WO2012003461A2 - Phloroglucinol synthases and methods of making and using the same - Google Patents

Abstract

The invention provides nucleic acids, amino acids, cells, and related methods for the biosynthetic production of phloroglucinol. In various embodiments, the invention includes an isolated or recombinant phloroglucinol synthase, which can be used in conjunction with an enzyme system for the biosynthetic production of phloroglucinol, mono-O-methyl phloroglucinol, di-O-methyl phloroglucinol and tri-O-methyl phloroglucinol. The synthesis can be from a renewable carbon source. The phloroglucinol can be used in subsequent reactions.

Description

PHLOROGLUCINOL SYNTHASES

AND METHODS OF MAKING AND USING THE SAME

RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional

Application Number 61/398,897, filed July 2, 2010, the entire contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

[0002] Aspects of this work was supported by Grant No. N000140710076 from

O.N.R. The United States government may have certain rights to this invention.

FIELD OF THE INVENTION

[0003] The invention relates generally to the biological synthesis of phloroglucinol, as well as its derivatives and precursors. The invention relates, more particularly, to nucleic acids, amino acids, cells, and related methods for the biosynthetic production of

phloroglucinol from a renewable carbon source.

BACKGROUND

[0004] Phloroglucinol (1,3,5-trihydroxybenzene) and its derivatives are widely used in commerce. Phloroglucinol and its derivatives (e.g., trimethylphloroglucinol) are used as pharmaceutical agents (e.g., as antispasmodics). Phloroglucinol is used as a starting material or intermediate in pharmaceutical, microbicide, and other organic syntheses. Phloroglucinol is also used as a stain for microscopy samples that contain lignin (e.g., wood samples) and in the manufacture of dyes (e.g., leather, textile, and hair dyes). Phloroglucinol is used in the manufacture of adhesives, as an epoxy resin curing agent, and in the preparation of explosives (e.g., the thermally- and shock-stable high explosive, l,3,4-triamino-2,4,6- trinitrobenzene or TATB). Phloroglucinol also functions as an antioxidant, stabilizer, and corrosion resistance agent, and is utilized as a coupling agent for photosensitive duplicating paper, as a substitute for silver iodide in rain-making, as a bone sample decalcifying agent, and as a floral preservative. Phloroglucinol can also be converted to resorcinol by catalytic hydrogenation.

[0005] Resorcinol (1,3-dihydroxybenzene) is a particularly useful derivative of phloroglucinol, although resorcinol is not currently produced by that route. Like phloroglucinol, resorcinol is used in the manufacture of dyes and adhesives, and as an epoxy resin curing agent. It is also used as a starting material and intermediate in pharmaceutical and other organic syntheses. Resorcinol and its derivatives are used, either alone or with other active ingredients such as sulfur, in cosmetics and in topical skin medicaments for treatment of conditions including acne, dandruff, eczema, and psoriasis (e.g., functioning, in part, as an antiseptic and antipruritic). Resorcinol is also used as a cross-linking agent for neoprene, as a tack-enhancing agent in rubber compositions, in bonding agents for organic polymers (e.g., melamine and rubber) and in the fabrication of fibrous and other composite materials. Resorcinol can be used in the manufacture of resins and resin adhesives (e.g., both as a monomer and as a UV absorbing agent), in the manufacture of explosives (e.g., energetic compounds such as styphnic acid, 2,4,6-trinitrobenzene-l,3-diol), and heavy metal styphnates, as well as in the synthesis of diazo dyes, plasticizers, hexyl resorcinol, and p- aminosalicylic acid.

[0006] Common resorcinol-based resins include resorcinol-aldehyde and resorcinol- phenol-aldehyde resins. These resorcinol-based resins are used, for example, as resin adhesives, composite material matrices, and as starting materials for rayon and nylon production. Examples of composite materials include resorcinol-formaldehyde carbon (or other organic) particle hydrogels, aerogels, and xerogels (e.g., which can be used as matrix materials for metallic and organometallic catalysts). Resorcinol-formaldehyde resins and particulate composites are also used in dentistry as a root canal filling material.

[0007] Resorcinol-aldehyde resin adhesives can be especially useful in applications requiring high bond strength (e.g., wooden trusses, joists, barrels, and boats, and aircraft). Modified resorcinol-aldehyde resin adhesives can also used as biological wound sealant compositions both on topical wounds and on internal wounds or surgical cuts (e.g., vascular incisions). Such medical uses are common in military field medicine (e.g., to minimize environmental exposure, reduce bleeding and fluid loss, and facilitate healing). Modified resin adhesives include gelatin-resorcinol-formaldehyde and gelatin-resorcinol- glutaraldehyde compositions. In such adhesives, the aldehyde can be maintained separately from the resorcinol-gelatin composition and mixed to form the sealant when needed.

[0008] Currently, both phloroglucinol and resorcinol are commercially produced by chemical organic synthesis using caustics and high temperatures, beginning with petroleum- derived starting materials and creating environmentally problematic waste and depleting fossil fuel and petroleum reserves. SUMMARY

[0009] The invention provides nucleic acids, amino acids, cells, and related methods for the biosynthetic production of phloroglucinol. In various embodiments, the invention includes an isolated or recombinant phloroglucinol synthase ("PhlD," e.g., the phloroglucinol synthase from Vibrio cholarae RC385, which is designated as PhlD^Vc, the phloroglucinol synthase from Yersinia mollaretii ATCC43969, which is designated as PhlD^Ym, as well as homologs, mutants, fragments, and modifications of these two enzymes). The phloroglucinol synthase can be used in or with an enzyme system for the biosynthetic production of phloroglucinol. The phloroglucinol synthase can also be used in or with an in vivo system such as a recombinant cell for the biosynthetic production of phloroglucinol. The synthesis can be from a renewable carbon source (e.g., malonyl-CoA, glucose, and the like). The phloroglucinol can be used in subsequent reactions, e.g., to produce other chemicals.

[0010] In one aspect, the invention features an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 and that encodes a functioning phloroglucinol synthase.

[0011] In another aspect, the invention features an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:4 and that encodes a functioning phloroglucinol synthase.

[0012] In still another aspect, the invention features an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5 and that encodes a functioning phloroglucinol synthase.

[0013] In yet another aspect, the invention features an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:6 and that encodes a functioning phloroglucinol synthase.

[0014] In another aspect, the invention features a system for converting malonyl-CoA to phloroglucinol. The system includes a PhlD⁺ isolated or recombinant enzyme system or a PhlD⁺ recombinant cell. The enzyme system or cell includes (i) an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 or SEQ ID NO:4 and that encodes a functioning phloroglucinol synthase or (ii) an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6 and that encodes a functioning phloroglucinol synthase.

[0015] In still another aspect, the invention features a method for producing anabolic phloroglucinol. The method includes providing a system for converting malonyl-CoA to phloroglucinol. The system includes a PhlD⁺ isolated or recombinant enzyme system or a PhlD recombinant cell. The enzyme system or cell includes (i) an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 or SEQ ID NO:4 and that encodes a functioning phloroglucinol synthase or (ii) an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6 and that encodes a functioning phloroglucinol synthase. The method also includes contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol.

[0016] In yet another aspect, the invention features a phlD⁺ isolated or recombinant nucleic acid vector that includes open reading frame (i) encoding an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 or SEQ ID NO:4 and from which a cell can express a functioning phloroglucinol synthase or (ii) corresponding to an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6 and from which a cell can express a functioning phloroglucinol synthase.

[0017] In another aspect, the invention features a phlD⁺ recombinant cell that includes a nucleic acid vector having open reading frame (i) encoding an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 or SEQ ID NO:4 and from which the recombinant cell can express a functioning phloroglucinol synthase or (ii) corresponding to an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO: 5 or SEQ ID NO: 6 and from which the recombinant cell can express a functioning phloroglucinol synthase.

[0018] In still another aspect, the invention features a method for producing a phlD⁺ recombinant cell. The method includes transforming a cell with a nucleic acid vector having open reading frame (i) encoding an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3 or SEQ ID NO:4 and from which the recombinant cell can express a functioning phloroglucinol synthase or (ii) corresponding to an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6 and from which the recombinant cell can express a functioning phloroglucinol synthase.

[0019] In other embodiments, any of the aspects above, or any composition of matter or method described herein, can include one or more of the following features.

[0020] In various embodiments, the isolated or recombinant nucleic acid sequence is at least 85% homologous. The isolated or recombinant nucleic acid sequence can be at least 90% homologous. The isolated or recombinant nucleic acid sequence can be at least 95% homologous. The isolated or recombinant nucleic acid sequence can be at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous.

[0021 ] In some embodiments, the isolated or recombinant amino acid sequence is at least 60% homologous. The isolated or recombinant amino acid sequence can be at least 70% homologous. The isolated or recombinant amino acid sequence can be at least 80% homologous. The isolated or recombinant amino acid sequence can be at least 85% homologous. The isolated or recombinant amino acid sequence can be at least 90% homologous. The isolated or recombinant amino acid sequence can be at least 95% homologous. The isolated or recombinant amino acid sequence can be at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous.

[0022] In certain embodiments, the enzyme system or cell is at least one of PhlA^",

PhlB^", and PhlC^". The enzyme system or cell can be PhlA^", PhlB^", and PhlC^". The cell can be genetically engineered to increase PhlD expression. The enzyme system or cell can include a malonyl-CoA synthesis enzyme. The recombinant cell can be an E. coli or P. fluorescens cell.

