 420 cccggcgcgg gcgtggccga cctggtcggc ctgagccgcc ggttcccgca ggtgagcttc 480 gtgctcgggc acagcggcgt cggcaacatc gacctctacg ccctgaccct gatccaggac 540 gagccgaaca tctcgctgga gacctccggc ggctacacct gcgtggccga ggcggcgcta 600 cgccgcctcg gcgacgaccg ggtggtgttc ggctccgagt acccgctgca gcacccggcc 660 gtggaactgg ccaagttcca ggcgttgcga ctgccgccgg agcggtggcg gcggatcgcc 720 tgggacaacg cgcatcgact gctaggagag gagaagcggt ga 762 <210> 48 <211> 438 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 48 Val Ser Glu Pro Ser Ser Ser Leu Pro Arg Leu Gly Gln Trp His Gly Leu Glu Asp Leu Arg Arg Leu Gln Glu Lys Gln Leu Ala Glu Thr Phe Thr Trp Ala Ala Arg Ser Pro Phe Tyr Arg Ala Arg Leu Ala Ser Gly Ala Pro Pro Val Thr Pro Ala Asp Leu Ala Asp Leu Pro Leu Thr Thr Lys Gln Asp Leu Arg Asp Asn Tyr Pro Phe Gly Met Leu Ala Val Pro Arg Glu Arg Leu Ala Thr Tyr His Glu Ser Ser Gly Thr Ala Gly Lys Pro Thr Pro Ser Tyr Tyr Thr Ala Glu Asp Trp Thr Asp Leu Ala Glu Arg Phe Ala Arg Lys Trp Ile Gly Met Ser Ala Asp Asp Val Phe Leu Val Arg Thr Pro Tyr Ala Leu Leu Leu Thr Gly His Leu Ala His Ala Ala Ala Arg Leu Arg Gly Ala Thr Val Val Pro Gly Asp Asn Arg Ser Leu Ala Met Pro Tyr Ala Arg Val Val Arg Val Met His Asp Leu Asp Val Thr Leu Thr Trp Ser Val Pro Thr Glu Cys Leu Ile Trp Ala Ala Ala Ala Ile Ala Ala Gly His Arg Pro Asp Ile Asp Phe Pro Ala Leu Arg Ala Leu Phe Val Gly Gly Glu Pro Met Thr Asp Ala Arg Arg Arg Arg Ile Ser Arg Leu Trp Gly Val Pro Val Ile Glu Glu Tyr Gly Ser Thr Glu Thr Gly Ser Leu Ala Gly Glu Cys Pro Glu Gly Arg Leu His Leu Trp Ala Asp Arg Ala Leu Phe Glu Val Tyr Asp Pro Asp Thr Gly Ala Val Arg Ala Asp Gly Asp Gly Gln Leu Val Val Thr Pro Leu Phe Arg Glu Ala Met Pro Leu Leu Arg Tyr Asn Leu Glu Asp Asn Val Ser Val Ser Tyr Asp Asp Cys Gly Cys Gly Trp Lys Leu Pro Thr Val Arg Val Leu Gly Arg Ser Ala Phe Gly Tyr Arg Val Gly Gly Thr Thr Ile Thr Gln His Gln Leu Glu Glu Leu Val Phe Ser Leu Pro Glu Ala His Arg Val Met Phe Trp Arg Ala Lys Ala Glu Pro Ala Leu Leu Arg Val Glu Ile Glu Val Ala Ala Ala His Arg Val Ala Ala Glu Ala Glu Leu Thr Ala Ala Ile Arg Ala Ala Phe Gly Val Asp Ser Glu Val Thr Gly Leu Ala Pro Gly Thr Leu Ile Pro Leu Asp Ala Leu Thr Ser Met Pro Asp Val Val Lys Pro Arg Ser Leu Phe Gly Pro Asp Glu Asp Trp Ser Lys Ala Leu Leu Tyr Tyr <210> 49 <211> 1317 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 49 gtgagcgagc caagttcgag cctgccccgg ctcggccagt ggcacggcct cgaggacctg 60 cggcgcctcc aggagaagca actggcggag acgttcacct gggcggcccg gtcgccgttc 120 taccgggcgc ggctggcctc cggcgcgccg ccggtgacgc ccgccgacct ggccgacctg 180 ccgctgacca ccaagcagga cctgcgggac aactacccct tcggcatgct cgccgtgccc 240 cgcgaacggc tggcgaccta ccacgagtcg agcgggaccg ccgggaagcc caccccctcc 300 tactacaccg cggaggactg gaccgacctg gcggagcgct tcgcccgcaa gtggatcggc 360 atgtccgccg acgacgtctt cctggtccgc acgccgtacg cgctgctgct gaccgggcat 420 ctcgcccacg ccgcagcccg gctgcgtggg gccacggtgg tacctggcga caaccggtcg 480 ctggcgatgc cgtacgcccg ggtggtccgg gtgatgcacg acctggacgt cacgctcacc 540 tggtcggtgc cgacggagtg cctgatctgg gccgccgcgg cgatcgcggc cgggcaccgg 600 cccgacatcg acttcccggc gctgcgcgcg ctgttcgtcg gcggcgagcc gatgaccgac 660 gcccgccggc ggcggatcag ccgcctgtgg ggggtgccgg tcatcgagga gtacggctcg 720 acggagaccg gcagcctggc cggggagtgc cccgagggac gcctgcacct gtgggccgac 780 cgggcgctgt tcgaggtgta cgacccggac accggcgccg tccgcgcgga cggcgacggc 840 cagctcgtgg tcacgccgct gttccgggag gcgatgccgc tgctgcggta caacctggag 900 gacaacgtgt cggtctccta cgacgactgc ggatgcggct ggaagctgcc caccgtgcgg 960 gtgctcggcc ggtcggcgtt cggctaccgg gtcggcggca ccaccatcac ccagcaccag 1020 ctggaggaac tggtcttctc cctgccggag gcgcaccggg tgatgttctg gcgggccaag 1080 gcggagccgg cgctgttgcg ggtcgagatc gaggtggccg ccgcgcaccg ggtcgccgcc 1140 gaggcggagc tgaccgccgc gatccgggcc gccttcggcg tggacagcga ggtcaccggc 1200 ctggcgccgg gaaccctgat cccgctcgac gcgctgacca gcatgccgga cgtggtgaag 1260 ccacgcagcc tgttcggtcc ggacgaggac tggagcaaag cgctcctcta ctactga 1317 <210> 50 <211> 396 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 50 Met Pro Gln Met Arg Val Ala Val Ala Gly Ala Gly Ile Ala Gly Leu Ala Phe Ala Ala Ala Leu Arg Arg Thr Gly Ile Asp Cys His Val Tyr Glu Gln Ala Asp Gln Leu Met Glu Val Gly Ala Gly Val Gln Val Ala Pro Asn Ala Thr Arg Leu Leu His Arg Leu Gly Leu Arg Asp Arg Leu Arg Thr Val Ala Va1 Ala Pro Gln Ala Ile Glu Met Arg Arg Trp Asp Asp Gly Thr Leu Leu Gln Arg Thr Gln Leu Gly Ser Val Cys Gly Arg Arg Phe Gly Ala Pro Tyr Tyr Val Val His Arg Ala Asp Leu His Ser Ser Leu Leu Ser Leu Val Pro Pro Asp Arg Val His Leu Gly Ala Arg 115 = 120 125 Leu Thr Ala Val Thr Gln Thr Ala Asp Glu Ala Tyr Leu His Leu Ser Asn Gly Thr Thr Val Ala Ala Asp Leu Val Val Gly Ala Asp Gly Ile His Ser Val Ala Arg Glu Gln Ile Val Ala Asp Arg Pro Arg Phe Ser Gly.Gln Ser Ile Tyr Arg Gly Leu Val Pro Ala Glu Arg Val Pro Phe Leu Leu Thr Glu Pro Arg Val Gln Leu Trp Phe Gly Pro Asp Gln His Cys Val Cys Tyr Pro Val Ser Ala Gly Arg Gln Val Ser Phe Gly Ala Thr Val Pro Ala Thr Asp Trp Arg Gln Glu Ser Trp Ser Gly Arg Gly Asp Val Thr Gln Leu Ala Ala Ala Tyr Ala Gly Trp His Pro Asp Val Thr Arg Leu Ile Ala Ala Ala Asp Arg Val Gly Arg Trp Ala Leu His Asp Arg Asp Ser Ile Asp Arg Leu Ser Ala Gly Arg Val Thr Leu Ile Gly Asp Ala Ala His Pro Met Leu Pro Phe Gln Ala Gln Gly Ala Asn Gln Ala Val Glu Asp Ala Val Val Leu Ala Val Cys Leu Ala Gly Val Glu Pro Ala Gly Leu Gly Ala Ala Leu Arg Arg Tyr Glu Arg Ile Arg Leu Pro Arg Thr Thr Arg Ile Gln Arg Gln Ser Arg Ala Asn Ala Glu Met Phe His Leu Ala Asp Gly Ala Asp Gln Arg Arg Arg Asp Val Ala Ala Gln Ser Ser Ser Gly Leu Asp Arg His Glu Trp Leu Phe Gly Tyr Asp Ala Glu Lys Ala Thr Thr Thr Ser Gly Ser Ala <210> 51 <211> 1191 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 51 atgccgcaga tgagggtcgc cgtggccggc gccggcatcg ccgggctcgc cttcgccgcc 60 gccctgcgcc ggaccgggat cgactgccac gtgtacgaac aggccgacca gctcatggag 120 gtgggcgcgg gcgtgcaggt cgcgccgaac gccacccggc tgctgcaccg gctgggcctg 180 cgtgaccgcc tgcgtacggt ggctgtcgcg ccgcaggcga tcgagatgcg ccgctgggac 240 gacggcacgc tgctgcaacg cacccagctg ggcagcgtgt gcggacgccg cttcggcgcg 300 ccgtactacg tggtgcaccg cgcggacctg cacagcagcc tgctgtcgct ggtgccgccg 360 gaccgggtgc acctgggcgc ccgcctcacc gccgtgacgc agaccgccga cgaggcgtac 420 ctgcacctgt ccaacggcac cacggtcgcg gcggatctcg tcgtgggcgc cgacggcatc 480 cactcggtcg cgcgggagca gatcgtggcg gaccggccgc gcttctccgg acagtccatc 540 taccgcgggc tggtgccggc cgagcgggtg ccgttcctgc tcaccgaacc ccgggtgcag 600 ttgtggttcg ggccggacca gcactgcgtc tgctacccgg tgtccgccgg ccggcaggtg 660 agcttcggcg cgacggtgcc cgccaccgac tggcggcagg agtcgtggtc gggccggggc 720 gacgtgacgc aactcgcggc cgcgtacgcg ggctggcacc cggacgtcac ccggctgatc 780 gccgcggccg accgggtcgg caggtgggcg ctgcacgacc gggacagcat cgaccggctc 840 agcgcgggac gggtgaccct gatcggcgac gccgcgcacc cgatgctgcc gttccaggcg 900 cagggcgcga accaggccgt cgaggacgcg gtggtgctcg cggtctgcct ggccggcgtg 960 gaaccggcgg gcctgggcgc cgcgctgcgc cgctacgaac ggatccgcct gccccggacc 1020 acccggatcc agcggcagtc ccgggccaac gccgagatgt tccacctggc cgacggcgcc 1080 gaccagcgcc gccgggacgt cgccgcacaa tcctcgtccg gcctggaccg ccacgaatgg 1140 ctcttcgggt acgacgccga gaaagccacc acgaccagcg ggagcgcctg a 1191 <210> 52 <211> 261 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 52 Met Glu Leu Thr Gly Ile Glu Ser Lys Val Ala Leu Val Thr Gly Ala Gly Gln Gly Ile Gly Ala Ala Val Ala Gly Val Leu Ala Arg Ala Gly Ala Gln Val Ala Ala Val Asp Arg Asn Ala Glu Ala Leu Thr Thr Val Val Thr Lys Leu Ala Ala Glu Gly Asp Ser Ala Arg Ala Tyr Cys Val Asp Val Cys Asp Ser Glu Ala Val Asp Ala Leu Val Arg Arg Val Glu Asp Glu Met Gly Pro Val Ala Ile Leu Val Asn Ala Ala Gly Val Leu His Thr Gly Arg Val Val Glu Leu Ser Asp Arg Gln Trp Arg Arg Thr Phe Ser Val Asn Ala Asp Gly Val Phe His Val Ser Arg Ala Val Ala Arg Arg Met Val Gly Arg Arg Arg Gly Ala Ile Val Thr Val Ala Ser Asn Ala Ala Gly Val Pro Arg Thr Glu Met Ala Ala Tyr Ala Ala Ser Lys Ala Ala Ser Ala Gln Phe Thr Arg Cys Leu Gly Leu Glu Leu Ser Gly Tyr Gly Ile Arg Cys Asn Val Val Ser Pro Gly Ser Thr Asp Thr Pro Met Leu Arg Ala Met Leu Gly Glu Gly Ala Asp Pro Ser Ala Val Ile Glu Gly Thr Pro Gly Ala Tyr Arg Val Gly Ile Pro Leu Arg Lys Leu Ala Gln Pro Arg Asp Val Ala Glu Ala Val Ala Tyr Leu Val Ser 225 . 230 235 240 Asp Gln Ala Gly His Val Thr Met His Asp Leu Tyr Val Asp Gly Gly Ala Ala Leu His Val <210> 53 <211> 786 <212> DNA
<213> Micromonospora sp. strain 046-EC01l <400> 53 atggaactga ccggaatcga gtcgaaggtc gccctggtca cgggcgcggg gcagggcatc 60 ggcgccgccg tggccggtgt cctggcgagg gcgggcgcgc aggtggcggc ggtggaccgc 120 aacgccgagg cgctgaccac cgtcgtgacg aagctcgccg ccgagggcga ctcggcgcgc 180 gcctactgcg tcgacgtgtg cgacagcgag gcggtggacg cgctggtgcg ccgggtcgag 240 gacgagatgg ggccggtcgc catcctggtc aacgccgccg gcgtgctgca caccggacgg 300 gtcgtcgagc tgtcggaccg gcagtggcgc cggaccttct cggtgaacgc cgacggcgtg 360 ttccacgtgt cccgggcggt ggcgcggcgg atggtgggcc gccgtcgtgg cgcgatcgtc 420 accgtggcgt cgaacgccgc cggggtgccg cgtaccgaga tggccgcgta cgccgcctcc 480 aaggccgcgt ccgcgcagtt cacccgctgc ctggggcttg agctgtccgg ctacggcatc 540 cggtgcaacg tggtctcgcc cggctccacc gacaccccca tgctgcgggc catgctcggc 600 gagggcgccg acccgagcgc ggtgatcgag ggcacgccgg gcgcgtaccg cgtcggcatc 660 ccgctgcgca agctggccca gccgcgcgac gtggccgagg cggtcgccta tctggtgtcc 720 gaccaggcgg gccacgtgac catgcacgac ctgtacgtcg acggcggcgc ggccctgcac 780 gtgtga 786 <210> 54 <211> 224 <212> PRT
<213> Micromonospora sp. strain 046-ECOll <400> 54 Met Ala Met Thr Pro Ile Ala Pro Tyr Arg Met Pro Gly Asp Gly Asp Leu Pro Gly Thr Ala Leu Pro Trp Arg Pro His Pro Asp Arg Ala Ala Val Leu Val His Asp Leu Gln Arg Tyr Phe Leu Arg Pro Phe Glu Ala Gly Glu Ser Pro Met Ala Glu Leu Leu Pro Asn Val Ala Lys Leu Leu Ala Thr Ala Arg Ala Ala Gly Val Pro Val Leu Tyr Thr Ala Gln Pro Gly Gly Met Ser Arg Gln Asp Arg Gly Leu Leu His Asp Leu Trp Gly Pro Gly Met Ser Ser Ala Glu Asp Asp Arg Gly Ile Val Asp Asp Val Ala Pro Gln Pro Gly Asp Thr Val Leu Thr Lys Trp Arg Tyr Ser Ala Phe Phe Arg Ser Asp Leu Glu Glu Arg Leu Arg Gly Ala Gly Arg Asp Gln Leu Val Val Cys Gly Val Tyr Ala His Met Gly Cys Leu Ile Thr Ala Cys Asp Ala Phe Ser Arg Asp Ile Glu Ala Phe Leu Val Ala Asp Ala Leu Ala Asp Leu Ser Arg Glu Asp His Leu Met Ala Leu Arg Tyr Ala Ala Asp Arg Cys Ala Val Pro Leu Trp Thr Ala Asp Val Leu Asp Gly Leu Ala Asp Ala Ala Gly Arg Pro Asp Gln Ser Ser Thr Gln Arg <210> 55 <211> 675 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 55 atggccatga ccccgatcgc gccgtaccgc atgcccggcg acggcgacct gcccggcacc 60 gcgctgccct ggcgtccgca cccggaccgg gccgccgtgc tggtgcacga cctgcaacgc 120 tacttcctgc gcccgttcga ggccggggag tccccgatgg ccgaactgct ccccaacgtc 180 gcgaagctgc tcgccacggc gcgggcggcc ggcgtgccgg tgctgtacac cgcgcagccc 240 ggcggcatga gccggcagga ccgcgggttg ctgcacgacc tgtggggccc cggcatgagc 300 agcgccgagg acgaccgggg catcgtcgac gacgtcgccc cgcagccggg cgacacggtg 360 ctgaccaagt ggcgctacag cgcgttcttc cgcagcgacc tggaggagcg actgcgcggt 420 gcgggacggg accagctcgt ggtctgcggc gtgtacgcgc acatggggtg cctgatcacc 480 gcctgcgacg cgttcagccg cgacatcgag gcgttcctgg tggcggacgc gctggccgac 540 ctatcgcgcg aggaccacct gatggcgctg cgctacgccg cggaccgctg cgcggtgccg 600 ttgtggacgg cggatgtgct ggacgggctg gcggacgccg ccgggcgtcc ggatcagagc 660 agcacccaac gatga 675 <210> 56 <211> 233 <212> PRT
<213> Micromonospora sp. strain 046-ECOll <400> 56 Met Ser Asp Arg Thr Arg Val Val Val Val Gly Gly Thr Ser Gly Ile Gly Arg His Phe Ala Arg Phe Cys Ala Glu Arg Gly Asp Asp Val Val Ile Thr Gly Arg Ser Ala Ala Arg Thr Lys Thr Val Ala Asp Glu Ile Gly Gly Arg Thr Arg Gly Leu Ala Leu Asp Leu Ala Glu Pro Glu Thr Ile Ala Asp Ala Leu Ala Asp Val Pro His Val Asp Arg Leu Val Val Ala Ala Leu Asp Arg Asp Tyr Asn Thr Val Arg Ala Tyr Arg Pro Gly Asp Ala Ala Arg Leu Leu Thr Va1.Lys Leu Val Gly Tyr Thr Ala Val Leu His Ala Leu Ala Pro Arg Met Thr Asp Glu Ser Ala Val Val Leu Leu Gly Gly Leu Ala Ser His Arg Pro Tyr Pro Gly Ser Thr Ser Val Thr Thr Ala Asn Gly Gly Ile Ser Ala Leu Val Arg Thr Leu Ala Val Glu Leu Ser Pro Val Arg Val Asn Ala Leu His Pro Ser Ile Val Ser Asp Thr Pro Phe Trp Ser Asp Lys Pro Ala Ala Arg Glu Ala Ala Ala Thr Arg Ala Leu Ser Arg Arg Pro Val Thr Met Gln Asp Cys Ala Glu Ala Ile Asp Phe Leu Leu Thr Asn Arg Ser Ile Asn Gly Val Asn Leu Asn Ile Asp Gly Gly Asp Val Leu Ile <210> 57 <211> 702 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 57 atgtcggatc ggacccgggt cgtggtcgtc ggcggaacct cggggatcgg gcggcacttc 60 gcccgattct gcgccgaacg cggagacgac gtggtgatca ccggccgttc ggcggcccgg 1.20 accaagaccg tggcggacga gatcggcggg cggacccgtg ggctcgctct cgacctggcc 180 gagccggaga cgatcgcgga cgcgctcgcc gacgtgccgc acgtcgaccg gctcgtggtc 240 gcggcgctgg accgcgacta caacaccgtc cgcgcgtacc ggccgggcga cgcggcgcgg 300 ctgctgaccg tcaagctggt cggctacacg gcggtcctgc acgccctcgc cccgcggatg 360 accgacgaga gcgcagtcgt gctgctcggc ggcctggcca gccaccggcc gtatcccggc 420 tccacctccg tcacgaccgc caacggcggg atcagcgcgc tggtgcggac cctggctgtg 480 gaactctcgc cggtccgggt caacgccctg cacccgagca tcgtctccga cacgccgttc 540 tggagcgaca agcccgccgc gcgggaggcc gccgcgaccc gcgcgctcag ccgacggccg 600 gtcaccatgc aggactgcgc cgaggcgatc gacttcctgc tgacgaaccg ctcgataaac 660 ggggtcaacc tgaacatcga cggcggggac gtgctcatct ga 702 <210> 58 <211> 246 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 58 Met Thr Ser Ala Leu Arg Thr Ser Ala Trp Thr Tyr Asp Asp Phe Thr Ser Arg Glu Leu Asp Pro Ala Arg Trp Ala Ile Met Ser Ile Ala Gly Ala Asp Gly Gln Thr His Ara Tyr Gln Asp Arg Asn Ala Gln Val Arg Thr Gly Asp Gly Arg Leu Glu Leu Thr Val Asp Pro Phe Thr Arg Phe His Asp Thr Asp Pro Arg Gln Asn Asn Ala Lys Gln Met Tyr Arg Ser Val Arg Arg Phe Ala Val Pro Ala Glu Gly Ser Leu Thr Val Glu Val Glu Met Gly Val Arg Thr Tyr Arg Gln Ile Pro His Asp Leu Leu Asp Ala Phe Gly Thr Val Asn Leu Phe Asp Leu Glu Thr Gly Val Val Phe Asn Ala Ala Ala Thr Asn Asp Thr Val Tyr Ala Thr Val Glu Arg Leu Val Leu Pro Gly Val Thr Gln Pro His Glu His Tyr Ile His Arg Val Val Leu Asp Val Pro Thr Glu Pro Gly Arg Ala His Gly Tyr Ala Ile Thr Tyr Arg Ala Pro Thr Ser Glu Val Glu Phe His Val Asp Gly Arg Leu Ala Tyr Trp Ala Arg Val Pro Val Pro Val Thr Gly Phe His Ala Gly Met Ala Leu Phe Ser Ala Arg Asp Leu Ala Arg Tyr Pro Arg Glu Gln Arg Glu His Gly Gln Gly Ala Thr Gly Trp Trp Gly Pro Trp Arg Ile Ala Ser Gly Val Arg <210> 59 <211> 741 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 59 atgacgtcgg cactgagaac cagcgcgtgg acgtacgacg acttcaccag ccgcgagctg 60 gaccccgccc gctgggcgat catgtcgatc gccggcgcgg acgggcagac ccacaggtac 120 caggaccgca acgcccaggt ccgcaccggc gacgggcggc tggagctgac cgtcgacccg 180 ttcacccgct tccacgacac cgatccccgg cagaacaacg ccaagcagat gtaccggtcg 240 gtgcggcgct tcgccgtgcc ggcggagggc tcgctgaccg tcgaggtgga gatgggcgtg 300 cggacgtacc ggcagatccc gcacgacctg ctggacgcgt tcggcacggt gaacctgttc 360 gacctggaga ccggcgtcgt gttcaacgcc gccgccacga acgacaccgt gtacgcgacg 420 gtcgagcgcc tggtgctgcc cggcgtgacc cagccgcacg agcactacat ccaccgggtg 480 gtcctggacg tgccgacgga gccgggccgg gcgcacggat acgccatcac ctaccgggcg 540 ccgacgtcgg aggtggagtt ccacgtcgac ggccggctcg cctactgggc gcgggtcccg 600 gtgccggtga ccggattcca cgccggcatg gcgctcttct ccgcccgcga cctggcccgg 660 tacccccgcg agcagcggga gcacgggcag ggcgcgaccg ggtggtgggg gccgtggcgg 720 atcgcctccg gcgtcagatg a 741 <210> 60 <211> 111 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 60 Met Asp Thr Ala Ala Pro Ala Thr Asp Gly Gly Arg Tyr Leu Ala Val His His Ser Ala Glu Phe Arg Glu Leu Arg Arg Arg Ser Ser Thr Phe Thr Leu Trp Ala Ser Val Ala Phe Phe Gly Trp Trp Phe Leu Gly Ser Leu Leu Ala Thr Tyr Ala Pro Asp Phe Phe Arg Glu Lys Val Ala Gly Pro Val Asn Val Gly Leu Leu Phe Val Phe Leu Ser Phe Ala Phe Val Val Thr Leu Ala Ala Phe Tyr Leu Arg Tyr Ala Arg Thr His Leu Asp Pro Leu Ser Glu Lys Ile Arg Ala Asp Leu Glu Gly Ala Ser Arg <210> 61 <211> 336 <212> DNA
<213> Micromonospora sp. strain 046-ECOll <400> 61 atggacacgg cagctccggc aacggacggc ggtcgctacc tcgccgtcca tcacagcgca 60 gagttcaggg aactacggcg acgatcgagc acgttcacgc tctgggccag cgtcgccttc 120 ttcggctggt ggttcctcgg cagcctgctc gccacctacg cgccggactt cttccgggag 180 aaggtggccg gcccggtcaa cgtgggtctg ctcttcgtct tcctgtcgtt cgccttcgtg 240 gtgacgctcg ccgccttcta cctgcgttac gcccgcacgc atctcgatcc gctcagcgag 300 aagatccgtg ccgacctgga aggagcgtcc cgatga 336 <210> 62 <211> 559 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 62 Met Ser Val Ile Leu Ala Asp Pro Pro Pro Pro Val Asp Asn Thr Trp Ala Thr Pro Ala Ile Ala Val Pro Val Thr Ile Val Leu Ala Leu Ala Val Leu Tyr Leu Val Arg Ser Ala Arg Ala Ser Thr Thr Thr Ala Asp Gly Phe Leu Leu Ala Asp Arg Arg Ile Gly Pro Val Gln Asn Ala Leu Ala Val Ala Ser Ala Pro Leu Met Tyr Ser Thr Met Tyr I].e Ile Thr Gly His Ile Ala Leu Ser Gly Tyr Asp Ala Ile Leu Leu Met Thr Ala Phe Thr Met Gly Thr Met Leu Ala Leu Phe Leu Phe Ala Gly Pro Val 100 105 11.0 Arg Asn Val Gly Gly Tyr Thr Leu Gly Asp Leu Leu Ala Val Arg Thr Arg Glu Arg Pro Ala Arg Ile Ala Ser Ala Val Leu Thr Leu Leu Thr Tyr Val Met Leu Thr Val Ile Met Met Ala Ala Ile Ala Phe Ile Phe Asn Arg Trp Phe Gly Val Asp Ala Leu Val Gly Leu Val Leu Pro Val Phe Val Val Gly Leu Ile Thr Val Gly Tyr Val Tyr Leu Gly Gly Met Leu Gly Val Thr Arg Ile Leu Val Phe Lys Leu Val Leu Ser Val Val Val Val Gly Val Leu Thr Ala Trp Val Leu Ala Arg Phe Asp Leu Asn Leu Phe Ser Leu Leu Glu Arg Ala Glu Ala Asn Ala Ala Pro Val Pro Ser Gly Ser Asp Leu Leu Gly Pro Gly Arg Leu Phe Gly Glu Gly Ala Thr Thr Leu Val His Leu Ser Lys Leu Phe Ala Ile Ala Val Gly Val Ala Ala Ile Pro Phe Leu Phe Met Arg Asn Phe Ala Va1 Thr Ser Gly Arg Asp Ala Arg Arg Ser Thr Gly Trp Ala Ser Met Ile Ile Val Gly Phe Tyr Leu Cys Leu Ser Val ',Ial Gly Leu Gly Ala Val Ala Ile Leu Gly Arg Asp Asn Ile Gly Val Ile Lys Ala His Arg Asp Ile Ser Phe Pro Lys Leu Ala Asp Glu Leu Gly Gly Pro Val Met Val Gly Ser Leu Ala Gly Val Ala Val Leu Thr Ile Val Gly Val Phe Ala Pro Leu Leu His Ser Ala Val Thr Thr Val Thr Lys Asp Leu Asn Val Ile Arg Gly Arg Arg Leu Asp Pro Ala Ala Glu Leu Arg Asp Ile Lys Arg Asn Thr Leu Ile Ile Gly Val Gly Ser Val Leu Leu Ala Val Val Met Leu Pro Val Arg Thr His 11e Phe Ile Pro Thr Ser Ile Asp Ile Ala Gly Ala Val Vai Leu Pro Ile Val Val Tyr Ala Leu Phe Trp Arg Arg Phe Asn Thr Arg Gly Leu Gln Trp Thr Val Tyr Gly Gly Leu Ala Leu Thr Ala Phe Leu Val Leu Phe Ser Asn Gly Val Ser Gly Glu Pro Asp Ala Ile Phe Pro Asp Arg Asn Phe Lys Phe Val Asp Val Glu Pro Ala Leu Ile Thr Val Pro Val Gly Phe Leu Leu Gly Tyr Leu Gly Ser Ile Thr Ser Arg Glu Arg Asp Asp Ala Ala Phe Ala Glu Met Gln Val Arg Ser Leu Thr Gly Ala Val Val Thr Gly Pro Pro Arg Pro Ala Ala Val Asp Asp Glu Asp Arg Asp Gly Arg Gln Asp Arg Ala Pro Ser Pro Val Ser <210> 63 <211> 1680 <212> DNA
<213> Micromonospora sp. strain 046-ECOl1 <400> 63 atgagcgtca tcctcgccga cccgccaccc ccggtcgaca acacgtgggc gacgcccgcg 60 atcgccgtgc cggtcaccat cgtcctcgcg ctcgcggtgc tctacctggt ccggtcggcg 120 cgcgccagca ccaccaccgc ggacggcttc ctgctggccg accggcggat cgggccggtg 180 cagaacgcgc tggcggtggc ctccgcgccg ctgatgtact cgacgatgta catcatcacc 240 ggccacatcg cgctcagcgg ctacgacgcc atcctgctga tgaccgcctt caccatgggc 300 accatgctcg cgctgttcct cttcgccggg ccggtgcgca acgtgggcgg ctacacgctc 360 ggtgacctgc tcgcggtccg tacccgggag cggccggcgc ggatcgcgtc ggcggtgctc 420 acgctgctga cgtacgtcat gctgacggtg atcatgatgg ccgccatcgc gttcatcttc 480 aaccgctggt tcggcgtcga cgccctcgtc ggcctggtcc tcccggtgtt cgtcgtcggt 540 ctgatcacgg tggggtacgt gtacctcggc gggatgctcg gggtcacccg catcctggtg 600 ttcaagctgg tgctgtcggt ggtcgtcgtg ggcgtgctga ccgcctgggt gctggcccgc 660 ttcgacctga acctcttcag cctgctggag cgggccgagg cgaacgcggc gccggtgccc 720 0ZL qpbqbapqbb aaqababaqq apqapabbqa qqbbaabbap abbaaeabqa abpapbbqaa 099 Pbbbaabpaa pa;qapabqb bqpaqqaaba bbbaabqaab baaabqbaeb aqqbbbbpbb 009 pbqbbaqpab bbqboabbba abaaqbbpaa bpbaqbabab baebabqbbb aqbbqbapqb 0lv5 pbbaqbbabq bbbaaqbbeb abbobbaapa Pba-ebaabaq aaqbabpaop aqbbbbbabo 08:~ Pbapbb-eaaa pbqabaqapb aqqbba-eaba bbaqaababq aal2baqbaqa aaab-ebbbab 0ZIV baab;abaaa bbbab~aaqb bao~-2bblao -ebaq-ebpaaa -qaqebba5-2a abbaaabi2ba 09E abbaabeaaa -eaBbapba-ei2 apab-ebaaba q-eaboababb qbebbl?abal2 b-4bbaqaaab OOE qabbbbb-4bb iqaaea~-ebaa b-2bqbbaa5b qaabbaapba qbbjaqpaqb apboabbbaa OvZ pabpba aaba baqbabbaab aabqbaaqba Pbapbaabaq aabbaqqbpa abaaqaaqqb 08T Pbbq-eobeab aaaba~~a~b a-eqapbb-4ab bbaqbaeb-eb ajqabbbpaa 0ZT -2baqba;a~~ babqaabajb abba,2a;baa abaqobabbq ababaa-eaqb bqeabbaqbb 09 aaabbbabab bbqbaqbbqa qqbbaa,2qbb aaq-ebbqb;a ~-e?abb-ebbba ~aaaa-ea-eaa f19 <00:~>
TZODH-9f,0 uz~a~s =ds i2zodsououioaazT4 <EZZ>
VNC <ZTZ>
096S <TZZ>
Tv9 <0TZ>
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i8 Ob9Z aPa54Paaaq PbabPbqabb qaqabqbaaa a123bbbbaba ~~b~bb~bba babaa~ba~~
08SZ baabaabqaa aqbpbbbaaq Pbbaaebbba abbbqebbab aaa~~b~aa~ ba~a~baabb OZSZ baaqpbabab babaaapbaq ababbpbab; bqbbbab;aa qbapbapbba aa~baaba~b 09:~ Z baabbba-4iRb ~,eab-Iabbaq bbaaae"eb-1 b b;bbae3a-4b qab-qpaabb:4 ba-eababbi?b 00:~ Z aabaalbbaa baaaebbabe bbbbb:;bbbq baabqaai23a a-ebaqbabb:j a3P-j02ab25 O:~ SZ abbbaiRai2bb aaa-ebbbaab aaqebqaaba aaqbL-ba~-4a 25a;baqbb-4 aaa5512ba-2a 08ZZ abp6a~bb-qb aabbbbebaa i2bbababaab qbabbaappa 5bbpbbbaa2 aaabababqa OZZZ a;bbbabeab qbbbaapbab Pbabb-ebap; babbbqbb;b qpabaaba;a aapbaabbpp 09TZ ababapqbba aababaabbq abpbaaqqpp aqpbqpapaa bbabaaq;aa qaaaaqpabp 00ZZ bqababal2b3 Pabbqb-ebba -25a5bi2aba~ bbaaaabi?-ea abaqqb-eabb baabb~-ebba OIV 0Z abbbqbbbab qab~ba-2pab abbbaapaqa b-ababeaqab baa2bbbbb-a a6baa12baql2 086t ajbbpbaq-eb i2-2aL~bba;a3 -eaeabbaaaa aaeab;beba ~-q aq aabL- b2 baababqaeb 0Z6T abbb:jqb-2ab jab:aa~abb bapbaaeba-3 babb~ab-2bb a-4bq-4b-3ab2 abbaaqqbbb 098Z bba~-qbb-eb6 b5aaa-2aaaq bbqabaabaa aabbaqbabb q--jbabb-eaqb qaa-2b-ebbab 008T ai2qbabbaaa Paaebb,2a5b -qaiRbbaa~,2a bpaa;bbabb aab-47eabbbu bbbqbaabbb Of,LZ aqa-eaaabLDa ba~-ebbabba ~-ea;qbbaaq -e"2abpabqeb -ea-ebbebbqa -ebaqaqal2bq 089Z abbaalbbaa bbaai2babai2 a~ababa~a~ bbbaaabbab baa,2a;baa5 qb0'2b3b3bb 0Z9Z Loqpbbabbjb bbbab-4baba b~aa~bb~ba aba-ebaaaa72 bbaabaabjb aaL-a~p-ebbb 095T babbqaabaq ba-eaa-2aa-eb baabbabqab :~aaqba:~aa-e baba~~2bbbq bq3b-e5;b3-00S'C abbTaaaaa-e baaebqbbqb aa2apaaa-ea qbbaaaqabb bbbababaab babbabbabb O'v'vZ qabqabaaqb baabaababb a-ebqbbaa"2a -zqaiRbbqba:j aaai2a-4;ba-q aabbaaa-eba 08ET ~-45baal2bo-4 qa~-eb-eab-ea bbbapbabL:>b -4ab-4aaqaab -2aaaboa-4ba aababbaaab OZ~T bqaa72ba-eba aaaba-eqbab bbabaaaeba L-bbaqaaab-e bbbbbaaabb b-25baabb-3a 09ZT -4abqbbaaba -qabbaa-qapq abaal2-Ia5ba qab-2ba:jPbb bbbaa-eqb-eb a~-ebpbabbb 00ZZ ba5-25aaban ba~aa~~aab ba5a-qbaa-15 aab~abaaaa babbabbaba bqaa~abpba Of,ZZ bbbabbbab-j aa2ab-lbabb b-jaaeabbaa L-aba;aapab ab:jeai2eab3 bbabbbbqbb 080T aaabaabapp baabaL-ba~-4 ab-eaebaqab qabbbaaaab babba:~-jabb bbaa;ba-;b:j 0Z0T abaabb-4ab-e Pbbqaabb-jb aL-baqbaaba i2babba:j 2ab baapa~-9b;i2 abba,2;baba 096 bbaa-2aaaaq ;b-4 2bbaai2a aabbaqaebq bbaabajab-4 abqbbbaabb aabz)baapqb 006 b-2aPbaaba:l ab~~a~~abb aaa-4babb-4a baba-4aa-e3~ abqabqbbp-e a-eba-jlebbab 0IV8 qabpbaa-eab aaqpba-ea-eb a;ab-aaaqqb ab5-255aaa"e baabbaabaa b-ebaPba~-jb 08L -eba,2-ebaaa72 ba~babba-ep aqbaLDababa a-ebb;aapa-e ba-qbb2ba-eb b-eaaaba;-ea ZO-90-600Z O6~996Z0 Fi'J