[0023] In various embodiments, the carbon source includes a saccharide, an aliphatic polyol, or both a saccharide and an aliphatic polyol.

[0024] In some embodiments, the contacting step includes an extractive fermentation.

The contacting step can include a dual temperature profile.

[0025] In certain embodiments, the method also includes contacting the anabolic phloroglucinol, hydrogen, and a rhodium catalyst under conditions allowing the

hydrogenation of the anabolic phloroglucinol, to produce resorcinol. The method can include producing a medicament, cosmetic, dye, polymer resin, rubber, adhesive, sealant, coating, composite material, laminated material, or bonded material from the anabolic phloroglucinol or resorcinol derived from the anabolic phloroglucinol. The method can include chemically modifying the anabolic phloroglucinol or resorcinol derived from the anabolic

phloroglucinol, to produce a propellant or explosive.

[0026] In various embodiments, the vector is at least one of phlA^~, phlB^~, and phlC.

The vector can be phlA^~, phlB^~, and phlC. The vector can be genetically engineered to increase PhlD expression. The vector can include a malonyl-CoA synthesis enzyme gene.

[0027] In certain embodiments, the method for producing anabolic O-methyl phloroglucinol comprises providing a system for converting malonyl-CoA to phloroglucinol comprising a PhlD isolated or recombinant enzyme system or a PhlD recombinant cell, wherein the enzyme system or cell comprises a nucleic acid sequence at least 80% , at least 85%, at least 90% homologous to SEQ ID NO:3 or SEQ ID NO:4, wherein the nucleic acid sequence encodes a functioning phloroglucinol synthase, or wherein the enzyme system or cell comprises an amino acid sequence at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6, wherein the system comprises the nucleic acid sequence necessary to express phloroglucinol O-methyl transferase (POMT); and contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol, the phloroglucinol being further transformed into O-methyl phloroglucinol. The enzyme system or cell is at least one of PhlA^", PhlB^", and PhlC^". The cell can be genetically engineered to increase PhlD expression. The enzyme system or cell can comprise a malonyl-CoA synthesis enzyme. The carbon source can include a saccharide, an aliphatic polyol, or both a saccharide and an aliphatic polyol. In some embodiments, the contacting step comprises an extractive fermentation and/ora dual temperature profile.

[0028] In certain embodiments, the method for producing anabolic O-methyl phloroglucinol and/or tri-O-methyl phloroglucinol comprises (a) providing a system for converting malonyl-CoA to phloroglucinol comprising a PhlD⁺ isolated or recombinant enzyme system or a PhlD⁺ recombinant cell, wherein the enzyme system or cell comprises the nucleic acid sequence at least 80%, at least 85%, at least 90% homologous to SEQ ID NO:3 or SEQ ID NO:4, or wherein the enzyme system or cell comprises an amino acid sequence at least 50% homologous to SEQ ID NO:5 or SEQ ID NO:6, wherein the system comprises the nucleic acid sequence necessary to express phloroglucinol O-methyl transferase (POMT), wherein the system comprises the nucleic acid sequence necessary to express orcinol O-methyl transferase (OOMT); (b)contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol, wherein the phloroglucinol is further transformed into O-methyl

phloroglucinol, wherein the O-methyl phloroglucinolis further transformed to di-O-methyl phloroglucinol; and (c) isolating the di-O-methyl phloroglucinol or transforming the di-O- methyl phloroglucinol to tri-O-methyl phloroglucinol.

[0029] Other aspects and advantages of the invention can become apparent from the following drawings and description, all of which illustrate principles of the invention, by way of example only. BRIEF DESCRIPTION OF DRAWINGS

[0030] The advantages of the invention described above, together with further advantages, may be better understood by referring to the following description taken in conjunction with the accompanying drawings. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention.

[0031] FIG. 1 presents Scheme 1, which illustrates literature -reported routes: (a) for acetylphloroglucinol biosynthesis without phloroglucinol as an intermediate, see M. G. Bangera & L. S. Thomashow, J Bact. 181(10):3155-63 (1999); and (c) for triacetic acid lactone biosynthesis, see S. Eckermann et al., Nature 396:387 (1998), J. M. Jez et al., Chem. Bio. 7:919 (2000); W. Zha et al., J. Am. Chem. Soc. 126:4534 (2004). Also shown are the routes (b) and (b') for acetylphloroglucinol biosynthesis with phloroglucinol as an intermediate. The correct biosynthetic pathway was later elucidated in J. Achkar et al., J. Am. Chem. Soc. 127:5332 (2005).

[0032] FIG. 2 presents Scheme 2, which illustrates: the common commercial chemical synthetic route (a, b, c) for phloroglucinol synthesis; a multi-step route (d, e, f, g) previously proposed for synthesis of phloroglucinol from glucose; a first, common commercial chemical synthetic route (i, j) for resorcinol synthesis; and a second, common commercial chemical synthetic route (k, 1) for resorcinol synthesis. Also illustrated with circled arrows are: (1) the fully biosynthetic route (indicated by a circled asterisk) for production of phloroglucinol; and (2) the chemical hydrogenation (h) of phloroglucinol to resorcinol. Specific reactions or reaction steps shown are: (a) Na₂Cr07, H2SO4; (b) Fe, HCl; (c) H2SO4, 108° C; (d) see W. Zha et al., J. Am. Chem. Soc. 126:4534 (2004); (e) Dowex 50 H⁺, MeOH; (f) Na, MeOH, 185° C; (g) 12 N HCl; (h) i) H₂, Rh on A1₂0₃, ii) 0.5 M H₂S0₄, reflux; (i) S0₃, H₂S0₄; (j) NaOH, 350° C; (k) HZSM-12 zeolite, propene; and (1) i) 0₂, ii) H₂0₂, iii) H⁺.

[0033] FIG. 3 presents putative reaction pathways, by which malonyl-CoA is biosynthetically converted to phloroglucinol by a phloroglucinol synthase, either via enzyme- activated 3,5-diketopimelate (3,5-diketoheptanedioate) or via enzyme-activated 3,5- diketohexanoate (3 ,5-diketocaproate).

[0034] FIG. 4 illustrates a variety of exemplary pathways for utilization of different carbon sources in a process for anabolic phloroglucinol synthesis. Dashed arrows show possible alternative carbon source utilization routes; square brackets enclose intermediates that can be absent in some pathways. [0035] FIG. 5 illustrates an example of a fermentor-controlled cultivation with a resin-based extraction.

[0036] FIG. 6 illustrates an example of phloroglucinol accumulation as a function of time in a fermentor-controlled cultivation with a resin-based extraction.

[0037] FIG. 7 illustrates an example of specific activity as a function of time in a fermentor-controlled cultivation with a resin-based extraction.

[0038] FIG. 8 illustrates an amino acid alignment between PhlD^Vc and PhlD^Ym.

[0039] FIG. 9 illustrates example plasmid constructs having PhlD^Vc and PhlD^Ym.

[0040] FIG. 10A illustrates an exemplary cultivation of E. coli

C41(DE3)serA/pKIT1.002 (PhlD^Vc) under fermentor-controlled conditions. FIG. 10A illustrates an exemplary cultivation of is. coli C41(DE3)serA/pKIT1.003 (PhlD^Ym) under fermentor-controlled conditions. Diamonds indicate dry cells weight and open bars indicate g/L phloroglucinol.

[0041] FIG. 11 illustrates exemplary PhlD^Vc, PhlD^Ym, and PhlD^pf"5 specific activities

^mol/min/mg) as a function of time during cultivation of E. coli C41(DE3)serA/pKIT1.002 (PhlD^Vc), £. co/ C41(DE3)serA/pKIT1.003 (PhlD^Ym), and £. coli C41(DE3)serA/pBC2.274 (PhlD^pf~5) under fermentor-controlled conditions.

[0042] FIG. 12 illustrates a sequential O-methylation of phloroglucinol by POMT

(Phloroglucinol O-methyl transferase), and OOMT (Orcinol 0-methyl transferase).

[0043] FIG. 13 illustrates an example of the production of O-methyl phloroglucinol from the fermentation of glucose in an organism having the genes necessary to produce phloroglucinol, plus POMT, operating in a fed-batch/resin extraction fermentation process.

[0044] Fig. 14A-B show the Pseudomonas fluorescens strain Pf-5 phlD nucleic acid

SEQ ID NO: 1 .

[0045] Fig. 15A-B show the Pseudomonas fluorescens strain Pf-5 PhlD amino acid

SEQ ID NO: 2.

[0046] Fig. 16 shows the Vibrio cholarae phlD^Vc optimized nucleotide sequence SEQ

ID NO: 3.

[0047] Fig. 17 shows the Yersinia mollaretii phlD^Ym optimized nucleotide sequence

SEQ ID NO: 4. DETAILED DESCRIPTION

[0048] The invention provides nucleic acids, amino acids, cells, and related methods for the synthesizing phloroglucinol. The phloroglucinol can be renewable (e.g., "green" or plant derived, as opposed to fossil-fuel or petroleum derived).

[0049] The invention includes a phloroglucinol synthase enzyme. The phloroglucinol synthase can correspond to, or can be homologous to, the amino acid sequences from Vibrio cholarae RC385 (designated as PhlD^Vc) or Yersinia mollaretii ATCC43969 (designated as PhlD^Ym). Examples of such enzymes include all of the functioning phloroglucinol synthases corresponding to the nucleic acid sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4, the amino acid sequences shown as SEQ ID NO: 5 and SEQ ID NO:6, and sequences that are homologous to SEQ ID NOS: 3, 4, 5, and 6 that each correspond to a functioning

phloroglucinol synthase.