OOSIV bba b3q5qbb bb53;3qP3b b3P-2Pb53bD 3bb3bbebqo q;Pa PPbqbb 3anY;ePb"2b 0~vlv bpbpbb~p-e3 Dqbbobbabq ;;q;p3p3bp bqbba ;qqqq pDqD3D6q3b Dnq2bDq;Do 08E~p qqbq-2Pq;bD bD53PPqP2b ;qD5bD3Pb5 ;bP;bPb3jP 500OPqDPOO b53b3;bqDP
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OOOE qqqboabboa bDbqq;aqbb oDboqbDpbq oobDDboqbb Pbba obbDbo bbDq;Doabb 0'v6Z oa;obobbob oa pbbboaob bo;bba pbob oapopbbppa obbbqbDbPP ba qPb,:~Dbpb 088Z DopoobbpD; bbbaaoppD; obbobooopb obqbqaba qo DbobbqDoeb OZ8Z bpbbpbobba obb;bboqqb bD5boqb35; bpboDpb;bD pba3i2oD5Do boqbbpbaqP
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ccgtcgccgc ggcgatcgcc ctggtggcgt cgctgacaaa cggtgtggcg gctgccccgc 4560 aggcgccgac cttcgacctc gacaacggga acgccctgac cgacgtcatc tacccggccc 4620 tcaacaccga gccgcgggtc gagtacagcg gccggcccgg gtcctgggcc gcggaccgcg 4680 ccatgctcat cgaactgccg tggttcgacg ccctggcggc gtaccacccc accgcggtcg 4740 gcatcttctc caccatcggc cgccgtcccg ccgaggagca cacgacgcgc aacaagaaca 4800 tcgccgtcat ctactcggcc tacacctcgc tcagcaagct ctacccccag cacgaggcga 4860 cctggcagcg gatgatggcc accgcgggcc tggacccggc cgtcaccgcg gaggaccgga 4920 ccaccgccag cggcatcggc atcctcgcct cgaagaacgc gatggcggcg cgccggaacg 4980 acggcacgaa ccgcgacggc gacgcgggcg gccgtcgcta caaccgtgag ccgtacgccg 5040 accacaccgg ctaccggccg gtcaacagcc cgtacgagct gcgcttcccg tcgcgctggc 5100 agccgaacac catctccaag cgcgaggtcg tcctgacgca ggagttcgcg acgccccagt 5160 tcggccgggt caagccgatc accttcgagc ggcccgagca gttccggctc accccgccgc 5220 cgaaccacca cctgttgaac ccgaagggct accggaagca ggccgacgag gtgctgcgcg 5280 cctcggcggg cctggacgac cgcaagaaga tgagcgcgga gatcttcagc gacaacatca 5340 cgccgtacgg cgccatcgcg cacacgctcc tgcggggccg gtacaacacc gaggactccg 5400 tccggttcat cgtgatgact gacgtcgccg ggttcgacgt ggcgatcgcg tcctggtact 5460 acatgcgcaa gtacgactcg gtgcagccgt tcagcgcgat ccgccacctg tacccgaaca 5520 agaagctgac cgcgtggggc ggcccgggcc ggggcaccgt caacgacatc accggcaccc 5580 agtggcgcag ctacctcagc tcggtcgcca tcgcggctcc ggattacccg tcggtcaacg 5640 cggcggtctg cgtcgcctac gcccaggtcg cgcgccggtt caccggcacg gacaagctga 5700 ccgtcgtgat cccggtccgc aagggctcct cgatcgtgga accgggcgtg accccggccg 5760 ccgacatgat gctcacctgg aacagctact cggagtgggc cgccgagtgc gggcagagcc 5820 gggtctgggc cggcgagaac ttccccgcct cggtcgcggc cgccgaccag tacgcgccgc 5880 agatcggcga ccgtgccttc gacttcgtcc agagcaagct gaacgggcgc tgacgcccgc 5940 gtaccggtcc gtgctgccgg 5960 <210> 65 <211> 532 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 65 Val Asp Pro Val Pro Val Leu Val Val Gly Ala Gly Pro Val Gly Met Val Thr Ala Leu Ala Leu Ala Arg His Gly Val Ala Cys Val Leu Val Asp Gln Gly Phe Glu Thr Ser Val His Pro Lys Leu Asp Tyr Val Asn Ala Arg Ser Met Glu Phe Leu Arg Gln Phe Gly Leu Ala Asp Asp Val Arg Ala Ala Gly Val Ala Pro Glu His Arg Ala Asp Val Ile Trp Ser Thr Gly Leu Ala Gly Glu Pro Ile Thr Arg Trp Gly Leu Pro Ser Val Thr Gln Glu Trp Arg Arg Ile Ala Glu His Asn Asp Gly Thr Gln Pro Ala Glu Pro Gly Gln Arg Ile Ser Gln Ile Asp Leu Glu Pro Val Leu Arg Ala Arg Cys Arg Arg Glu Pro Leu Val Asp Leu Arg Leu Gly Val Arg Phe Asp Ser Leu Thr Gln Asp Asp Ala Gly Val Thr Ser Val Leu Ala Asp Asp Thr Gly Gly Glu Val Arg Val Arg Ser Glu Tyr Val Val Gly Cys Asp Gly Ala Ser Ser Gln Val Arg Arg Ala Val Gly Ile Gly Glu Glu Gly Phe Asp Val Pro Gly Leu Pro Gly Ala Phe Met Val His Phe Thr Ser Arg Asp Leu Asp Ser Leu His Arg His Gly Arg Phe Trp His Tyr Phe Ala Phe Arg Tyr Val Ile Ile Ala Gln Asp Glu Val Asp Thr Trp Thr Ala His Val Asn Gly Val Asp Pro Asn Glu Phe Asp Glu Pro Pro Ala Asp Pro Glu Ala Phe Leu Leu Asp Thr Ile Arg Thr Glu Leu Arg Ile Asp Lys Val Leu Leu Thr Ser Arg Trp Arg Pro Gly Phe Met Leu Ala Asp Arg Tyr Arg Ala Gly Arg Val Leu Leu Ala Gly Asp Ser Ala His Arg Met Phe Pro Thr Gly Ala Tyr Gly Met Asn Thr Gly Ile Gly Asp Ala Val Asp Val Ala Trp Lys Leu Ala Ala Val Val Arg Gly Phe Gly Gly Pro Gly Leu Leu Asp Ser Tyr Asp Ala Glu Arg Arg Pro Val Gly Arg Arg Asn Met Arg Thr Ser His Arg His Leu Gly Val His Leu Arg Ala Gly Glu Leu Leu Arg Gly Gly Ala Pro Leu Pro Ser Val Ala Ala Phe Leu Asp Ala Glu Arg Gly Glu Asn Glu Tyr Arg Gly Ile Glu Leu Gly Tyr Arg Tyr Ser Gly Ser Pro Val Leu Trp Pro Glu Gly Pro Gly Glu Pro Ser Asp Asp Pro Arg Ala Tyr Ala Pro Thr Thr Trp Pro Gly Ala Arg Pro Pro Ser Leu Leu Leu Ser Asp Gly Gln Gln Ile Phe Asp Arg Phe Asp Pro Ala Ser Phe Thr Leu Val Asp Phe Thr Gly Asp Gly Ala Ala Gly Pro Leu Leu Ala Ala Ala Ala Ala Arg Gly Leu Pro Val Thr His Thr Val Val Thr Asp Pro Arg Ala Arg Glu Leu Trp Glu Arg Asp Leu Val Leu Leu Arg Pro Asp His His Val Ala Trp Arg Gly Asn Thr Val Pro Pro Asp Pro Asp Ala Val Val Gln Arg Val Arg Gly Gly Gly <210> 66 <211> 1599 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 66 gtggatccgg taccggttct ggtcgtgggc gcgggcccgg tcggcatggt caccgcgctg 60 gcgctcgccc gtcacggcgt cgcctgcgtc ctcgtcgacc agggcttcga gacgtcggtc 120 catcccaagc tggactacgt caacgcccgc agcatggagt tcctccgcca gttcggcctc 180 gccgacgacg tccgtgccgc cggcgtcgcg cccgagcacc gggccgacgt catctggtcg 240 accggcctgg ccggtgagcc gatcaccagg tgggggctgc cctcggtgac gcaggagtgg 300 cgccgcatcg ccgagcacaa cgacggcacc cagccggccg agcccggcca gcggatctcc 360 cagatcgacc tggaaccggt cctgcgggcc cgctgccggc gggagcccct tgtcgacctg 420 cgcctcggcg tacggttcga ctcgctgacc caggacgacg cgggggtcac cagcgtcctc 480 gccgacgaca ccggcggcga ggtccgggtg cggtcggagt acgtggtcgg gtgcgacggc 540 gcgtcgagcc aggtccgccg ggccgzgggc atcggtgagg aggggttcga cgtgcccggc 600 ctgccgggcg ccttcatggt gcacttcacc agccgggacc tggacagcct gcaccggcac 660 ggccggttct ggcactactt cgcgttccgg tacgtgatca tcgcccagga cgaggtcgac 720 acctggaccg cgcacgtcaa cggcgtcgac ccgaacgagt tcgacgagcc gccggccgac 780 ccggaggcgt tcctgctcga cacgatccgc accgagctgc ggatcgacaa ggtgctgctc 840 acctcgcgct ggcgtcccgg cttcatgctc gccgacaggt accgcgccgg ccgggtgctg 900 ctcgccggtg actcggccca ccggatgttc cccaccggcg cgtacggcat gaacaccggc 960 atcggcgacg ccgtcgacgt ggcctggaag ctggccgctg tcgtccgggg cttcggcggc 1020 cccgggctgc tcgacagcta cgacgccgaa cgccgcccgg tggggcggcg caacatgcgc 1080 acctcgcacc ggcacctggg cgtgcacctg cgggcgggcg agctcctgcg cggcggcgcc 1140 ccgctgccgt ccgtcgcggc cttcctcgac gccgagcggg gcgagaacga gtaccggggg 1200 atcgagctcg gctaccgcta ctccggctcg ccggtgctct ggccggaggg cccgggggag 1260 ccctcggacg acccgcgggc gtacgccccg acgacctggc ccggcgcccg tccgcccagc 1320 ctcctgctga gcgacgggca gcagatcttc gaccggttcg acccggcctc gttcaccctc 1380 gtggacttca ccggtgacgg cgccgccggt ccgctgctgg cggcggcggc cgcgcggggg 1440 ctcccggtca cccacaccgt ggtgaccgac ccccgggctc gtgagctgtg ggaacgcgac 1500 ctcgtcctgc tgcggccgga ccaccacgtc gcctggcggg gaaacaccgt gccgccggac 1560 cccgacgccg tggtccagcg cgtgcggggt ggcggatag 1599 <210> 67 <211> 423 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 67 Met Gln Gln Ser Gly Ser Thr Ala Glu Arg Ser Pro Leu Gly Pro Trp Glu Gly Met Pro Ala Val Gln Gln Pro Asp Trp Gln Asp His Pro Ala Tyr Ala Glu Thr Cys Gln Ala Leu Ala Ser Ala Pro Pro Leu Val Pro Pro Gly Glu Val Arg Gly Phe Arg Gln Leu Leu Ser Glu Leu Ala Ser Thr Asp Gly Leu Leu Leu Gln Leu Gly Asp Cys Ala Glu Ser Leu Tyr Glu Cys Thr Pro Arg His Thr Ser Asp Lys Ile Glu Val Ile Asp Arg Leu G1y Asp Arg Leu Ser Glu Leu Thr Gly Arg Asn Val Leu Arg Val Gly Arg Met Ala Gly Gln Phe Ala Lys Pro Arg Ser Gln Ala Thr Glu Trp His Asp Ala Leu Ser Ile Pro Ser Phe Arg Gly His Met Ile Asn Ser Glu Leu Ala Ala Pro Gly Thr Arg Lys Ala Asp Pro Arg Arg Met Trp Trp Ala Tyr Glu Ala Ser Asp Arg Val Gln Arg Val Leu Arg Ala His Arg Glu Gly Asn Arg Arg Ala Ala Arg Thr Glu Gly Pro Trp Ser Ser His Glu Ala Leu Val Val Asp Tyr Glu Ser Arg Leu Ile Arg Arg Asp Pro Asp Thr Gly Glu His Tyr Leu Ala Ser Thr His Leu Pro Trp Val Gly Glu Arg Thr Arg Arg Ser Ala Glu Ala His Val Ala Met Leu Ser Thr Val Val Asn Pro Val Gly Cys Lys Ile Gly Pro Asp Ala Asp Pro Asp Asp Val Leu Arg Val Cys Glu Ala Leu Asp Pro Arg Arg Asp Pro Gly Arg Leu Val Leu Ile Pro Arg Met Gly Arg Asp Arg Ile Arg Glu Ser Leu Pro Pro Ile Val Arg Ala Val Val Asn Ala Gly His Pro Val Leu Trp Leu Ser Asp Pro Met His Gly Asn Thr Val Lys Ala Ser Val G1y Leu Lys Thr Arg His Leu Ser Asp Val Val Thr Glu Ala Leu Trp Phe Arg Asp Ile Leu Asp Gln Gln Arg Gln His Ala Ala Gly Leu His Ile Glu Val Ala Ala Thr Asp Val Thr Glu Cys Va1 Gly Gly Ser Val Ala Gly Glu Glu Asp Leu Ala Arg His Tyr Thr Ser Leu Cys Asp Pro Arg Leu Asn Pro Gly Gln Ala Thr Glu Leu Ile Glu Ala Trp Ala Lys Asp Thr Ala Thr Val Gly Pro Gly Pro Arg Arg Ser Gly Pro Ser Ala Arg Pro Glu Val Ala Ala <210> 68 <211> 1272 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 68 na1 dsV sTH 4aW PTV PTy naq naq baV aTI STH nTE) TPA usv nZ~ ~ZS
SIV 0b S~
PTV naq baV ozd aaS dsV Y~TS ozd nari naZ PTV aaS TPA Old TPA auw 0~ SZ OZ
nTE) ,ZS na2 TPA zaS bjV dsK nTE) 3us PTV IPTy dsV ozd naZ ozd aud bzV nTf) dsV usV rTS sAZ TPA nTE) na7 auy usV aag JaS AT5 dzs jaN
69 <00T7>
TTODE-9V0 uTP-TIs -ds PzodsououioaoTN <~TZ>
s'd d <ZTZ>
0V~ <TTZ>
69 <OTZ>

ZLZT Pb ;oobooboqb 09ZT bPbboobba b a bboqqooob booqabobbo b3a -2bbbooo bbojbbaPbo ba o2oP5bP2 00ZT oobbblbobp Pboqpbqa b-e ba aeoobbpo qbbboa aP-eo qabba baoo'e bobqbqoboq O'vTT ooPoP-4oPof5 bobo.6b-qoo-2 bbpbbabobb oobbqbboqq bbobbo;bob qb-eboo-ebqb 080T oabooPoobo oboqbbpbo; aopabqobbb ooboobopab PobbobPa b,2 oa-eboqooqP
0Z0T o2ba booqqb b;bqobobba baoPa qbbqb o-eba aqo;oo Pooboba PbP Pbqoobboqb 096 baqoobb-epo qboa eoPPob bo-2a bq-2oa o q-ebobPb:~ob bqoqobqboo ooaobbbbob 006 oPi2bqbb;bb oboboa qboq Pboa boobqa ooqb-2bbboo qPbbaoPbbb oobbbqPbba 0IV8 booojPb;oo ?bo;oqboob bbaoq72babo bboboa oPbo ;obabb-ebob qbqbbbobqo 08L oqbo-eboebb ooa -26a aboe bboobbboq-e bPuobqa bbo qbba oo~-ebq bbqbbo12ao;
0ZL bqob;2oobb qbaPobabb-e boobooqbbo oboaoPbbob -ebbbbbqbbb qboobqoo-eo 099 ooeboqbobb qooPqo-23b-e bobb5ovo,25 baoo125bboa ba a;-ebqoob oaoqb-eb32q 009 o-ebojba jbb jooabbi2boP oobPbo;bbq boobbbbPbo opbboboboa bqbobbooP-e 0lvS abbbebbboo aooobobobq oaqbbbobPo b;bbba oebo bPbobbPbop qbobbbqbbq 08IF bq-eobooboq oaoPboobbP PobaboPqbb oooboboobb ;abPboo;qP Po;i2b;Po-eo 0Zfi obboba aq;o oqoaao;-eob abqoboba-eb oPabbqbPbb a Pbobb2obo qbboooobPe 09~ ooboqqb-2ob bboabb;-ebb oobbbjbbbo bqob;boPPo bobbboo-23; a bPbobPoqo 00~ bba opbbbbb qobbooebo; PoqbbPba jP b-eeo-ej5boqo oPo-eabba oo oooPobqbHb O:~ Z aPqoqoobPb Pboobobqo-e bobbbqqbPo bqobjooqob bbopboaebo ~bobb;a bPb 08T bo;bqqbqob Pobbooqqbb bbboPqbb,2b bbbooaPooo qbbqoboobo ooabboqbob 0ZT b;qbobb-23q bqoaPb,2bba bopqbobba o oP- oopbb-e ob bqo-ebbooaI2 abpooqbbab 09 boa bqPobbb i2bbb;boo5b baqo-eooob12 oboPebbobb oP-eaq;bboo ~P-23bPobq2 ZO-90-600Z O6~996Z0 Fi'J