[0050] The invention also includes systems (e.g., cells and enzyme systems) for synthesizing phloroglucinol. For example, such systems include cells having a recombinant nucleic acid that encodes a polypeptide having phloroglucinol synthase activity. Such systems also include transformed or recombinant cells having a nucleic acid vector with an open reading frame encoding a nucleic acid that encodes a polypeptide having phloroglucinol synthase activity.

[0051] Further, the invention includes isolated or recombinant nucleic acid vectors having an open reading frame encoding a nucleic acid that encodes a polypeptide having phloroglucinol synthase activity. Such vectors can be used in methods for producing a transformed or recombinant cell having an open reading frame encoding a nucleic acid that encodes a polypeptide having phloroglucinol synthase activity.

[0052] Furthermore, the invention includes methods for producing renewable phloroglucinol. The methods include providing a system for producing phloroglucinol from a renewable carbon source and contacting the system and the carbon source, to produce renewable phloroglucinol.

[0053] The phloroglucinol can be used in subsequent reactions. For example, the phloroglucinol and resorcinol can be used in or to prepare medicaments, cosmetics, dyes, polymer resins, rubbers, adhesives, sealants, coatings, propellants, explosives, composite materials, and laminated or bonded materials. Sequence Homology

[0054] In various embodiments, the invention includes an isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO: 3 or SEQ ID NO: 4 and that encodes a functioning phloroglucinol synthase. For example, the sequence can be at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 92, 94, 95, 96, 97, 98, 99, or 100% homologous to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sequence can be less than 80% homologous to SEQ ID NO: 3 provided that it encodes a functioning phloroglucinol synthase.

[0055] In various embodiments, the invention includes an isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO: 5 or SEQ ID NO: 6 and that encodes a functioning phloroglucinol synthase. For example, the sequence can be at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 92, 94, 95, 96, 97, 98, 99, or 100 % homologous to SEQ ID NO: 4 or SEQ ID NO: 6. In some embodiments, the sequence can be less than 50% homologous to SEQ ID NO: 3 provided that it encodes a functioning phloroglucinol synthase.

[0056] Sequence homology can refer to the degree of identity between two sequences of amino acid residues, or between two sequences of nucleobases. Homology can be determined by visual comparison of two sequences, or by use of bioinformatic algorithms that align sequences for comparison or that determine percent homology among compared sequences. Automated algorithms are available, for example, in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (available from Genetics Computer Group, Madison, Wis., USA). The alignment algorithms automated in these modules include the Needleman & Wunsch, the Pearson & Lipman, and the Smith & Waterman sequence alignment algorithms. Other useful algorithms for sequence alignment and homology determination are automated in software including: FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL V. See, e.g., N. P. Brown et al., Bioinformatics: Applications Note, 1998, 14:380-81; the U.S. National Center for

Biotechnology Information at http://www.ncbi.nlm.nih.gov/Tools/index.html; and U.S. Pat. No. 6,790,639, Brown et al., issued Sep. 14, 2004, which provides software parameter settings useful for homology determination.

[0057] The sequence homology exhibited by nucleobase polymers, such as nucleic acids and nucleic acid analogs, can be determined by hybridization assays between a first sequence and the complement of a second sequence. Any of the well known hybridization assays can be used for this purpose, and examples of these include those described in U.S. Pat. No. 6,767,744, Koffas et al., issued Jul. 27, 2004, and U.S. Pat. No. 6,783,758, Wands et al., issued Aug. 31, 2004.

Conservative Substitutions

[0058] In addition, conservative amino acid substitutions can be found in a polypeptide according to the invention. The term conservative amino acid substitution can indicate any amino acid substitution for a given amino acid residue, where the substitute residue is so chemically similar to that of the given residue that no substantial decrease in polypeptide function (e.g., enzymatic activity) results. Conservative amino acid substitutions are well known (see, e.g., U.S. Pat. No. 6,790,639, Brown et al., issued Sep. 14, 2004; U.S. Pat. No. 6,774,107, Strittmatter et al., issued Aug. 10, 2004; U.S. Pat. No. 6,194,167, Browse et al., issued Feb. 27, 2001; or U.S. Pat. No. 5,350,576, Payne et al, issued Sep. 27, 1994). In one embodiment, a conservative amino acid substitution can be any one that occurs within one of the following six groups:

1. Small aliphatic, substantially non-polar residues: Ala, Gly, Pro, Ser, and Thr;

2. Large aliphatic, non-polar residues: He, Leu, Met and Val;

3. Polar, negatively charged residues and their amides: Asp and Glu;

4. Amides of polar, negatively charged residues: Asn, His and Gin;

5. Polar, positively charged residues: Arg, His and Lys; and

6. Large aromatic residues: Trp, Phe and Tyr.

[0059] In one embodiment, a conservative amino acid substitution can be any one of the following, which are listed as Native Residue (Conservative Substitutions) pairs: Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gin); He (Leu; Val); Leu (He; Val); Lys (Arg; Gin; Glu); Met (Leu; He); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); and Val (He; Leu).

[0060] Just as a polypeptide can contain conservative amino acid substitution(s), a polynucleotide can contain conservative codon substitution(s). A codon substitution is considered conservative if, when expressed, it produces a conservative amino acid substitution. Degenerate codon substitution, which results in no amino acid substitution, are also possible. For example, a polynucleotide encoding a selected polypeptide can be mutated by degenerate codon substitution in order to approximate the codon usage frequency exhibited by an expression host cell, or to otherwise improve the expression. Biosynthetic pathway of Phloroglucinol

[0061] As illustrated in FIG. 3, the mechanism by which phloroglucinol synthase catalyzes phloroglucinol synthesis proceeds according to the following series of steps, or via an alternative mechanism in which the first malonyl-CoA providing the group that is transferred to form the illustrated thioester (— SR) linkage, provides a malonyl, rather than an acetyl group:

1. Acetyl Activation— The first step involves activation of an acetyl group. This occurs by decarboxylation of malonyl-CoA to transfer an acetyl group to the enzyme, thus forming an enzyme-activated acetyl thioester (R in FIG. 3 represents the enzyme or a moiety attached thereto); in an alternative embodiment, the first step involves activation of an entire malonyl group to form an enzyme-activated malonyl thioester;

2. Chain Extension— The next phase involves two successive malonyl-CoA decarboxylations to transfer further acetyl groups to form an enzyme-activated 3- ketobutanoate thioester and then an enzyme-activated 3,5-diketohexanoate thioester; in an alternate embodiment, successive transfers form enzyme-activated: 3-ketoglutarate thioester and 3,5-diketopimelate thioester; and

3. Cyclization— The final step involves cyclization of the 3,5-diketohexanoate thioester intermediate to form phloroglucinol; in an alternative embodiment, a

decarboxylation of 3,5-diketopimelate takes place to permit cyclization to phloroglucinol. All three steps are catalyzed by phloroglucinol synthase.

Biosynthesis of pholorglucinol

[0062] In various embodiments the system includes at least one phloroglucinol synthase. The phloroglucinol synthase can correspond to, or can be homologous to, the amino acid sequences from Vibrio cholarae RC385 (designated as PhlD^Vc) and Yersinia mollaretii ATCC43969 (designated as PhlD^Ym). Examples of such enzymes include all of the functioning phloroglucinol synthases corresponding to the nucleic acid sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4, the amino acid sequences shown as SEQ ID NO: 5 and SEQ ID NO:6, and sequences that are homologous to SEQ ID NOS. 3, 4, 5, and 6 that correspond to a functioning phloroglucinol synthase.

[0063] In one embodiment, an enzyme system can also include at least one enzyme capable, either solely or jointly with other enzyme(s), of catalyzing the formation of malonyl- CoA. Malonyl-CoA can be biosynthetically produced (e.g., from acetyl-CoA by a malonyl- CoA synthesis enzyme). Example include the malonyl-CoA synthetase (MatB) from Rhizobium leguminosarum (see GenBank Accession AAC83455 [gi:3982573]), which converts malonate to malonyl-CoA; the malonyl-CoA decarboxylase (MatA) from Rhizobium leguminosarum (see GenBank Accession AAC83456 [gi:3982574]), which converts malonic semialdehyde to malonyl-CoA; and the transcarboxylase activity of acetyl-CoA carboxylase (EC 6.4.1.2), which carboxylates acetyl-CoA to form malonyl-CoA. The malonic acid, malonic semialdehyde, or acetyl-CoA starting material can be biosynthetic. For example, the acetyl-CoA can be biosynthetically derived from a biological source such as glucose, photosynthetic 3-phosphoglycerate, and the like.

[0064] A system according to the invention can be in vitro or in vivo. Where a malonyl-CoA synthesis enzyme is not provided, malonyl-CoA can be supplied to the medium in contact with the cells and/or enzymes. In one embodiment, a phloroglucinol synthase- encoding nucleic acid can be transformed into a cell of an organism capable of synthesizing malonyl CoA. Examples of organisms synthesizing malonyl CoA include plants, algae, animals, and humans. In vitro systems include batch enzyme suspensions, (adsorbed or covalently) immobilized enzyme bioreactors, and the like. In vivo systems include immobilized cell bioreactors, continuous fermentations, batch fermentations, and the like. Fermentation can indicate cultured cell growth under any effective conditions and is not limited to anaerobic conditions or anaerobic metabolism. A source of malonyl-CoA can be provided to the phloroglucinol synthase, whether or not that source is added (e.g., exogenous) malonyl-CoA or in situ biosynthesized (e.g., endogenous).