Pro Pro Ile Leu Val Gln Arg Gly Thr Met Arg Val Ile Asp Gly Met His Arg Leu Arg Ala Ala Lys Leu Arg Gly Asp Glu Thr Val Arg Val Thr Phe Phe Asp Gly Asp Asp Ala Ala Ala Phe Leu Leu Ser Val Asp Ala Asn Ile Lys His Gly Leu Pro Leu Ser Arg Ala Asp Arg Glu Ala Ala Ala Thr Arg Ile Leu Arg Leu Tyr Pro Gln Trp Ser Asp Arg Ala Val Ala Ala Ala Ala Gly Leu Ser Pro Thr Thr Ala Ser Gly Ile Arg Arg Arg Leu Leu Gln Pro Ala Ala Arg Glu Gly Ser Arg Val Gly Arg Asp Gly Arg Val Arg Pro Leu Asp Gly Ser Ala G1y Arg Arg Arg Ala Ser Ala Val Ile Ala Leu Arg Pro Asp Ala Pro Leu Arg Ala Ile Ala Gln Glu Ala Gly Val Ser Val Gly Thr Ala Arg Asp Val Arg Ala Arg Leu Gln Ala Gly Arg Asp Pro Val Leu Thr Ser Gln Arg Pro Ala Ala Glu Pro Glu Pro Ala Ala Asp Asp Gly Pro Glu Ala Arg Arg Arg Arg Leu Gly Gln Pro Ser Val Pro Pro Val Asp Trp Pro Ala Val Arg Gly Asn Leu Ile Arg Asp Pro Ala Val Lys Tyr Ala Glu Leu Gly Arg Ala Phe Val Arg Trp Ala Asp Gly His Val Val Asp Pro Ala Ala Trp Arg Glu Phe Val Asp Ala Val Pro Pro Tyr Trp Arg Lys Ser Val Ala Glu Leu Ala Arg Ser Cys Ala Ser Ala Trp Leu Ala Phe Ala Gln Glu Leu Glu Asp Arg Ala <210> 70 <211> 1023 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 70 atgtggggca gctcaaacac gctggaagtg aagggcaacg acgagagatt ccccctgccc 60 gatgcagcta cggaggatcg gtctgtgctt ggcgagacgg ttccggtttc cgcgctgctg 120 cccggtgact ccccgcggct ggcgggcgag aacgtcgagc acatccggct gctggccgcg 180 atgcacgacc tcccgccgat cctggtgcaa cgcggcacga tgcgggtgat cgacggcatg 240 caccggctgc gggccgccaa gctgcgcggc gacgagaccg tgcgggtgac gttcttcgac 300 ggggacgacg ccgcggcgtt cctgctctcg gtcgacgcca acatcaaaca cgggctgccg 360 ttgtcccgcg ccgaccggga ggccgccgcc acccgcatcc tgcggttgta tccgcagtgg 420 tcggaccgcg ccgtcgccgc ggcggccggg ctgtcaccga ccacggcgag cggcatccgg 480 cgccgcctgc tgcaaccggc ggcgcgggag ggcagccggg tgggacggga cgggcgggtg 540 cgcccgctgg acggctcggc gggccgacgg cgggccagcg cggtcatcgc gctccggccg 600 gacgcgcccc tgcgtgccat cgcgcaggag gccggggtgt cggtgggcac ggcgcgggac 660 gtgcgcgccc ggttgcaggc gggccgggac cccgtcctga cctcgcagcg accggcggcc 720 gagcccgagc cggccgccga cgacgggccg gaggcgcgca gacgccggct cggccagccc 780 tccgtgccgc ctgtcgactg gccggcggta cggggcaacc tgatccggga ccccgcggtg 840 aagtacgccg agctgggccg ggccttcgtc cgctgggccg acgggcacgt ggtggatccg 900 gcggcctggc gcgagttcgt cgacgccgtg ccgccgtact ggcgcaaatc ggtggccgag 960 ctggcccgtt cgtgcgccag cgcctggctg gcgttcgccc aggaactgga ggaccgggcg 1020 tga 1023 <210> 71 <211> 493 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 71 Val Asn Ile Leu Arg Arg Pro Arg Lys Arg His Leu Gly Gly Val Ala Ala Val Ala Ala Ala Ile Ala Leu Val Ala Ser Leu Thr Asn Gly Val Ala Ala Ala Pro Gln Ala Pro Thr Phe Asp Leu Asp Asn Gly Asn Ala Leu Thr Asp Val Ile Tyr Pro Ala Leu Asn Thr Glu Pro Arg Val Glu Tyr Ser Gly Arg Pro Gly Ser Trp Ala Ala Asp Arg Ala Met Leu Ile Glu Leu Pro Trp Phe Asp Ala Leu Ala Ala Tyr His Pro Thr Ala Val Gly Ile Phe Ser Thr Ile Gly Arg Arg Pro Ala Glu Glu His Thr Thr Arg Asn Lys Asn Ile Ala Val Ile Tyr Ser Ala Tyr Thr Ser Leu Ser Lys Leu Tyr Pro Gln His Glu Ala Thr Trp Gln Arg Met Met Ala Thr Ala Gly Leu Asp Pro Ala Val Thr Ala Glu Asp Arg Thr Thr Ala Ser Gly Ile Gly Ile Leu Ala Ser Lys Asn Ala Met Ala Ala Arg Arg Asn Asp Gly Thr Asn Arg Asp Gly Asp Ala Gly.Gly Arg Arg Tyr Asn Arg Glu Pro Tyr Ala Asp His Thr Gly Tyr Arg Pro Val Asn Ser Pro Tyr Glu Leu Arg Phe Pro Ser Arg Trp Gln Pro Asn Thr Ile Ser Lys Arg Glu Val Val Leu Thr G1n Glu Phe Ala Thr Pro Gln Phe Gly Arg Val Lys Pro Ile Thr Phe Glu Arg Pro Glu G1n Phe Arg Leu Thr Pro Pro Pro Asn His His Leu Leu Asn Pro Lys Gly Tyr Arg Lys Gln Ala Asp Glu Val Leu Arg Ala Ser Ala Gly Leu Asp Asp Arg Lys Lys Met Ser Ala Glu Ile Phe Ser Asp Asn Ile Thr Pro Tyr Gly Ala Ile Ala His Thr Leu Leu Arg Gly Arg Tyr Asn Thr Glu Asp Ser Val Arg Phe Ile Val Met Thr Asp Val Ala Gly Phe Asp Val Ala Ile Ala Ser Trp Tyr Tyr Met Arg Lys Tyr Asp Ser Val Gln Pro Phe Ser Ala Ile Arg His Leu Tyr Pro Asn Lys Lys Leu Thr Ala Trp Gly Gly Pro Gly Arg Gly Thr Val Asn Asp Ile Thr Gly Thr Gin Trp Arg Ser Tyr Leu Ser Ser Val Ala Ile Ala Ala Pro Asp 'Pyr Pro Ser Val Asn Ala Ala Val Cys Val Ala Tyr Ala Gln Val Ala Arg Arg Phe Thr Gly Thr Asp Lys Leu Thr Val Val Ile Pro Val Arg Lys Gly Ser Ser Ile Val Glu Pro Gly Val Thr Pro Ala Ala Asp Met Met Leu Thr Trp Asn Ser Tyr Ser Glu O'v'vT opbobboqpb poboobobop qbpoa pboob oobboboqbb oqoobooooq qoppbpbobb 08~Z oobbbja qbb bo3b-eb,2obb bobqbpboob oobbbqbpbb o-joeqob-eop ~Bb~oo~o~o 0Z~T oboobboooo -ebqbobbboo Psbbqba qpb oqoo;a bbb-e ea boa qbboo 09ZT oq-ebqboqbo oebqob~-2312 bbo-eobboo-2 oqqbboobob oboqbbpooo bo7eloobolb 00ZZ obqoqbbobb a bo~-2aqbbo qbo3o~~q,2b booqobba ba qeooboqbbo q3beoqoa 2q O'vTZ ob-ea ba bbqb eooa -2obboo -eoqpopbopp oqboopobbb boobbbooob ba bbbbqbob 080T ao-ebqob-ePb i2i2Db2boooP qbqa o-eooba oILDbobob-eo qqboob-eoba bb3qol2boeq 0Z0T bp,2oba bqpo -eqo-2qbbq3o qba boq-eba b bqbo-ebo;qb bbooboqbae bqopbqebqb 096 0qeoqqbboo qba Dqo-ebbp boa po~-eo-2q bboobbbbob qoa qa bo,23"2 oba boqeoob 006 obbopqboob oeoqPoP-eop bo52oqqaqp b72bb3bobpb qi2S~-ebe-eob oo-ebaebbqo 0fi8 obbba bboqa obobobqa bq bb,2boe6o3b b-23b~-ebboo -eqobbbi?-eba ~oi2-e5jqbja 08L o-2ooL-oo-el2b ooba aboooo poqobbooqq b-eobi2booa b bob,2boqqoo 2oq-2boob*ep 0ZL oqbbboobbo qqbeoaaa bo -2bobo-4qb-eb bpobo2bqoo qboqbb-ebob ob~-2ooqoqp 099 oo2o~-eboob -eobbqoboba qboooqqobo bqa beboeqb ooob,2oi2,2oq bboobboa ,2q 009 3bboo-2oeoo pboobo-eqbo obpbqboap-e o~~a bo;boo bbobbboba p bobbo-2bobo OfiS o~-2bo2abbo '2bo"eebbDob obobbobbqp bobo~2i?5e-eb oqooba qao; Pa bboq"eobb 08V obpoobooea o,25boa pbb-e bboboo-23qb oobbooo-25b qaobbboboa -eoobbqebqi2 0Z17 bbob-eobbqo o~ebobbpbo-e ob-eooooopq oqob,2-eob-eo qoboloo-eo~ -loobboqoeq 09~ oqpa qbooba q-23P,2b-2ea e Pa boba Pbo-e o-eob,215b-2bo obooa qboob oobboq-ea oLo 0 0 ~ aoqaqqoq-eD bboqbboboo Poaoo~=,eq bobbobbloo obopbojqbb jboobqo~-2b O:~ Z oq-eaqabqeo a ba boo-ebbo boobbb-13oq bbboa obboo bbobpoejbP boqbbboboo 08T b-eboopoi2po ~ooobbooo~ qo~-eoqbo-eb oo-ebqooobo ~-ebbbopeo-e boqoo72boqj 0ZT oo-eboobabb Looboooobqo bbobbqbqbb oLo,2-eo-ebqob oqbobbqbbq oooboqLobob 09 bobooboqbo a bboboqbqb bbbboqoq,23 bbo~~-ebbob oobbobbpbq oqqpo~-ebqb ZL <00~,>
TZODa-9-v0 uTezqs - ds ~.~iodsouovsoaozw <~TZ>
VNQ <ZTZ>
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~6 0Z9T oDbpobpboq ob~b~obb~o bpbqbbbb3o bbbobpbaqj o;bbqpbqbb qoboea oeoq 09ST qbooob,2o5,e bqjooqobpo bobbboa qbq PobobqbDbLo ba -eooqboob -eebobbj"2bb 00ST booqbobo-eo obpobqbobo a boq-ebb-eob oboqoa qobb boebboobqo 3jo-jboq2oo O'v'vZ bbobobqboq bobbobboob oqbbbobqoo bboPbbbbob bpqobboobb oobobbqboo 08E T bbbbboaoPb qDbbpbbooo a abboobobo ooppbboDbb oa obbDoboo obbooobopb OZ~T oboobDabab bobbopboqb oa bbbDbDjD oboo-eopqbo bqDbDobobb oqobobbqoo 09ZT pqbqobbbob boqqbqobbq oqqoaboobo qqbqoa qqob bobobpboob b~~bo~o~ob OOZZ obpqpoDbbq aboba bbDob bj3bobD325 Iboobopqob 5o3bb533qp bbbopqoobo OPZZ bqbqobobob baobboboqo boabbboobo qpbb;bqobo bbobobboqb bbqobboqbo 080Z bbaqbboqa b bqbboobboq boboqbbobb qbbqobqba b bDDobqpbob bqba qqpbb:l OZOT bboopb;oqp qoba bbqbbq bbDpbbqbbD obpbba ba bb a bqooobbbq boobqbaqpb 096 obbobbobbb qoobbqpbqD boba qoboDD qqopqa pabq Dobbbopbpb obboobbobo 006 bqaoba qqbq -ebopbqaoqb bobbobDbbo obD0qbbopq bbbboobqba pobbbooobo 0~V8 oabobbqboq oba oboboob bDboqboqob a bboDbqobq obapo0obqa boboqppbba 08L ooobboqobb oobooqooqb obbbpobpbo bboDa obba o bbpa oI-eboo bpbbqbboDb 0ZL qbpoboDqbo obqabqoo;q Dbpbqbobqb qbooboqpbq obqbobbaqb bobbqbbobo 099 obbqooaqbq ooo2bboobb oobbbooobb ooDboDpbbp ooobboobqo bbbbqobpbb 009 Pboboba bbp a bbooobbqb opbbbobabo a bobboqoob booboqooqp bpbopoa obo Of'S bpbbapbbob eopboqoa ob opba a boqpa Pobobobooq bqpbba boqb oqpbpbbpbb 081V obbopobboo qboooqbopb 3345qqbpbb oboPboqabo bobobqa qpq oopb5qb3bb OZP obbebboooj oobba Pbopb a bpbqpobpp bbpbbbbbqp bqoopbopbb qbopobDobp 09E ooqpbpbbPD bbqbqbbobb oboqqb;bbb obqooeobDb bbobbbbpoo Pboa paqpba 00~ bopqopqbbo bbDoobopab bbbpobpboD ooobbqbbbq bbooopbpob IboD135bao 0tlZ bbpobbobqp obooDbOJob jaboD0pqbq DobpDbbbpb obpboboD72b loqbbobooq 08T booboqobbq Deboabbqop pbbqoobbop qbqpopbobo bbobDqoqob loaobDqa bq 0Zti bobqa pqbpb bqobqba bbb PpDboDqpov pboplopalp bbobpbqbop obbbbopbbq 09 pobq-ebobqq oqpqbbppob eqpqbqqobb qpbppobaaa ooa pbooqqb obbopoobeo 8L <00'v>
TZOOa-9'v0 uzPz4s =ds PzodsouoUJoaozN <~ZZ>
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agggcggaca ggccggcggt cgcgaacgac gcgtgcacca gcgcggcgac gaacagcccg 9240 gccagcacca cgaacccggc ccgccggccg tacctgacgg tctgccgggt gacgagcgcg 9300 aagtcgacgc ccggcacgat gatgatgagc aggctggcgg cgacgaaact gatgatctgg 9360 atgtcagaca cgacgccggc tctcctgtcc tccggcgagc gccggcactg cctcctcgat 9420 gacggagacg ccgctgtcct ggcgtggtcc gtgccggcgc cactgttccc gcagccggat 9480 ccggccgtcc ggcagccgtt cgggccggga ctcgcactcg ccgatgacta tggtgccgtc 9540 ggtgagcacc tccaggtagg cgaagcgcac gacgccctgc gcgtcgcagg tgccggccag 9600 ccggccgtgc cggaccgggc cgccggtgat ctccgcccag accaggtcgc cacgctggtg 9660 gtagtgcccc cgcagcggct cggcgccgtc accggcgtcg tggtccaccg agacgaagac 9720 gcggccgtcg tagtcgaatg tcgtcatcgc gctcacgccc ac 9762 <210> 74 <211> 112 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 74 Met Asp Gly Ala Arg Glu Arg Ile Thr Thr Asn Ile Arg Lys Gly Val Leu Glu Tyr Cys Val Leu Ala Leu Leu Ser Arg Arg Asp Met Tyr Gly Leu Glu Leu Ala Asp Trp Leu Ala Val Arg Gly Leu Thr Ala Ser Glu Gly Ser Leu Tyr Pro Leu Leu Ala Arg Met Arg Gln Ala Gly Ser Val Gln Thr Arg Trp Val Ala Pro Glu Gln Gly His Ala Arg Arg Tyr Tyr Ala Ile Thr Asp Gln Gly Arg Ala His Leu Arg Val Phe Ala Ala Val Trp Gln Glu Ile Gln Pro His Val Asp Asp Leu Met Gly Glu Glu Ala <210> 75 <211> 339 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 75 atggacgggg cacgtgagcg gatcactacg aacatccgca agggcgtgct ggagtactgc 60 gtgctcgccc tgctctcgcg gcgcgacatg tacggcctgg aactggccga ctggctcgcc 120 gtccgcggtc tgaccgcgag cgagggcagc ctgtatccgc tgctcgcccg catgcggcag 180 gccggctccg tgcagacccg gtgggtggcc cccgagcagg ggcacgcccg gcggtactac 240 gcgatcaccg accaggggcg ggcgcacctg cgggtgttcg cggcggtgtg gcaggagatc 300 cagccgcacg tggacgacct gatgggggag gaagcatga 339 <210> 76 <211> 325 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 76 Met Ser Asp Asp Gly Leu Pro Glu Ala Ala Trp Thr Tyr Leu Arg Ala Leu Asp Ala Glu Leu Ser Asp Val Pro Ser Gly Thr Ala Glu Glu Ile Val Ala Asp Val Arg Ala His Ile Ala Asp Ala Leu Asp Ser Gly Arg Ser Ala His Glu Ile Leu Ala Gly Leu Gly Ala Ala Arg Asp Val Ala Arg Gln Ala Arg Glu Glu Leu Gly Leu Pro Ala Gln Asp Arg Pro Ala Arg Ala Gly Arg Thr Leu Ser Leu Ala Ala Val Ala Val Gly Val Leu Ile Ala Val Cys Val Ser Phe Leu Leu Pro Ser Ala Val Pro Val Glu Pro I1e Gln Ala Gly Pro Gly Glu Gln Gly Val Leu Arg Arg Leu Gly Pro Gly I1e Ala Leu Leu Thr Leu Leu Pro Ala Leu Val Ala Ala Ala Pro Leu Val Ala Pro Ala Arg Ala Arg Ala Gly Val Arg Phe Ala Gly Ala Ala Val Leu Thr Met Phe Ala Cys Ala Ala Gly Glu Thr Gly Leu Tyr Tyr Phe Pro Leu Ala Leu Met Ala Trp Ala Ala Ala Ile Val Pro Trp Ala Leu Arg Arg Gly Ala Gly Gly Arg Trp Trp Arg Tyr Leu Thr Gly Gly Phe Val Ala Met Pro Gly Val Leu Val Ala Val Ala Ser Ala Gly Gly Ser Val Gly Val Gly Trp Val Gly Ala Ala Leu Trp Ile Ala Gly Pro Leu Ala Ala Gly Ala Leu Cys Ala Tyr Gly Ile Arg Ala Gly Tyr Ala Val Thr Ala Leu Ala Gly Ala Leu Ala Ile Ala Leu Ser Met Ala Glu Arg Gly Phe Leu Phe Ala Ala Phe Trp Leu Phe Gly Gly Leu Tyr Leu Ala Leu Gly Ala Ala Ala Tyr Thr Ala Ser Arg Ala Val Asp Gly Asp Ala Ala Ala Thr Pro Gly Pro Pro Ala Arg Pro Glu Pro Ala Pro Ala Pro Gly Gly <210> 77 <211> 978 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 77 atgagcgacg acggcctccc ggaggcggcg tggacctatc tgcgcgcgct cgacgcggag 60 ttgtccgacg tcccgtccgg cacggcggag gagatcgtcg cggatgtccg cgcgcacatc 120 gccgacgccc tcgacagcgg acggagcgcc cacgagatcc tcgccggcct cggcgccgcg 180 cgggacgtgg cccggcaggc gcgcgaggag ctggggctgc cggcccagga ccgcccggcc 240 cgggccggcc ggaccctgtc cctggccgcg gtggcggtcg gcgtgctgat cgccgtgtgc 300 gtgagcttcc tgctgccgtc cgcagtgccg gtggagccga tccaggccgg ccccggcgag 360 cagggcgtcc tccgccggct cggccccgga atcgcgctgc tcacgctgct gccggcgctc 420 gtcgcggccg cgccgctcgt ggcgcccgcc cgggcacgtg ccggggtacg gttcgccggc 480 gcggcggtcc tgacgatgtt cgcctgcgcg gccggcgaga cgggcctgta ctacttcccg 540 ctcgcgctga tggcctgggc ggcggcgatc gtgccgtggg ccctgcggcg cggagccggt 600 ggacggtggt ggcgctatct gaccggtgga ttcgtggcga tgcccggcgt gctggtggcg 660 gtcgcgtcgg ccggtggctc ggtcggcgtc ggctgggtcg gcgcggcgct gtggatcgcc 720 gggccgctcg cggccggcgc gctgtgcgcc tacgggatcc gggccggcta cgccgtgacc 780 gcgctggccg gcgcgctggc catagcgctc tcgatggccg agcgcggctt cctgttcgcc 840 gccttctggc tgttcggcgg gctgtacctg gcgctcggcg ccgctgcgta caccgcctcg 900 cgggccgtcg acggcgacgc cgccgcgacg cccggcccgc cggcccggcc ggaacccgcg 960 ccggcccccg gaggctga 978 <210> 78 <211> 663 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 78 Met Leu Asp His Ala Ser Gly Arg Ile Asp Val Thr Arg Leu Arg Glu Ala Leu Asp Gly Arg Trp Ala Glu Val Arg Arg Ala His Arg Glu His Leu Asp Glu Arg Phe Leu Pro Val Tyr Gly Glu Thr Gly Asp Gln Ala Arg Glu Arg Ile Thr Arg Leu Leu Ser Glu Leu Pro Val Glu Leu Gly Ile Ala Ser Gly Phe Pro Ala Glu Tyr Gly Gly Arg Glv Asp Val Gly Ala Ser Ile Val Ala Thr Glu Met Leu Ala Gln Val Asp Leu Ser Leu Met Val Lys Ala Gly Val Gln Trp Gly Leu Phe Gly Gly Ala Val Ala Ala Leu Gly Thr Lys Arg His His Asp Ala Tyr Leu Arg Asp Ile Val Ala Gly Arg Leu Phe Gly Cys Phe Ala Met Thr Glu Thr Gly His Gly Ser Asp Val Gln Gln Leu Arg Thr Thr Cys Val Tyr Asp Pro Gln Thr Gln Thr Phe Asp Leu His Thr Pro His Glu Ala Ala Arg Lys Asp Tyr Ile Gly Asn Ala Ala Arg Asp Gly Arg Met Ala Val Val Phe Ala Gln Leu Val Thr Gly Gly Arg Arg His Gly Val His Ala Trp Leu Val Pro Ile Arg Asp Glu His Gly Lys Pro Met Pro Gly Val Thr I1e Gly Asp Ala Gly Pro Lys Ala Gly Leu Leu Gly Val Asp Asn Gly Arg Leu Ser Phe Asp His Val Arg Val Pro Arg Glu Met Leu Leu Asp Gln Tyr Ala G1n Val Ala Glu Asp Gly Thr Tyr Ser Ser Pro Ile Glu Asn Asp Ser Arg Arg Phe Phe Thr Met Leu Gly Thr Leu Val Arg Gly Arg Val Ser Val Gly Gly Ala Ala Ser Ala Ala Thr Lys Ser Ala Leu Ala Ile Ala Val Arg Tyr Gly Asp Ile Arg Arg Gln Phe Ala Asp Ala Asp Gly Asp Arg Glu Val Leu Leu Asn Asp Tyr Leu Ala His Gln Arg Lys Leu Leu Pro Ala Leu Ala Thr Thr Tyr Ala Leu Thr Phe Ala Gln Ala Glu Leu Val Ala Ala Leu Asp Asp Ile Gln Gly Gly Asp Gly Pro Val Asp Glu His Arg Gln Arg Glu Leu Glu Ser Arg Ala Ala Gly Leu Lys Ala Ala Gln Thr Trp His Ala Thr Arg Thr Ile G1n Ile Cys Arg Glu Ala Cys Gly Gly Ala Gly Tyr Leu Ser Glu Asn Arg Leu Pro Ser Leu Lys Ala Asp Thr Asp Val Phe Thr Thr Phe Glu Gly Asp Asn Thr Val Leu Leu Gln Leu Val Ala Lys Gly Leu Leu Thr Gly Tyr Arg Asp Glu Phe Gly Ser Leu Asp Gly Trp Gly Arg Ala Ser Phe Val Ala Glu Gln Val Arg Glu Met Val Leu Glu Arg Thr Ala Ala Arg Ala Leu Ile Ala Arg Leu Val Ser Ala Val Pro Gly Arg Asp Asp Glu Val Ala Val Thr Asp Arg Gly Trp Gin Leu Lys Leu Phe Glu Asp Arg Glu Glu His Leu Leu Asp Ser Ala Val Arg Arg Leu Arg Gly Gly Ala Ser Thr Lys Lys Asp Arg Pro Phe Asp Ile Phe Asn Asp Val Gln Asp His Val Leu Ala Val Ala Ala Ala His Ile Asp Arg Val Thr Leu Glu Ala Phe Val Ala Gly Ile Asp Ala Ile Ala Asp Pro Ala Val Lys Glu Leu Leu Ser Arg Val Cys Asp Leu Tyr Ala Leu Thr Val Ile Glu Ala Asn Lys Gly Trp Leu Leu Clu His Gly Arg Leu Thr Pro Ala Arg Ser Lys Thr Ile Thr Ser Val Val Asn Gly Leu Leu Lys Glu Leu Arg Pro Asp Met Arg Thr Leu Val Asp Gly Phe Ala Ile Pro Asp Ala Trp Leu His Ala Ala Ile Leu Arg Glu Glu Pro Val Arg Gln Glu Thr Met Ala Ala His Asp Ala Ala Gly Asp Pro Gln Ala Val Pro Ala <210> 79 <211> 1992 <212> DNA