[0065] Recombinant cells according to the invention can express at least one phloroglucinol synthase and, optionally, at least one malonyl-CoA synthesis enzyme.

However, the cells expressing a phloroglucinol synthase should not express an entire phlABCD operon or all three oiphlA, phlB, and phlC genes involved in the microbial acetylphloroglucinol pathway, e.g., phlABCDEF. In one embodiment, the recombinant cell can be a walled cell. Examples of walled cells include plant (including avascular plants such as moss), yeast, fungal, bacterial, and Archaea cells, as well as some protists (e.g., algae). In one embodiment, the recombinant cell can be a microbe (e.g., a bacterial cell, proteobacterial cell, and the like). The recombinant cell can lack the ability to express functional enzymes from phlABC, phlE, and phlF genes. The cell can be a phlABC, phlK, and phlF cell.

Recombinant host cells can contain at least one nucleic acid encoding a phloroglucinol synthase. The nucleic acid can be in the form of a vector, such as a plasmid or transposon.

[0066] In one embodiment, a cell that is both phlD⁺ as well as phlA⁺, phlB⁺, and/or phlC⁺, can be made phlA^~, phlB^~, and/or phlC by any gene inactivation or knockout techniques generally known in the art (e.g., any gene excision or mutation technique that results in the cell's inability to make the functioning expression product encoded by the wild- type or pre-knocked-out gene). In one embodiment, all of the phlA, phlB, and phlC genes in the cell are inactivated or knocked out. The resulting cell can retain its phlD⁺ phenotype. Optionally, phlE and/or phlF genes present in the cell can also be knocked out. In one embodiment, a phlABCD⁺ cell can be made into a phlABC cell. In one embodiment, a cell that is both phlD^~ and phlA^', phlB^', and/or phlC can be made phlD⁺ by inserting an expressible PhlD-encoding nucleic acid into the cell (e.g., into the genomic DNA and/or as part of an extrachromosomal unit such as a plasmid). In one embodiment, a phlABCD^' cell can be made into a phlD⁺ cell.

[0067] In some embodiments, a native or recombinant cell that is PhlD⁺ can be supplemented with one or more additional phlD genes (e.g., by transformation with nucleic acid comprising one or more expressible open reading frames encoding a phloroglucinol synthase). The PhlD cell can be a PhlA^", PhlB^", and/or PhlC^" cell (e.g., a phlA^', phlB^', and/or phlC, or a phlABC cell), or it may be a PhlA⁺, PhlB⁺, and/or PhlC⁺ cell, such as a phlA⁺, phlB⁺, and/or phlC⁺ cell (e.g., a phlABCD⁺ cell). The resulting recombinant cell, which is capable of expressing the additional phlD gene(s), can exhibit enhanced phloroglucinol synthesis capability.

[0068] Similar to recombinant cells, isolated or recombinant enzyme systems according to the invention can include at least one phloroglucinol synthase and, optionally, at least one malonyl-CoA synthesis enzyme or enzyme set. In one embodiment, an enzyme systems including at least one phloroglucinol synthase does not also include all three of PhlA, PhlB, and PhlC enzymes. An enzyme systems including at least one phloroglucinol synthase can include none of PhlA, PhlB, and PhlC enzymes.

[0069] It is generally known that stepwise acetylation of phloroglucinol performed by

PhlABC leads to monoacetylphloroglucinol (MAPG) and 2,4-DAPG, respectively. The level of produced DAPG is regulated mainly by degradation through the specific hydrolase PhlG, which converts DAPG into MAPG, and by the regulatory proteins PhlF and PhlH. PhlF represses the expression of the phlABCD operon by binding to two conserved sites in the phlA leader region. DAPG itself is able to dissociate the repressor PhlF from the phlA promotor, hence acting as an autoinducer of it own biosynthesis. PhlH, the second pathway- associated transcriptional regulator, is hypothesized to antagonize the repressive effect of PhlF. In addition, PhlE regulates the efflux of DAPG. [0070] U.S. Publication No. 2007/0178571, now U.S. Patent No. 7,943,362, which is incorporated by reference, includes additional examples and background regarding phloroglucinol biosynthesis.

[0071] It should be understood that according to generally accepted gene/protein nomenclature guidelines, gene symbols are italicized, whereas protein designations are not italicized and the first letter is in upper case. "+" and "-" signs indicate the presence and absence, respectively, of a functional gene or protein. For example, "phlD" represents the phloroglucinol synthase gene, "PhlD" represents the phloroglucinol synthase protein, "p/z/ ⁺" indicates that the gene is present and functional, and "phlD^~" indicates that the gene is inactive or knocked out.

Whole-Cell Fermentation Modes

[0072] Whole cell fermentations of recombinant cells can be performed in essentially any culture mode (e.g., batch, fed-batch, and continuous, semi-continuous, reseeding, and the like). In some embodiments, phloroglucinol-containing growth medium can be processed to extract phloroglucinol after the cells complete producing phloroglucinol . However, phloroglucinol can exert toxicity against the cultured cells when it reaches a threshold concentration in the growth medium (e.g., end-product inhibition). Such toxicity can decrease cellular production of phloroglucinol and reduce cell viability. Thus, in some embodiments, phloroglucinol (and in some cases phloroglucinol derivative) extraction can be performed when the cells are actively producing phloroglucinol (e.g., an extractive fermentation).

[0073] Extractive fermentation can be performed by known methods. For example, some embodiments employ a dispersed extractive fermentation mode where an extractive, absorbent, or adsorbent liquid or particle phase is introduced into the growth medium. The liquid or particles phase can remove phloroglucinol from the growth medium. Such removal can be non-specific, preferential, or specific for phloroglucinol. In various examples, maintaining culture medium phloroglucinol concentration below about 1 g/L can help maintain a robust culture.

[0074] FIG. 5 illustrates an example setup for a fermentor-controlled cultivation with a resin-based extraction. The setup includes a filter unit (1), a column unit (2), and a fermentation vessel (3). Fermentation broth is pumped from the fermentation vessel to the filter unit (e.g., an ultrafiltration unit including 100 kD slice cassettes mounted on a jet pump, both available, for example, from Sartorius Corporation), where cells and other large particles that can impair column function are removed. The retentate can be returned to the fermentation vessel and the permeate can be pumped to the column unit (e.g., a column packed with an AG- 1 resin) where phloroglucinol is removed. The post-resin permeate can then be returned to the fermentation vessel. In various examples, such in-line tangential flow filtration can be used to separate cells and phloroglucinol can be used to mitigate clogging of the extraction resin (e.g., by lysed cell debris).

[0075] The liquid or particle phase can be removed from the culture medium after becoming loaded with phloroglucinol (e.g., by centrifugation, filtration, magnetic collection of magnetic or magnetizable particles, by phase separation where the extractive phase rises above or sinks below the bulk of the growth medium, and the like). In some embodiments, a counter-current or cross-current extraction can be used to extract phloroglucinol from the growth medium (e.g., where the stream that is counter-current or cross-current to the culture medium stream includes an extractive, absorbent, or adsorbent liquid or particle phase).

[0076] In some embodiments, a membrane extractive fermentation can be performed by passing the growth medium over an extraction membrane (e.g., an ion exchange membrane). In some embodiments, a column extractive fermentation can performed by passing the culture medium through an extraction column (e.g., a hollow fiber membrane extractor or a fibrous or bead resin column). The cells in the growth medium can pass through the column, or some or all of the cells may be removed (e.g., by filtration) before the medium is passed through the column. The extractive fermentation can be performed once, multiple times, or continuously during fermentation. Growth medium from which at least some of the phloroglucinol has been removed can be returned to the fermentation vessel.

[0077] A column extractive fermentation can be employed to remove phloroglucinol from the culture medium during biosynthesis. Column extractive fermentations can employ anion exchange media (e.g., anion exchange beads, membranes, particles, fibers, hollow fibers, and the like) in a fluidized or stationary bed mode. Anion exchange media can include an organic or inorganic support that includes or is attached (e.g., covalently) to an anion exchange group. Organic supports can include styrene-divinylbenzene, polystyrene, polyvinyl, acrylic, phenol-formaldehyde, organosilicon, or cellulose polymer backbone attached to an anion exchange group.

[0078] Anion exchange groups can be a cationic group (e.g., a non-metal cationic group such as organic ammonium, sulfonium, and phosphonium). Cationic groups can be organic (e.g., tertiary ammonium such as diethylaminoethyl cellulose), quaternary ammonium, pyridinium, tertiary sulfonium, and quaternary phosphonium groups. In one embodiment, the anion exchange groups of the anion exchange medium can be a quaternary ammonium or pyridinium group. Examples of quaternary ammonium-type resins include AG-1 X8 resin (from Bio-Rad Laboratories Inc., Hercules, Calif, USA) and DOWEX 1 resin (from The Dow Chemical Co., Midland, Mich., USA). Examples of pyridinium-type resins include polyvinyl-alkyl-pyridinium resins obtainable by alkyl halide treatment of polyvinyl- pyridine resins, such as REILLEX HP (from Reilly Industries, Inc., Indianapolis, Ind., USA), or obtained directly from commercial sources, such as poly(4-vinyl N-methyl pyridinium iodide) (from Polymer Source Inc., Montreal, QC, CA).

[0079] In one embodiment, the anion exchange medium is treated before use to prepare a phosphate complex with cationic groups in the medium. Where an anion exchange medium is re -used (e.g., without phloroglucinol removal) or is in continuous contact with a fermentation, it can be replaced with new or renewed anion exchange medium frequently enough that the phloroglucinol concentration of the culture medium does not rise to a level at which a substantial degree of end-product-inhibition occurs (e.g., above about 2 g/L or 1.5 g/L phloroglucinol).

[0080] An anion exchange medium that has already been loaded with phloroglucinol

(e.g., by extractive fermentation or post-fermentation extraction) can be treated to removal phloroglucinol by washing with water, acidified water, and/or acidified alcohol (e.g., acidic ethanol). Water washing followed by acidified alcohol washing is one example technique. After phloroglucinol removal, the anion exchange medium can be prepared for re-use (e.g., by equilibrating it with a phosphate solution to form cationic group-phosphate complexes). Phloroglucinol separated by extractive fermentation can be further purified known techniques (e.g., phase separation, solvent evaporation, and the like).

Whole-Cell Fermentation Conditions

[0081 ] Cultures of whole cells producing phloroglucinol can utilize conditions that are supportive of both cell growth and anabolic phloroglucinol production. In some methods, a phloroglucinol synthase can be expressed throughout the cell culture period (e.g., constitutively). In other methods, phloroglucinol synthase can be expressed

only/predominantly near the end of the exponential growth phase (EGP). Where a later expression is desired, a phloroglucinol synthase coding sequence that is under the control of a regulated promoter generally can be activated or derepressed when about 70 to 100%, about 70 to 90%, or about 70 to 80% of EGP has elapsed. Examples of promoters useful for this purpose include the tac, T5, and T7 promoters (e.g., Pn). Induction can be made using lactose or a gratuitous inducer such as IPTG (isopropyl-beta-D-thiogalactopyranoside).

[0082] In some embodiments, a recombinant microbial cell, such as a recombinant bacterial host cell can be used as a whole cell biocatalyst. Bacterial host cells can include Proteobacteria (e.g., the gamma proteobacteria, such as enterobacteria and pseudomonads), Escherichia (e.g., E. coli), and Pseudomonas (e.g., P. fluorescens). Host cells can lack, or be treated to decrease or eliminate, protease activity that can degrade a phloroglucinol synthase and/or malonyl-CoA synthesis enzymes. In bacteria, Lon and OmpT are two such proteases that can be absent or otherwise decrease or eliminated (e.g., by mutation). E. coli strains BL21 and W31 10 are examples of phlABCD⁺ cells for insertion of phlD gene(s). P.

fluorescens strain Pf-5 is an example of a phlABCD⁺ cell for inactivation of phlA, phlB, and/or phlC, with or without insertion of further phlD gene(s), or for inactivation of phlABCD, with insertion of further phlD gene(s), or for supplementation with additional phlD gene(s). E. coli strain BL21 can be obtained as: BL21 STAR (DE3) ONE SHOT (Invitrogen Corp., Carlsbad, Calif, USA) or ULTRA BL21 (DE3) (Edge BioSystems, Gaithersburg, Md., USA). E. coli strain W31 10 can be obtained as ATCC No. 27325 (American Type Culture Collection, Manassas, Va., USA). P. fluorescens strain Pf-5 can be obtained as ATCC No. BAA-477.

[0083] In the case of E. coli, fermentation temperatures can be from about 20 to about

37° C, about 25 to about 37° C, or about 30 to about 37° C. In anabolic phloroglucinol synthesis, a combination of a higher temperature during EGP or during the pre-induction portion of EGP, and a lower temperature during at least part of the remaining culture period (e.g., throughout all or part of the post-induction or all or part of the maintenance phase) can facilitate phloroglucinol production. Thus, recombinant E. coli cells can be grown at about 35-37° C, about 36-37° C, or about 36° C during EGP or during pre -induced EGP, and at about 30-34° C, about 30-33° C, about 33° C, or about 30° C during maintenance phase or during post-induction.

[0084] In some embodiments, the switch to a lower temperature can occur well into the maintenance phase (e.g., up to about 15 hours after EGP ends). Thus, in the case of a cell culture in which EGR ends at about 15 hours from the start of culturing (e.g., E. coli), the switch from a higher to a lower temperature for a two-temperature fermentation profile can occur at about 1 1 or about 12 hours (e.g., at approximately the same time as a 70% or 80% EGR induction point), or at about 15 hours, or even up to about 30 hours from the start of culturing. In the case of P. fluorescens, temperatures can be from about 20 to about 30° C, with the higher temperatures being from about 27 to about 30° C and the lower temperatures being from about 24 to about 27° C.

Carbon Sources

[0085] The systems and methods for producing phloroglucinol according to the invention can utilize a wide variety of carbon sources including carbohydrates (C6), celluloses (C5), and glycerides. For example, a carbon source can include one or more of glucose, xylose, arabanose, glycerol, a starch, a cellulose, a hemicellulose, and a plant oil.

[0086] In various embodiments, a carbon source can be a simple carbon source.

Simple carbon sources can contain from 0% to about 5%, 0% to about 2%, 0% to about 1%, 0% to about 0.5%, or about 0% by weight secondary metabolites and larger or complex organics. In other example, simple carbon sources can be free or substantially free of secondary metabolites and larger/complex organics. In some embodiments, a simple carbon source can include primary metabolite-type compound(s). Examples of primary metabolite- type compounds include saccharides (e.g., mono- and/or di-saccharides) and polyols (e.g., glycerol). Useful monosaccharides include glucose, xylose, and arabinose. In one embodiment, glucose, xylose, and/or arabinose can be used as the carbon source (e.g., as the carbon source throughout both the exponential growth phase and the maintenance phase of the cell culture). In one embodiment a combination of a monosaccharides (e.g., glucose, xylose, and/or arabinose) and glycerol can be used (e.g., at a 1 : 1 or 2: 1 weight ratio). Such a combination can be used during the maintenance phase, with monosaccharides (without glycerol) being used during the exponential growth phase.

[0087] FIG. 4 illustrates a number of representative routes for anabolic synthesis of phloroglucinol from carbon sources (e.g., malonyl-CoA or a malonyl-CoA precursor). Suitable carbon sources can include biomolecules that can be catabolized by the system as well as simpler organic molecules that can be fixed by the system (see FIG. 4 and U.S. Publication No. 2007/0178571).

Renewable phloroglucinol

[0088] The biosynthesized phloroglucinol includes carbon from the atmospheric carbon dioxide incorporated by plants (e.g., from a carbon source such as glucose, malonyl- CoA, or malonyl-CoA precursor). Therefore, the biosynthesized phloroglucinol includes renewable carbon rather than fossil fuel-based or petroleum-based carbon. Accordingly, the biosynthetic phloroglucinol and associated products will have a less of an environmental impact than similar compositions produced by conventional methods because they do not deplete fossil fuel or petroleum reserves and because they do not increase the amount of carbon in the carbon cycle (e.g., increase greenhouse gases). Additionally, the biosynthetic phloroglucinol and associated products will have a less of an environmental impact because the biosynthesis does not require the toxic chemicals required by conventional synthetic methods.

[0089] The biosynthetic phloroglucinol can be distinguished from similar compounds produced from a fossil fuel or petrochemical carbon source by dual carbon-isotopic finger printing. This method can distinguish chemically-identical materials, and apportions carbon in the copolymer by source (and possibly year) of growth of the biospheric (plant) component using the information contained in the ¹⁴C and ¹³C isotope ratios. The radiocarbon dating isotope (¹⁴C), with its nuclear half life of 5730 years, allows one to apportion specimen carbon between fossil (dead) and biospheric (alive) feedstocks (See Currie, L. A. "Source Apportionment of Atmospheric Particles," Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74). The basic assumption in radiocarbon dating is that the constancy of ¹⁴C concentration in the atmosphere leads to the constancy of ¹⁴C in living organisms.

[0090] When dealing with an isolated sample, the age of a sample can be deduced approximately by the relationship t = (-5730/0.693)ln(A/A_o) where t = age, 5730 years is the half-life of radiocarbon, and A and A₀ are the specific ¹⁴C activity of the sample and of the modern standard, respectively (Hsieh, Y., Soil ScL Soc. Am J., 56, 460, (1992)). However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, ¹⁴C has acquired a second, geochemical time characteristic. Its concentration in atmospheric C0₂, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (¹⁴C/¹²C) of ca. 1.2 x 10^"12, with an approximate relaxation half-life of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric ¹⁴C since the onset of the nuclear age.) It is this latter biospheric ¹⁴C time characteristic that holds out the promise of annual dating of recent biospheric carbon. ¹⁴C can be measured by accelerator mass spectrometry (AMS), with results given in units of fraction of modern carbon (ΪΜ). ΪΜ is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-lndustrial Revolution wood. For the current living biosphere (plant material), f_M « 1.1.