~OT
008T aabbaaaa~a qabbaabbap abpbaqabqa bbqabbbp-ea Ppbabb72bal PbqbaaPaIa OTPLT babapqbqaa -2b35qaqbbb aaaqbqabqa Ppbbppaqbb abbaaapbaa baqpaabapb 089T aq-ebbbaaba qbaqqbabbp bbqabapbqb bb3apb3q-2a Pababbabaa baqbaabaqa 0Z9t aqba-eaaebb -eaaqba-eba~ -eajqaq-eaLob aqqaaaabaa -ebb~-eb-epaa -eaaqbababb 09SZ qbbababqaa baabaaqbba bab-eai?baja bqaapab-2bb -ebabaa,2bb-2 baqqaqabpp 00SZ aqab,2abbqa bbbbaal2baa pajbaabaqb bpbapba-eba babbbaaabq baababeaqb 0T717Z b~ajba-eaba q,2bq3babbb aba5aabaa,2 abaL-Pbbqab qbbq-eb-ebab a2qbbeabeb 08~Z aabbqbaqqa aqaababa-eb bbbjabba-eb aqabaqabba qqbl2bapbbb aaeqabbaa-e OZ~T bqabqabbbb ~~aaba~bb~ aPPabqabqa bqbba2a~-ea Pb3bbb-2baq qaaPaapaqq 09ZT aqbqebaa-ea -ebaabb-epaq aabPaaabla abaa-ei2b-eba aqbqaap;ab baababbabb OOZT qbqbabbebb baabqaq-eb-e aaq-eaa-eoba aa-eaaba-eaS bqaa-eb,2aba bbabb~-ebqa 0fZT qbbaabaabb baaaqbi2bbq ab-ebbbab-ea bbaa-pabeba 2baqbbaabb ba-ebabbabb 080Z b-eaaq-eaeba -ebaqababba ba;bbqab-eb babb-eaaaba qqaapbqaba bapqbaLoaa-e OZOT aabbqababa aabqabqabe -2abab,23aea babbqaa-eqa ebai2,eaqabq a5qbb,25aba 096 a2babbaeba abaebaabaq qb-eabbaaba aqi2a-ebabba Loqababjbba baj*eaabbja 006 babbaqbpea aeaabbabba qbabaababb abbbqbabpb qbbbaabbbb aaqbbqaaa-e N18 abbbqabq-ea a-2aqqaqqab abbaaajaeb a-eeb-ebaq-eb a3ab-eaaqa-2 jba-eabba-eb 08L b-eboabaqbb 2ababa-eqb-e aapbbqabja bq-2bebbbab aabqbbbabq ba-eaaebaqq OZL ab~a~abbab bba-eba-2bbq babbaqabqa abbaabbp-2a aabbbaabap babbaq2aa-e 099 bqbabbaaab qpbaab~-eab baPab-eba-eb abaaq-2baab qbbqabbqaa ba-2aBqbbbb 009 a-eaabaabab bbabbaapaq baqab-eaaab aqqbqbbqbq abbq'25babb bal2bbbaa05 OIV S 5abae"2abba q,2aLD;a-ebbe -eabababaab bl2ba-2aba33 a'ea'2ablaa-e baqI33-eb20 081V boLDbeobaaa e5ai2jaqbab Iaa-eaa-2aba bqaepabpab qbaebbaqab ba-eaabbaae 0ZIV bebaapbq-eb abaqqabqab baqqaqabba abbbabaqba ql2a,2bbbabq aa-eqaabaeb 09~ aeaapabbab -epba,2abbaq aaabaaba-ib baba.6babba ~;b-4aabbbb -4bpab-qb3bb 00~ aabb~-ebqbb q,2bqal2aq5q aapbbqbbea aabbqabq-eb -ebaa,eaabaq baq2baqaab 0IV Z abbbqba-2ba bbabaabbab baeqb-ebaab aaaaq;qbba aqaabaql2ab bbqabi2baqb 08Z aaaaqdp2ba aqbqabqabb aaa"2a;paba b-ebabaaabb 2a3e5qbbaa 7eb-ebabba-eq 0ZT bqbbaaaqaa 1; aba~7eba-e baq3apa~,2b abaa-2ab3b15 baabaaqbb-e baabbbqbba 09 abbaL-baqab 3bei2bbbabq aaba-ea-eaqb a-e5aq-2abaa bba3q-2ab3-e 3qeb3qabq2 6L <00'v>
ZTOOH-9TV0 uz72zqs -ds -e zodsouovzozazk <~TZ>
ZO-90-600Z O6~996Z0 FiJ