[0091] The stable carbon isotope ratio (¹³C/¹²C) provides a complementary route to source discrimination and apportionment. The ¹³C/¹²C ratio in a given biosourced material is a consequence of the ¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed and also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C₄ plants (the grasses), and marine carbonates all show significant differences in ¹³C/¹²C and the corresponding δ ¹³C values. Furthermore, lipid matter of C3 and C₄ plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, ¹³C shows large variations due to isotopic fractionation effects, the most significant of which for the instant invention is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (e.g., the initial fixation of atmospheric CO2). Two large classes of vegetation are those that incorporate the C₃ (or Calvin-Benson) photosynthetic cycle and those that incorporate the C₄ (or Hatch-Slack) photosynthetic cycle. C₃ plants, such as hardwoods and conifers, are dominant in the temperate climate zones. In C₃ plants, the primary CO2 fixation or carboxylation reaction involves the enzyme ribulose-l,5-diphosphate carboxylase and the first stable product is a 3- carbon compound. C₄ plants, on the other hand, include such plants as tropical grasses, corn and sugar cane. In C₄ plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid, which is subsequently decarboxylated. The CO2 thus released is refixed by the C₃ cycle.

[0092] Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, but typical values are ca. -10 to -14 per mil (C₄) and -21 to -26 per mil (C₃) (Weber et al., J. Aqric. Food Chem., 45, 2942 (1997)). Coal and petroleum fall generally in this latter range. The ¹³C measurement scale was originally defined by a zero set by pee dee belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The 5¹³C values are in parts per thousand (per mil), abbreviated %₀, and are calculated as follows: 5^IjC≡ (¹³C/¹²C)sample - ("C/^Qstandard / ("C/^Qstandard x 1000%_o Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is 5¹³C. Measurements are made on CO₂ by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45 and 46.

[0093] Therefore, the biosynthesized phloroglucinol and compositions including biosynthesized phloroglucinol can be distinguished from their fossil-fuel and petrochemical derived counterparts on the basis of ¹⁴C (ΪΜ) and dual carbon-isotopic fingerprinting, indicating new compositions of matter (e.g., U.S. Patent Nos. 7,169,588, 7,531,593, and 6,428,767). The ability to distinguish these products is beneficial in tracking these materials in commerce. For example, products comprising both new and old carbon isotope profiles may be distinguished from products made only of old materials. Hence, the biosynthetic phloroglucinol and derivative materials can be followed in commerce on the basis of their unique profile.

EXAMPLES

Example 1 - Expression of PhlD in E. coli and Resulting Phloroglucinol Synthesis

[0094] Plasmid pJA3.131A (Kan^R, lacI^Q, Ρτν-phlD, serA) is transfected into chromosomally serA ^. coli strains BL21(DE3), W3110(DE3), and JWF1(DE3) [i.e., RB791serA^"(DE3)], and into strain KL3(DE3) [i.e., AB2834(serA::aroB)]. E. coli strains RB791 and AB2834 are available from the E. coli Genetic Stock Center, New Haven, Conn., USA. All DE3 strains are obtained by integration of λϋΕ3 prophage into the cell chromosomes. Cells are cultured in fed-batch conditions under mineral salts and limited glucose. Although all transformed strains express substantial levels of phloroglucinol, the BL21 and W3110 strains produce superior titers of 3.0 and 3.1 g/L phloroglucinol, respectively. Relative to the amounts of glucose supplied to the cultures, these strains produce a superior phloroglucinol yields of 4.4 and 3.1 moles phloroglucinol per 100 moles of glucose (% mol/mol).

[0095] These tests also compare phloroglucinol expression levels in BL21 strains similarly transformed with a plasmid in which phlD is under the control of Ptac or Ρχ5. Ρτ₇ is found to provide superior results (data not shown). In these tests, phloroglucinol accumulation for all strains stops increasing during the stationary (or maintenance) phase. Peak phloroglucinol concentration is achieved about 6 hours and about 12 hours after initiation of induction (i.e., the first IPTG addition) for BL21 and W31 10, respectively. End- product inhibition is observed. Further tests demonstrate that phloroglucinol is responsible for the inhibition when the concentration is at or above about 2 g/L (data not shown).

Example 2 - Extractive Phloroglucinol Fermentations

[0096] In one example, an anion-exchange resin column-based extractive

fermentation is employed to remove phloroglucinol during fermentation, which

reduces/eliminates phloroglucinol cytotoxicity and phloroglucinol synthesis repression. A stirred tank reactor is equipped with tubing leading through an anion exchange column and returning to the tank. The tubing is equipped with a peristaltic pump in order to circulate the medium through the column. Bio-Rad Econo columns (25x200 mm) packed with 80 mL (bed volume) AG 1-X8 resin are rinsed with 15 bed volumes of KH₂P0₄ (0.8 M) to convert the tertiary ammonium salts to their phosphate form before the in situ extraction. A total of 3 to 5 columns are used for each fermentation. Each column is used for about 6-12 h before being replaced with another column, to keep the culture medium phloroglucinol

concentration below about 1.5 g/L. All columns are operated in a fluidized-bed mode with a circulation flow rate of about 8-12 mL/min.

[0097] To recover the phloroglucinol adsorbed on the AG 1-X8 resin, the column is washed in a fluidized-bed mode with 10 bed volumes of distilled, deionized water to remove residual cells. The washing also recovers about 15% of the phloroglucinol from the resin. Then, the column is rinsed in a fixed-bed mode with 15 bed volumes of acidic ethanol (10% v/v acetic acid, 75% v/v ethanol, and 15% v/v H₂0), to recover remaining phloroglucinol from the resin. After phloroglucinol recovery, the column is regenerated by further rinses of 15 bed volumes of KH₂P0₄ (0.8 M), 2 bed volumes of ethanol (70%), and 5 bed volumes of sterilized distilled, deionized water.

[0098] To purify the recovered phloroglucinol, cells in the resulting water solution are removed by centrifugation. The solution is then concentrated to about 1/10 of the original volume, to produce a concentrated aqueous solution. Separately, the acidic ethanol solution is concentrated to dryness, to produce a residue that is redissolved with the concentrated aqueous solution. The resulting aqueous phase is then extracted three times with an equal volume of ethyl acetate. The organic phases are combined, dried over MgS0₄, mixed with silicone gel, concentrated to dryness, and loaded onto a flash column. Phloroglucinol is separated form other brown impurities by rinsing with 1 : 1 hexane:acetate. The phloroglucinol fraction is identified by TLC and then concentrated to dryness and dried under high vacuum, to produce phloroglucinol as pale crystals.

[0099] Other examples were carried out using variations on this general process, which illustrates that the product yield varies with variations in the recombinant

microorganism, the filtration, and the resin-based extraction.

[00100] One setup using an unfiltered, extractive fermentation with multiple fluidized bed columns in connection with an E. coli including a codon optimized phlD

(PGl/pBC2.274) produced phloroglucinol at about 8 g/L (approximately 3.0% yield).

[00101] Another setup using a microfiltered, extractive fermentation with multiple fluidized bed columns in connection with an E. coli including phlD* (PGl/pBC2.274) produced phloroglucinol at about 12 g/L (approximately 4.5% yield).

[00102] Still another setup using a microfiltered, extractive fermentation with multiple fluidized bed columns in connection with an E. coli including phlD* and groESL

(PGl/pKITl 0.045) produced phloroglucinol at about 15 g/L (approximately 5.6% yield).

[00103] Yet another setup using a microfiltered, extractive fermentation with multiple fixed bed columns in connection with an E. coli including phlD* and groESL

(PGl/pKITl 0.045) produced phloroglucinol at about 18 g/L (approximately 6.8% yield).

[00104] Still yet a another setup using a microfiltered, extractive fermentation with a four changes of resin in a single fixed bed column in connection with an E. coli including phlD* in plasmid (PGl/pKITl 1.080) produced phloroglucinol at about 28 g/L. GC analysis of the fermentation samples showed that a total of 28 g of phloroglucinol were made during the peak production seen at 92 h giving a 9 % yield. The GC analysis of the fermentation samples also shows that the column of AG-1 resin was able to keep the concentration of phloroglucinol from getting over 1 g/L in the culture. FIG. 6 illustrates the previous example of phloroglucinol accumulation as a function of time in the fermentor-controlled cultivation with a resin-based extraction producing phloroglucinol at about 28 g/L. The diamonds indicate dry cell weight in g/L (right side y-axis), the bars before 41 hours and the lower portion (filled) of the later bars (at and after 41 hours) indicate culture medium

phloroglucinol in g/L, and the upper portion (open) of the bars at hour 41 and later indicate resin-bound phloroglucinol in g/L.

[00105] Enzyme activity for the run described immediately above showed a steady rise in enzyme activity during the first 9 h post induction followed by a steady and prolonged decrease seen through the time of harvest. [00106] FIG. 7 illustrates an example of specific activity as a function of time in the fermentor-controlled cultivation with a resin-based extraction producing phloroglucinol at about 28 g/L. The experiment shows that maintaining culture medium phloroglucinol concentration below about 1 g/L can help maintain a robust culture.

Example 3 - Optimization of Phloroglucinol Fermentation

[00107] A variety of dual temperature fermentation profiles are used in extractive and non-extractive fermentations of the transformed W3110 strain described above. Glucose is steadily fed by ρ(¾ cascade control and the exhausted CO₂ level is maintained at a steady level until the end of the fermentation. In both extractive and non-extractive of

fermentations, lowering the temperature during fermentation (e.g., from an initial 36° C) increase the titer and yield of phloroglucinol (with extractive the fermentation results being greater than the non-extractive fermentation results). Temperature shifts to 30° C are performed in separate fermentations at 12 h (e.g., the time of the first induction by IPTG), 15 h (e.g., the beginning of the maintenance phase), or 30 h. Superior results are obtained when the temperature shift occurs at 15 h and the extractive fermentation is permitted to proceed for a total of 60 h. Under these conditions, the W3110serA^"(DE3)/pJA3.131A synthesizes 15 g/L phloroglucinol in a yield of 11% (mol/mol). In comparison with the non-extractive fermentation, the extractive fermentation is found to provide undiminished phloroglucinol production throughout the fermentation, a steady PhlD specific activity, maintained cell viability, and longer maximum fermentation times.