cgctcgaaga ccatcaccag cgtggtgaac gggctgctca aggagctgcg cccggacatg 1860 cgcacgctcg tggacggctt cgccatcccg gacgcgtggc tgcacgcggc gatcctgcgc 1920 gaggagcccg tccggcagga gacgatggcc gcgcacgacg ccgccggcga cccgcaggcc 1980 gtccccgcct ag 1992 <210> 80 <211> 573 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 80 Val Ser Pro Leu Pro Pro Gly Ser Ala Val Thr Ala Arg His Val Leu Arg Gln Ala Leu Arg Arg Gln Arg Arg Pro Val Leu Ile Gly Val Thr Leu Leu Gly Leu His Gln Val Thr Glu Ala Leu Val Pro Val Ala Ile Gly Val Ile Iie Asp Arg Ala Val Val Thr Gly Asp Pro Trp Ala Leu Ala Tyr Ser Val Ala Gly Leu Ala Ala Leu Phe Thr Val Leu Ala Phe Ala Tyr Arg Asn Gly Ala Arg Gln Ala Phe Ala Ala Val Glu Arg Glu Ala His Leu Leu Arg Val Glu Leu Ala Glu Arg Ala Leu Asp Pro Arg Gly His Arg Ser Gly Leu Arg Asp Gly Glu Leu Leu Ser Val Ala Ala Ser Asp Ala Glu Leu Ser Ala Tyr Val Val Arg Val Ala Gly Phe Gly Val Ala Ala Val Ser Ala Leu Thr Val Ala Ala Val Ala Leu Leu Val Ile Asp Val Pro Leu Gly Leu Gly Val Leu Ile Gly Val Pro Val Leu Val Leu Ala Leu Gln Arg Met Ala Pro Leu Leu Ser Arg Arg Ser Ala Ser Gln Gln Glu Ala Leu Ala Glu Thr Thr Ala Leu Ala Val Asp Leu Val Ser Gly Leu Arg Val Leu Arg Gly Ile Gly Ala G7.~n His His Ala Ala Gly Arg Tyr Ala Glu Ala Ser Arg Arg Ala Leu Ala Val Thr Leu Arg Ala Ala Asn Thr Lys Gly Leu His Leu Gly Leu Thr Thr Ala Ala Asn Gly Leu Phe Leu Ala Ala Val Ala Gly Val Ala Gly Trp Leu Ala Leu Arg Gly Arg Leu Thr I1e Gly Glu Leu Val Thr Val Val Gly Leu Ala Gln Phe Val Ala Glu Pro Val Gln Thr Leu Gly Tyr Cys Val Gln Leu Phe Ala Met Ala Arg Ala Ser Ala Ala Arg Val Gly Arg Val Leu Gly Ala Glu Pro Leu Thr Arg Pro Gly Ser Ala Pro Arg Pro Asp Arg Thr Asp Gly Pro Arg Leu Val Leu Asp His Val Gly His Ala Ala Leu Asp Giy Val Cys Leu Arg Val Asp Pro Gly Glu Ile Val Gly Val Leu Ala Tyr Asp Pro Ala Asp Ala Asp Ala Leu Val Ala Leu Leu Ser Gly Arg Val Pro Ala Asp Arg Arg Arg Gly Thr Val Arg Val Asp Gly Val Pro Ala Asp Asp Leu Asp Val Asp Ala Leu Arg Gly Ala Val Leu Val Glu Pro His Asp Val Thr Leu Phe Glu Gly Thr Val Ala Ala Asn Leu Ala Ala Gly Ser Arg Thr Glu Glu Gly Arg Leu Arg Ala Ala Val Arg Ala Ala Ala Ala Asp Asp Val Val Asp Ala His Pro Gly Gly Leu Gly His Arg Leu Val Glu Arg Gly Ala Asn Leu Ser Gly Gly Gln Arg Gln Arg Leu Gly Leu Ala Arg Ala Leu His Ala Asp Pro Pro Val Leu Val Leu His Asp Pro Thr Thr Ala Val Asp Ala Ala Thr Glu Ala Gln Leu Ala Asp Gly Leu Ala Gly Ala Arg Arg Glu Ala Pro Arg Gly Thr Leu Leu Val Thr Ser Ser Pro Ala Leu Leu Arg Ile Thr Asp Arg Val Val Val Ile Ala Asp Gly Arg Val Thr Ala G1u Gly Thr His Glu His Leu Leu Ala Thr Asp Ala Arg Tyr Arg Glu Glu Thr Leu Arg <210> 81 <211> 1722 ZZLZ pb '16baba0pae bpbbpbabaa pqabaaabale baaPaabbqa 089T bqaa-2abpba eabapbbbbe baabaapbqb bbaabbapbo abaji2bqbbq bbqbbbaa,eb 0Z9T aaeaqpbbab qabqaaaboa aab,2ab-eaa-e aqbbqabqab aeabbbbaaa abab~-ebaba 09ST ababababba abbqa-ebba,2 baabaqa,2-ea 3abbi2baaea abbabaebbq baabaa,eao-2 00ST aaao-ebaeab qabqbbqabq bbaobaaa-eb aaba-eabqab abb5ababb0 abbbaqabba 0'v'vti b-eaababeab bbabbaaqaq aaepaababb bbabl2baqba qabbaal2aab baqaabbabb 08~Z oaa3-2aboba i2bbqbbqbai2 bapbbabbab aabbabbbaa lbbobaabob ab;aababbb OZ~T bpbbpbaapb beabL>bbbaa baabaqaapp aabaabbqba a~~bbb~ba~ qbqabasbqb 09ZZ apbapabaab pba;bbqaaq baababbaba b;ababapba qbapbbqaap bapbaabaaa 00ZZ Pqbbbbapba qbabapqbba pabbbbaaba bbaanbbaba aabqbbbabb baaqbqabqa O'vZZ babbqbbqab abapbbabap baabbaaaPb apjbabbqao qbabbaqbaq Pbpbpbbbaa 080T apbaqbabab qaabqbqbbb bopbbqabab aabaeaabba qbapaapbaq oaqbaqabba 0Z0T baabbbapbb apabaapbba abbaaaabab abPabbbaab baaaPbqaba abPbaababb 096 aqobqbabab bbaqbbbaaa baabaaqaab abaaabbqpb abaqqbqabp abqbabqapq 006 abbbqabapb Pa51b53abp baabaqbaq; bpababaqab bbaqbbqbaa PaqbbqabPb 0b8 abbaqeaaPa qabbaabbob abqababaqa bbqabbaaba qbbbbaabaq baabaabaqa 08L aqjoqaabba Ppbabaabaa paapaiabbb oqaapabqaa bbbppaapap paabaababa OZL bjobapbqba abaqaaabab apbaabi2aab bpbaabapqb baabbaabaa bapaapabPa 099 aobabbaqPa bbababqabq bababqaabb aaqaqbaqaa pbbqbaabaq ababbapaap 009 bpbbabaqaa abbpbbpabp aaaqaobab2 ababbaaaqb Iabqabaaba bbqpbbaPPa 0~v5 bqababbjaa qbbjabjbba apqbabbaqp aqabqbabba qapbbaloba aaqbapbaqp 08IP aqbbqabqab abaqbbabba baqbaapbqa bababpbqbb abaabalbab baqqabbaab 0Zlv bIbbbaaqbb lbapqbabaa qaqappbaab apboaqaaba abaqbbaqaq abjabvbabb 09~ a-ebabobqaa bbaalabaa-2 abbbababaa a-ebaqababa babebaabbq abpbaqbbba 00~ bqabqaaPab abb-ebbbapp bbqbbabbab aqqbabbeaa baaababbap Pabaapqaab OlvZ aqqbabbqab qbaaPaIIbq aaabaabaqa abbaabaqba aqapqbabaq ababbbqbaa 08Z apbabbaaPb qbbqbaabbb aapbaqpaqp aqbabbaqpb abbqbbaabq baqobabbpb 0ZT aaVajbbpaa Labqabbbaq abqaaapbqb abboypbqab qbbaaabaab abpaabaaba 09 bqababbeaa baaqabqbap abbaaabaap aqbaababpa bbaaaaaaqq abaaoaqbqb Z8 <00'v>
TTODS-9:~0 u?P31s =ds Pzodsouov1030zW <~ZZ>
VNQ <ZZZ>
ZO-90-600Z O6~996Z0 Fi'J

<210> 82 <211> 596 <212> PRT
<213> Micromonospora sp. strain 046-ECOll <400> 82 Val Thr Ala Asp Pro Arg Thr Ala Glu Pro Thr Arg Val Leu Leu Pro Thr Ala Thr Ala Arg Arg Thr Trp Thr Thr Leu Gly Ala Glu Phe Arg Arg Arg Pro Gly Leu Ser Ala Ala Ala Thr Ala Val Leu Val Ala Ala Ala Thr Gly Gly Leu Val Ala Pro Trp Val Leu Gly Arg Leu Val Asp Asp Val Ile Ala Asp Ala Pro Val Ser Arg Ile Ala Gly Arg Val Ala Val Ile Ala Gly Ala Ala Val Leu Thr Gly Leu Leu Thr Ala Ala Gly Ala Ala Leu Ala Ser Arg Leu Gly Glu Thr Val Leu Ala Arg Leu Arg Glu Arg Val Leu Asp Arg Ala Leu His Leu Pro Ser Ala Thr Leu Glu Arg Ala Gly Thr Gly Asp Leu Leu Ala Arg Val Gly Asp Asp Val Ala Val Val Thr Asn Val Ile Ala Val Ser Gly Pro Ala Phe Val Gly Ala Leu Leu Ser Val Val Leu Thr Val Phe Gly Leu Val Ala Leu Asp Trp Arg Leu Gly Leu Ala Gly Leu Val Ala Ala Pro Ala Tyr Ala Leu Ala Leu Arg Trp Tyr Leu Arg Arg Ser Ala Pro Tyr Tyr Ala Arg Glu Arg Val Ala Thr Gly Glia. Arg Thr Gln Ala Met Ala Gly Ala Leu Arg Gly Ala Ala Thr Val Arg Ala Tyr Arg Thr Glu Asp Ala His Val Ala Ala Ile Ala Glu Arg Ser Gly Val Ala Arg Asp Leu Ser Leu Glu Ile Phe Asn Leu His Thr Arg Phe Gly Leu Arg Ile Asn Arg Ser Glu Phe Leu Gly Leu Ala Ala Val Leu Val Ala Gly Phe Phe Leu Val Arg Ala Asp Leu Val Thr Val Gly Ala Ala Thr Thr Ala Ala Leu Tyr Phe His Arg Leu Phe Asn Pro Ile Gly Leu Leu Leu Met Glu Ser Asp Ser Val Leu Gln Ala Gly Ala Ser Leu Ala Arg Leu Val Gly Val Ala Thr Leu Pro Asp Thr Ala Pro Ser Gly Pro Ala Pro Ser Ala Ala Gly Arg Arg Gly Pro Ala Ala Leu Asp Val Thr Val Arg Arg His Arg Tyr Asp Asp Asp Gly Pro Leu Val Leu Ala Asp Val Asp Leu Arg Leu Ala Pro Gly Glu Arg Val Ala Leu Val Gly Ala Ser Gly Ala Gly Lys Ser Thr Leu Ala Gly Ile Ala Ala Gly Ile Ile Ala Pro Thr Asp Gly Ser Val Arg Leu Gly Gly Val Pro Leu Thr Glu Arg Gly Glu His Ala Val Arg Arg Asp Val Ala Leu Val Ser Gln Glu Val His Val Phe Ala Gly Pro Leu Ala Glu Asp Leu Arg Leu Ala Ala Pro Asp Ala Thr Asp Ala Glu Leu Leu Asp Ala Leu Asp Arg Val Gly Ala Thr Thr Trp Leu Arg Ala Leu Pro Asp Gly Leu Ala Thr Ala Val Gly Glu Gly Gly His Arg Leu Thr Ala Ala Gln Ala Gln Gln Va1 Ala Leu Ala Arg Leu Val Leu Ala Ala Pro Ala Val Ala Val Leu Asp Glu Ala Thr Ala Glu Ala Gly Ser Ala Gly Ala Arg Asp Leu Asp Arg Ala Ala Leu Ala Ala Thr Glu Gly Arg Thr Thr Leu Ile Val Ala His Arg Leu Ser Gln Ala Val Ala Ala Asp Arg Ile Val Leu Leu Asp His Gly Arg Ile Val Glu Gln Gly Thr His Ser Glu Leu Leu Ala Ala Asp Gly Arg Tyr Gly His Leu Trp Arg Ser Trp Ser Val Pro Val <210> 83 <211> 1791 <212> DNA
<213> Micromonospora sp. strain 046-EC011 ~8 <oiZ>
T6LZ -e bq-eqbbaaaq bab,2bbqoaq ababbqbqaq -eabbbai2qbb aabbal2baab 0:~LT aabaqabqa~ -ebbaqa2aba -2abbb-eab*eb bjbajebbab bba2aaebaq abqaaqbaq'e 089T bbaa-ebaaba aba~bbabb-e aab-e aq abba a~ababb~ba q,2bjaba'eaa -ebba72bbbPb 0Z9Z aaeaabaabb qababbabbb aaebbqaa-2b -4babab-2bba abab2abbaa bbpbaabaa-e 09SZ aabbeba-25b -q ab-4 baaB a~ baabaaabab aabb-jabjbb :~abbaaabbq aaabaqbbpa OOST b-eaaabbL>ab abaabaa~a~ abbaa-eaabb ;Dbbbpbabba qbbab-2apaa b5qa5bba-25 O'v 'v Z baabjababa babqabbqaa Paa,2aababb 31bbbaa2bb qababaeba-4 abqal2Pbaab 08~Z ai2baai2aaba -ebbaaaabqa bbqaababqa ~-ebb-2baaba qabaal2bbqa ba;aa-453-ea 0Z~T b;bbsbbLoaa b,2aqbbqaba ba-qbaPbaba bbab~baaba -2abebabbbb ab,2baa2bqa 09ZT baabqbabba bbbqaaba-eq bbaqbbba-eb aaeaaababa apaqpbbbaa baaba-jpabb 00ZT aaba-qaba-ea beb~-2abbba babbab-eaab abbbabaqab abaqbbbabp babbbaaaab ZfIZZ bqaababqaa P5ajba-2baa bbqaaqbbqa laaabba2ba p5apbap-135 aapabbaaba 0802 aqbbapaqba pbbqababba bbaaabbaba bbabbbaabb abbaqbaaba baaabbbaaq OZOZ baaaabaava pbaaabqaba paabbqbabb aqbblabbaa abaqaabvba babbaabbpa 096 bqabqbbaqa pbaaqbpbbq pbqabqabqa abbaqebaaa ppaqqbqabb aaPaaqqapq 006 aqababaaba apaapbabba babbbqbpap aqbbqaapba ababaaqbbq aaqqaqqbbb 0fi8 aabaqbaqab qbbabaabbq aabbaqaaqq bpbbaqbbpa -eeaqpbbabq abbbailbba 08L aaeaPabqaa ppaqqaqpbp bb-4ab3jbja apbabababb qbabba0406 abpbaabaqp 0ZL babbabaqba Pababapbbp baapbbaapq babababqba apaabbabab bqbabqabab 099 abbaabbqPb abbpabapbb abebabbaap aabaqbabab pbabaaabap qapqbaabab 009 baqbbaabab qaapqbbqab abqababbqa baba-eqaaba aababaabaq bbqabbbaab O:~S aqaabbaqab babb~a~ba~ ababaqbbqa bbbaqqbqba apbqabqbbq baaqbqabqa 081V bababba:~ba qqbabbaaab babpaqbbab aqpbqbapeb aP54bbqbba bbqbapbaPb 0Zil abbaqbbbaa abbqabqaap babbaa~abb aabbba~~bb ~aba~babba ~aaab~aa~a 09~ blababbbaa -ebaqaaqbbb abpbababqa bbaaabbqab qbbapbpbbb bbqaabaaal 00~ babaqababa abbbbaabaa baaPaqabqa pbbaapaqab qbpabbabab 500baq2bqb OlvZ babbqbbbaa bbaabaqpbb aaalaqbbaa aabaPbaaba IPa4baIebaP b04b3qaaba 08T abbaqabqbb bqaaaba baq bbqabbbabb aa-20abaaba abaIbaqaba baabaaPbab 0ZZ aabaababpa qaabbaaabb abbaabaaqq bPbbababba qabapbapbb qaa,ebbabba 09 aabaapbaba aPaaabqabq qbqbbbaaae aaappbaaba aeqbabaaae bqabaapbqb ~8 <00v>
ZO-90-600Z O6~996Z0 Fi'J