[00108] An identical fermentation profile, with the same extractive fermentation conditions, is also used to test phloroglucinol production by the BL21serA^"(DE3)/pJA3.131A strain described above. Equivalent results to those of the W3110 fermentation are obtained. Another dual temperature profile, where in which the initial 36° C temperature is shifted at 15 h to 33° C, is found to increase recovery of phloroglucinol from BL21 yet further, giving a 17.3 g/L titer and a 12.3% (mol/mol) yield.

[00109] In addition, expression of recombinant phlD in yeast (e.g., S. cerevisiae) is successful, although yields are from 0.5 to about 1.5 mg/L under test conditions (data not shown). Phloroglucinol fermentations like those described in Examples 1-3 can also be carried out using the phloroglucinol synthase from Vibrio cholarae RC385, which is designated as PhlD^Vc, the phloroglucinol synthase from Yersinia mollaretii ATCC43969, which is designated as PhlD^Ym, as well as homologous enzymes. Example 4 - Phloroglucinol synthases PhlD^vc and PhlD* ^m

[00110] A number of full length PhlD sequences are known. PhlD from Pseudomonas fluorescens Q2-87 (designated as PhlD^Q2~87) is 83% identical at the protein sequence level with PhlD^pf"5. PhlD from Pseudomonas fluorescens HP72 (designated as PhlD^HP72) is 85% identical at the protein sequence level with PhlD^pf"5. PhlD from Pseudomonas fluorescens 2P24 (designated as PhlD^2P24) is 84% identical at the protein sequence level with PhlD^pf"5. In addition, numerous partial PhlD sequences are known (e.g., listed in NCBI GenBank). As with the full length PhlD sequences, the partial PhlD sequences are similar and share 85% or greater sequence identity with PhlD^pf"5.

[00111] The invention includes phloroglucinol synthases corresponding to amino acid sequences from Vibrio cholarae RC385 (designated as PhlD^Vc) and Yersinia mollaretii ATCC43969 (designated as PhlD^Ym). The genes encoding PhlD^Vc and PhlD^Ym were synthesized and shown to exhibit in vitro enzyme activity in the presence of substrate malonylCoA. See Table 1. Plasmid-localized phlD^Vc and phlD^Ym were also transformed into an Escherichia coli host and the intact constructs evaluated for synthesis of phloroglucinol under fermentor-controlled conditions. See FIG. 10. Specific activities of PhlD ^Vc and PhlD^Ym were determined over the course of these fermentor runs. See FIG. 11.

[00112] FIG. 8 illustrates an amino acid alignment of PhlD^pf"5 (SEQ ID NO: 2) with PhlD^Vc (SEQ ID NO: 5) and PhlD^Ym (SEQ ID NO: 6). The amino acid alignment was produced using ClustalW (available through the European Bioinformatics Institute, or directly on the World Wide Web via http://www.ebi.ac.uk/Tools/clustalw/index.html). The PhlD^Vc protein sequence is only 46% identical with PhlD^pf"5. The PhlD^Ym protein sequence is only 43% identical with PhlD^pf"5. PhlD^Vc and PhlD^Ym are also very different from one another - they share only 46% identity at the protein sequence level. Unlike the phlACBDE gene cluster in which phlD^pf"5 resides in Pseudomonas fluorescens Pf-5, phlD^Vc and phlD^Ym are not part of a phlACBDE biosynthetic gene cluster or any other apparent gene cluster.

[00113] The %GC content of the wild-type nucleotide sequences of PhlD ^Vc and

PhlD ^m are each about 40%. PhlD^Vc and PhlD^Ym also have a significant difference in codon usage relative to E. coli. Therefore, direct heterologous expression of the wild-type nucleotide sequences was not performed. The amino acid sequences PhlD^Vc and PhlD^Ym were codon optimized for E. coli expression using Gene Designer software (DNA2.0). The first amino acid of the PhlD^Ym sequence was manually changed from valine to methionine based on the alignment shown in FIG. 8. The resulting back-translated nucleotide sequences were synthesized at DNA2.0 and are shown in SEQ ID NO: 3 (PhlD^Vc) and SEQ ID NO: 3 (PhlD^Ym).

[00114] Expression plasmid pKIT 1.001 was prepared by li gating a blunt-ended DNA fragment that contained the serA gene with plasmid pET27b (Novagen), which had been previously linearized by sequential treatment with Sphl and Klenow (FIG. 9, left). The two optimized phloroglucinol synthase candidate genes were received as inserts in plasmids pJ201-19397 (phlD^Vc) and pJ201-19398 (phlD^Ym). The two genes (phlD^Vc and phlD^Ym) were excised from pJ201-19397 (phlD^Vc) and pJ201-19398 (phlD^Ym) using Ndel and Xhol enzymes and li gated with pKIT 1.001, which had been previously treated with Ndel and Xhol to yield plasmids pKIT 1.002 (containing phlD^Vc) and pKIT 1.003 (containing phlD^Ym). The resulting clones were transformed into E. coli C41(DE3)ser_^4 for evaluation.

[00115] Table 1 shows the result of the enzyme activity assays using E. coli

C41(DE3 er^/pKIT 1.002 (PhlD^Vc) and £. coli C41(DE3 er^/pKIT 1.003 (PhlD^Ym) cultured under shake flask conditions. Enzyme activity assays using crude cell lysate with PhlD^Vc activity gives specific activity of 0.001 /mg, while using crude cell lysate from PhlD^Ym construct gives 0.01 U/mg. For comparison, P. fluorescens Pf-5 PhlD^pf"5 has a specific activity of 0.04 U/mg. One unit (U) of activity is defined as Ι μιηοΐ of phloroglucinol product formed per minute from malonylCoA substrate. PhlD^Ym is more active than PhlD^Vc in this experiment.

Table 1. Specific Activities of PhlD^Vc and PhlD^Ym with malonylCoA in crude cell lysate.

Construct Gene origin Specific activity (U/mg)

C41 (DE3 )serA/pKn 1.002 Vibrio cholarae 0.001

C41 (DE3 er^/pKIT 1.003 Yersinia mollaretii 0.01

[00116] FIG. 10A illustrates an example cultivation of E. coli

C41(DE3)serA/pKIT1.002 (PhlD^Vc) under fermentor-controlled conditions. FIG. 10A illustrates an example cultivation of is. coli C41(DE3)serA/pKIT1.003 (PhlD^Ym) under fermentor-controlled conditions. Diamonds indicate dry cells weight and open bars indicate g/L phloroglucinol.

[00117] E. coli C41(DE3 er^i/pKIT 1.002 (PhlD^Vc) and E. coli

C41(DE3 er^i/pKIT 1.003 (PhlD^Ym) were also cultured under fermentor controlled conditions. Peak phloroglucinol synthesis of 0.03 g/L was observed (FIG. 10A) for is. coli C41(DE3)ser^/pKIT 1.002 (PhlD^Vc). This compares with Peak phloroglucinol synthesis (FIG. 10B) of 4.0 g/L for E. coli C41(DE3 er^/pKIT1.003 (PhlD^Ym).

[00118] FIG. 11 illustrates example PhlD^Vc, PhlD^Ym, and PhlD^pf"5 specific activities

^mol/min/mg) as a function of time during cultivation of E. coli C41(DE3)serA/pKIT1.002 (PhlD^Vc), £. co/ C41(DE3)serA/pKIT1.003 (PhlD^Ym), and £. coli C41(DE3)serA/pBC2.274 (PhlD^pf~5) under fermentor-controlled conditions.

[00119] The specific activity of PhlD over the course of fermentor runs were determined for E. coli C41(DE3 er^i/pKIT1.002 (PhlD^Vc) and £. coli

C41(DE3 er^i/pKIT 1.003 (PhlD^Ym) for comparison (FIG. 11) with E. coli

C41(DE3 er^i/pBC2.274 (PhlD^pf~5). Phloroglucinol synthase was detectable for a short period of time during cultivation of is. coli C41(DE3 er^i/pKIT 1.002 (PhlD^Vc) under fermentor-controlled conditions (FIG. 11). Although the peak specific activity for E. coli C41(DE3 er^i/pKIT 1.003 (PhlD^Ym) was not as high as for E. coli C41(DE3 er^i/pBC2.274 (PhlD^pf~5) in this experiment, phloroglucinol synthase activity was maintained for a longer period of time (FIG. 11) for E. coli C41(DE3 er^i/pKIT 1.003 (PhlD^Ym) relative to E. coli C41(DE3 er^/pBC2.274 (PhlD^pf"5).

[00120] The toxicity of phloroglucinol can be mitigated by O-methylation. Such O- methylation can proceed step-wise to giwe phloroglucinol derivatives that have O- methylation in one, two, or three positions. Further, such O-methylation of phloroglucinol can yield phloroglucinol derivatives of enhanced utility in certain chemical reactions, for example, in the synthesis of explosives such as tetra amino trinitro benzene (TABT).