<211> 507 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 84 Met Thr Asp Ala Pro Ala Arg Phe Val Leu Phe Pro Gly Arg His His Leu Leu Thr Arg Phe Gln Ala Asp Tyr Leu Arg Arg Leu Ala Gly Asp Asp Ala Thr Val Val Trp Ala Val Thr Ser Ala Asn His Glu Asn Thr Arg Arg Asn Pro Val Pro Tyr His Arg Arg Glu Ala Ala Ile Glu Arg Phe Ser Val Leu Ser Gly Leu Arg Ser Val Val Val Pro Ile Phe Asp Thr Ala Tyr Thr Asp Ala Phe Ala Glu Val Thr Leu Lys Ser I1e Ala Val Ala Thr Gly Leu Glu Leu Thr Pro Ala Asp Thr Val Leu Ala Cys Ser Thr Pro Glu Val Ala Lys Leu Tyr Glu Gln Leu Gly Phe Ser Ile Ala Pro Val Glu Ala Asp Pro Asp Leu Pro Glu Pro Pro Glu Arg Pro Trp Asp Val Leu Leu Arg Leu Ala Ala Gly Asp Glu Thr Trp Arg Ala Leu Thr His Pro Ala Thr Ile Asp Val Phe Glu Arg Tyr Arg Leu Val Glu Ser Ile Arg Ser Val Val Asn Asp Pro Leu Val Gly Asp Glu Gly Gly Leu Thr Val Thr Arg Asp Tyr Arg Thr Tyr Val Glu Ala Phe Ala Thr Ala Ala Gln Arg Lys Trp Asp Ser Val Arg Arg Tyr Val Gln Pro Gly Arg Ile Val Asp Ile Gly Cys Gly Ala Gly Ala Val Leu Glu Leu Ala Asp Arg Glu Ala Ala Leu Arg Glu Ser Asp Leu Ile Gly Val Glu Val Ala Arg His Leu Tyr Gln Glu Cys Leu His Lys Lys Ala Gln Gly Va1 Phe Arg Asn Ala Asn Val Tyr Phe Phe His Arg Asn Val Leu Gly Gly Ala Val Phe Lys Asp Arg Ser Val Asp Thr Thr Leu Thr Phe Ala Leu Thr His Glu Ile Trp Ser Tyr Gly Arg Arg Arg Glu Ser Leu Leu Gln Phe Ala Arg Arg Ile His Asp His Thr Val Pro Gly Gly Val Trp Ile Asn Ser Asp Val Cys Gly Pro Asp Asp Pro Arg Arg Gln Val Leu Leu Arg Leu Ser Thr Asp Asp Gly Asp Asn Pro Ala Ala Pro Arg Pro Asp Leu Ala Glu Leu Thr Ser Ala Glu Val Arg Arg Tyr Val Gly Gly Leu Ser Thr Arg Ala Arg Leu Asp Gln Phe Ala Val Asp Phe Ala Phe Asp Phe Asp Tyr Glu Pro Leu Pro Asp Gly Ala Val Arg Leu Thr Leu Gly Ala Ala Met Asp Tyr Leu Thr Arg Lys Asp Tyr Thr Asp Asn Trp Leu Ser Glu Thr Gln Glu Gln Phe Cys Gly Leu Ser Ph.e Ala Asp Trp Thr Asp Leu Leu Thr Glu Ala Gly Phe Glu Ile Gly Pro Ala Ser Ala Pro Val Arg Asn Glu Trp Val Ile Asp Asn Arg Ile Ala Pro Val Ala Ser Leu Thr Asp Leu Asp Gly Arg Pro Leu Asp Trp Pro Thr Thr His Val Leu Thr Val Ala His Arg Pro Arg Asn Gln <210> 85 <211> 1524 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 85 atgaccgacg cgccggcccg cttcgtgctc ttcccggggc ggcaccacct gctgacccgg 60 ttccaggccg actacctgcg gcggctggcc ggggacgacg ccacagtggt ctgggcggtg 120 acgtcggcca accacgagaa caccaggcgc aacccggtgc cctaccaccg gcgggaggcc 180 gcgatcgaac gattcagcgt gctgagcggg ctgcgctcgg tggtggtgcc gatcttcgac 240 accgcgtaca ccgacgcgtt cgccgaggtg acgctgaagt ccatcgcggt ggccaccggg 300 ctcgaactca cccccgccga caccgtgctg gcctgctcca cgccggaggt cgcgaagctg 360 tacgagcagc tcggcttttc gatcgcgccg gtcgaggcgg acccggacct gcccgagccg 420 cccgaacggc cgtgggacgt gctgctgcgc ctggccgccg gggacgagac ctggcgcgcg 480 ctcacccacc cggccaccat cgacgtgttc gagcgctacc gcctggtcga gtcgatccgg 540 tcggtggtga acgacccgct cgtcggcgac gagggcggtc tcacagtgac ccgcgactac 600 cggacctacg tcgaggcgtt cgccacggcc gcgcagcgca agtgggactc ggtacgccgg 660 tacgtgcagc ccggccgcat cgtggacatc ggctgcggcg cgggcgccgt cctggaactc 720 gccgaccggg aggccgcgct gcgtgagagc gacctgatcg gcgtggaggt cgcccgccac 780 ctctaccagg agtgcctgca caagaaggcg cagggcgtgt tccgcaacgc caacgtctac 840 ttcttccacc gcaacgtcct cggcggcgcg gtgttcaagg accgctcggt cgacaccacg 900 ctcacgttcg cgctgaccca cgagatctgg tcgtacgggc ggcggcggga gtcgctgctg 960 cagttcgccc gccgcatcca cgaccacacg gtgcccggcg gcgtctggat caacagcgac 1020 gtgtgcggtc cggacgaccc ccggcggcag gtgctcctgc gactgtccac cgacgacggc 1080 gacaacccgg ccgcgccccg ccccgacctc gccgagctga cctcggcgga ggtccggcgt 1140 tacgtcggcg ggctgtcgac gcgggcgcgg ctggaccagt tcgccgtcga cttcgcgttc 1200 gacttcgact acgagccgct ccccgacggc gcggtacgcc tgacgctggg cgccgcgatg 1260 gactacctga cccgcaagga ctacacggac aactggctgt cggagacgca ggagcagttc 1320 tgcggcctga gcttcgccga ctggacggac ctgctcaccg aggcggggtt cgagatcggc 1380 ccggcgtcgg cgccggtgcg caacgagtgg gtgatcgaca accggatcgc gccagtcgcg 1440 tccctcaccg acctcgacgg ccggccgctg gactggccga ccacccacgt cctcaccgtc 1500 gcccaccgcc cccgcaacca gtga 1524 <210> 86 <211> 232 <212> PRT
<213> Micromonospora sp. strain 046-ECO11 <400> 86 Val Ser Asp Ile Gln Ile Ile Ser Phe Val Ala Ala Ser Leu Leu Ile Ile Ile Val Pro Gly Val Asp Phe Ala Leu Val Thr Arg Gln Thr Val Arg Tyr Gly Arg Arg Ala Gly Phe Val Val Leu Ala G' y Leu Phe Val Ala Ala Leu Val His Ala Ser Phe Ala Thr Ala Gly Leu Ser Ala Leu Leu Val Ser Ser Pro Thr Leu Tyr Thr Val Leu Arg Val Ala Gly Ala Leu Tyr Leu Leu Tyr Leu Gly Gly Thr Ile Leu Trp Ala Thr Arg Pro Arg Arg Thr Val Pro Ala Ala Gln Pro Val Thr Val Gly Ala Gly Gly Ala Gly Pro Asp Thr Asp Thr Gly Pro Ala Pro Val Pro Asp Thr Pro Ala Ala Asp Glu Pro His Val Ala Arg Arg Ser Phe Val Met Gly Val Thr Ser Gln Leu Leu Asn Val Lys Val Val Val Phe Tyr Val Ser Phe Val Pro Gln Phe Val Lys Pro Gly Glu Gly Ala Ala Ala Arg Thr Ala Val Leu Ala Ala Thr Phe Ile Gly Leu Ala Val Leu Trp Trp Ala Cys Tyr Ile Met Leu Ile Asp Arg Leu Gln Pro Trp Leu Thr Arg Pro Ser Val Leu Leu Val Ile Glu Arg Leu Thr Gly Leu Ile Leu I1e Val Leu Ala Ile Arg Ile Ala Leu Ser Arg <210> 87 <211> 699 <212> DNA
<213> Micromonospora sp. strain 046-EC011 <400> 87 gtgtctgaca tccagatcat cagtttcgtc gccgccagcc tgctcatcat catcgtgccg 60 ggcgtcgact tcgcgctcgt cacccggcag accgtcaggt acggccggcg ggccgggttc 120 gtggtgctgg ccgggctgtt cgtcgccgcg ctggtgcacg cgtcgttcgc gaccgccggc 180 ctgtccgccc tgctggtctc ctcgccgacg ctctacacgg tgctgcgcgt cgccggcgcg 240 ctgtacctgc tctacctggg cggcacgatc ctctgggcga cccggccgcg ccggacggtc 300 ccggcggcgc agccggtcac tgtcggcgcg ggcggcgccg ggccggacac ggacaccggc 360 cccgcgccgg tgccggacac cccggccgcc gacgagccgc acgtggcccg ccgctcgttc 420 gtcatgggcg tcaccagcca gctgctgaac gtcaaggtgg tcgtcttcta cgtctcgttc 480 gtgccgcagt tcgtcaagcc cggcgagggg gcggcggccc gtacggcggt gctcgccgcc 540 acgttcatcg gcctcgcggt gctctggtgg gcctgctaca tcatgctcat cgacaggttg 600 cagccctggc tgacccggcc gtccgtgctg ctggtgatcg aacggctgac cgggctcatc 660 ctgatcgtcc tggcgatccg gatcgcgctg agccggtga 699 <210> 88 <211> 132 <212> PRT
<213> Micromonospora sp. strain 046-EC011 <400> 88 Val Gly Val Ser Ala Met Thr Thr Phe Asp Tyr Asp Gly Arg Val Phe Val Ser Val Asp His Asp Ala Gly Asp Gly Ala Glu Pro Leu Arg Gly His Tyr His Gln Arg Gly Asp Leu Val Trp Ala Glu Ile Thr Gly Gly Pro Val Arg His Gly Arg Leu Ala Gly Thr Cys Asp Ala Gln Gly Val Val Arg Phe Ala Tyr Leu Glu Val Leu Thr Asp Gly Thr Ile Va1 Ile Gly Glu Cys Glu Ser Arg Pro Glu Arg Leu Pro Asp Gly Arg Ile Arg Leu Arg Glu Gln Trp Arg Arg His Gly Pro Arg Gln Asp Ser Gly Val Ser Val Ile Glu Glu Ala Val Pro Ala Leu Ala Gly Gly Gln Glu Ser Arg Arg Arg Val <210> 89 <211> 399 <212> DNA
<213> Micromonospora sp. strain 046-ECO11 <400> 89 gtgggcgtga gcgcgatgac gacattcgac tacgacggcc gcgtcttcgt ctcggtggac 60 cacgacgccg gtgacggcgc cgagccgctg cgggggcact accaccagcg tggcgacctg 120 gtctgggcgg agatcaccgg cggcccggtc cggcacggcc ggctggccgg cacctgcgac 180 gcgcagggcg tcgtgcgctt cgcctacctg gaggtgctca ccgacggcac catagtcatc 240 ggcgagtgcg agtcccggcc cgaacggctg ccggacggcc ggatccggct gcgggaacag 300 tggcgccggc acggaccacg ccaggacagc ggcgtctccg tcatcgagga ggcagtgccg 360 gcgctcgccg gaggacagga gagccggcgt cgtgtctga 399

Claims (18)

We claim:

1. A compound of Formula II

2. A pharmaceutical composition comprising a compound of Formula II
together with a pharmaceutically acceptable carrier.

3. A farnesyl dibenzodiazepinone of Formula II
obtained by a method comprising:
a) cultivating Micromonospora sp. 046-ECO11, [S01]046 or a farnesyl dibenzodiazepinone producing mutant of 046-ECO11 or [S01]046, wherein said cultivation is performed under aerobic conditions in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms;
and b) isolating the farnesyl dibenzodiazepinone from the bacteria cultivated in step (a).

4. The farnesyl dibenzodiazepinone of claim 3, wherein the Micromonospora sp.
is strain [S01]046.

5. The farnesyl dibenzodiazepinone of claim 3 that generates NMR spectra essentially as shown in Figure 3.

6. A process for making the compound of claim 1, comprising cultivation of Micromonospora sp. 046-ECO11, [S01]046 or a farnesyl dibenzodiazepinone producing mutant of 046-ECO11 or [S01]046, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of said compound.

7. A process for making a compound of claim 1, comprising cultivation of Micromonospora sp. strain [S01]046 in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of said compound.

8. The process of claim 6 or 7, wherein said cultivation occurs under aerobic conditions.

9. The process of any one of claims 6 to 8, wherein said cultivation is carried out at a temperature ranging from 18°C to 40°C.

10. The process of any one of claims 6 to 9, wherein said cultivation is carried out at a pH ranging from 6 to 9

11. Micromonospora sp. having IDAC Accession No. 231203-01.

12. Micromonospora sp. having IDAC Accession No. 070303-01.

13. Use of a compound of claim 1 to inhibit the growth of a cancer cell.

14. Use of a compound of claim 1 in the treatment of a pre-cancerous or cancerous condition in a mammal.

15. Use of the pharmaceutical composition of claim 2 to inhibit the growth of a cancer cell in a mammal.

16. Use of a compound of claim 1 in the preparation of a medicament to treat a pre-cancerous or cancerous condition in a mammal.

17. Use of a compound of claim 1 in the preparation of a medicament to treat a bacterial infection in a mammal.

18. Use of a compound of claim 1 to inhibit 5 lipoxygenase in human cells.