[00121] As illustated in Fig. 12, Phloroglucinol O-methyl transferese (POMT) then catalyzes the reaction of S-adenosylmethionine with phloroglucinol to form 5- methoxyresorcinol (mono-O-methylphloroglucinol). Orcinol O-methyl transferase (OOMT) catalyzes the following two sequential methylations leading to 3,5-dimethoxyphenol (di-O- methylphloroglucinol), and 1,3,5-trimethoxybenzene (tri-O-methylphloroglucinol).

[00122] De novo synthesized, codon-optimized POMT was inserted into plasmid pET22b ( ovagen) and expressed from a T7 promoter and having a C-terminal his-tag, which can assist with purification of POMT to homogeneity if necessary. After

transformation, the resulting E. coli containing the plasmid-localized, codon-optimized POMT insert was cultured and the cells harvested, lysed in a French press. After centrifugation, the clarified lysate had a specific activity for POMT of 0.02 U/mg. POMT activity was assayed with phloroglucinol (2 niM), dithiothreitol (10 niM), S- adenosylmethionine (5 niM), glycerol (10 mM) incubated together in 50 mM sodium phosphate buffer at pH 7 at ambient temperature. The assay was initiated by the addition of crude lysate of the E. coli construct to give a total volume of 5 mL for the reaction mixture. A 0.9 mL sample was taken at 10, 20 and 30 min and quenched with 0.1 mL trichloroacetic acid (10%). The sample was centrifuged and the supernatant was dried under reduced pressure. The synthesized mono-O-methylphloroglucinol was derivatized with BSTFA and quantified using GC. One unit of mono-O-methylphloroglucinol forming activity was defined as the catalyzed formation of 1 μιηοΐ mono-O-methylphloroglucinol per min.

[00123] An E. coli strain that can synthesize mono-O-methylphloroglucinol from glucose has been constrcuted. DNA2.0 optimized phloroglucinol 0-methyltransferase gene (POMT) was excised from plasmid pET22b-pPOMT using Ndel and Xhol restriction enzymes. The resulting 0.7 kb gene was ligated with pKIT10.080, which had been previously treated with Ndel, Xhol and CIAP to afford pKIT 1.007. A 1.6 kb serA locus was excised from plasmid pRC1.55B by digestion with Smal and ligated to the plasmid pKIT 1.007, which had been previously treated with Seal and CIAP. The ligation mixture was transformed into E. coli PG1. Transformants carrying the serA insert were selected on M9 medium plates. In the resulting construct E. coli PGl/pKITl.008, the serA, 0_POMT and phlD* genes are transcribed in the same direction. Cultivtion of PGl/pKITl.008 under fermentor controlled conditions synthesized 0.7 g/L mono-O-methylphloroglucinol. GC- MSD was used to characterize medium samples taken during fermentation.

[00124] Supplementation of the fermentation medium with methionine was found out to have a positive effect in mono-0-methylphloroglucinol production using PGl/pKITl.008. The bar chart on the left side in Figure 7 shows a control fermentation of PGl/pKIT 1.008 without methionine supplementation. The one on the right side shows a fermentation of PGl/pKIT 1.008 with 0.4 g/L methionine supplemented at the time when IPTG was added to induce protein production. A similar production of mono-0-methylphlorglucinol was observed when compared to the control run.

[00125] It is believed that mono-O-methylphloroglucinol is toxic to E. coli cells. To circumvent this issue, attempts had been made to evaluate this microbe using resin-based extractive fermentation (Figure 5). By coupling our methionine feeding strategy and a strong anion-exchange resin column, E. coli PGl/pKIT 1.008 synthesized 2 g/L mono-0- methylphloroglucinol in 70 h after inoculation (Figure 13), which accounts to another 45% increase in mono-O-methylphloroglucinol production. As illustrated in Fig. 13, optimized fed-batch fermentation runs of E. coli PGl/pKITl.008. Methionine (0.2 g/L) was added every 6 h after IPTG induction. The peak phloroglucinol titer at 29 h is 3.3 g/L. The final phloroglucinol titer at 44 his 1.7 g/L. The peak mono-O-methylphloroglucinol titer at 44 h is 1.1 g/L. The final mono-O-methylphloroglucinol titer at 44 h is 1.1 g/L.

[00126] The protein sequence of orcinol O-methyltransferase (OOMT) from Rosa hybrida was codon optimized and back-translated into DNA sequence for E. coli expression using Gene Designer (DNA2.0). The resulting OOMT1 gene was synthesized and cloned into expression vector pET22b (Novagen) under a T7 promoter. The resulting plasmid pOOMTl was transformed into E. coli and was assayed for methylase activity using mono- O-methylphloroglucinol as substrate. The crude extract specific activity of this construct was found to be 0.006 U/mg. The production of di-O-methylphloroglucinol in the reaction mixture was characterized by GC/FID and GC/MS.

[00127] The citation of references does not constitute an admission that any references are prior art or have any particular relevance to the patentability of the invention. All references cited in the Description section of this specification are hereby incorporated by reference in their entirety.

[00128] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMS What is claimed is:

1. An isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:3, wherein the nucleic acid sequence encodes a functioning phloroglucinol synthase (PhlD).

2. An isolated or recombinant nucleic acid sequence that is at least 80% homologous to SEQ ID NO:4, wherein the nucleic acid sequence encodes a functioning phloroglucinol synthase (PhlD).

3. The isolated or recombinant nucleic acid sequence of claim 1 or 2, wherein the sequence is at least 85% homologous to SEQ ID NO:3 or SEQ ID NO:4, respectively.

4. The isolated or recombinant nucleic acid sequence of claim 1 or 2, wherein the sequence is at least 90% homologous to SEQ ID NO:3 or SEQ ID NO:4, respectively.

5. The isolated or recombinant nucleic acid sequence of claim 1 or 2, wherein the sequence is at least 95% homologous to SEQ ID NO:3 or SEQ ID NO:4, respectively.

6. The isolated or recombinant nucleic acid sequence of claim 1 or 2, wherein the sequence is at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to SEQ ID NO:3 or SEQ ID NO:4, respectively.

7. An isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO:5, wherein the amino acid sequence encodes a functioning phloroglucinol synthase (PhlD).

8. An isolated or recombinant amino acid sequence that is at least 50% homologous to SEQ ID NO: 6, wherein the amino acid sequence encodes a functioning phloroglucinol synthase (PhlD).

9. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 60% homologous to SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

10. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 70% homologous to SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

11. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 80% homologous to SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

12. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 85% homologous to SEQ ID NO:5 or SEQ ID NO:6, respectively.

13. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 90% homologous to SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

14. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 95% homologous to SEQ ID NO:5 or SEQ ID NO:6, respectively.

15. The isolated or recombinant amino acid sequence of claim 7 or 8, where the amino acid sequence is at least 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to SEQ ID NO:5 or SEQ ID NO:6, respectively.

16. A system for converting malonyl-CoA to phloroglucinol comprising:

an isolated or recombinant enzyme system or a recombinant cell comprising a phloroglucinol synthase (PhlD⁺),

wherein the enzyme system or cell comprises the nucleic acid sequence of claim 1 or 2, and/or the amino acid sequence of claim 7 or 8.

17. The system of claim 16, wherein the enzyme system or cell is at least one of PhlA^", PhlB^", and PhlC^".

18. The system of claim 16, wherein the enzyme system or cell is PhlA^", PhlB^", and PhlC^".

19. The system of claim 16, wherein the cell is genetically engineered to increase PhlD expression.

20. The system of claim 16, further comprising a malonyl-CoA synthesis enzyme.

21. The system claim 16, wherein the recombinant cell is E. coli or P. fluorescens.

22. A method for producing anabolic phloroglucinol comprising:

providing a system for converting malonyl-CoA to phloroglucinol comprising an isolated or recombinant enzyme system or a recombinant cell having a phloroglucinol synthase (PhlD⁺), wherein the enzyme system or cell comprises the nucleic acid sequence of claim 1 or 2, and/or the amino acid sequence of claim 3 or 4; and

contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol.

23. The method of claim 22, wherein the enzyme system or cell is at least one of PhlA^", PhlB^" and PhlC^".

24. The method of claim 22, wherein the enzyme system or cell is PhlA^", PhlB^" and PhlC^".

25. The method of claim 22, wherein the cell is genetically engineered to increase PhlD expression.

26. The method of claim 22, wherein the enzyme system or cell comprises a malonyl- CoA synthesis enzyme.

27. The method of claim 22, wherein the carbon source comprises a saccharide, an aliphatic polyol, or both a saccharide and an aliphatic polyol.

28. The method of claim 22, wherein the contacting step comprises an extractive fermentation.

29. The method of claim 22, wherein the contacting step comprise a dual temperature profile.

The method of claim 22, further comprising: contacting the anabolic phloroglucinol, hydrogen, and a rhodium catalyst under conditions allowing the hydrogenation of the anabolic phloroglucinol, to produce resorcinol.

31. The method of claim 22, further comprising:

producing a medicament, cosmetic, dye, polymer resin, rubber, adhesive, sealant, coating, composite material, laminated material, or bonded material from the anabolic phloroglucinol or resorcinol derived from the anabolic phloroglucinol.

32. The method of claim 22, further comprising:

chemically modifying the anabolic phloroglucinol or resorcinol derived from the anabolic phloroglucinol, to produce a propellant or explosive.

33. An isolated or recombinant nucleic acid vector comprising:

an open reading frame encoding the nucleic acid sequence of claim 1 or 2, or corresponding to the amino acid sequence of claim 7 or 8, from which a cell can express a functioning phloroglucinol synthase.

28. The nucleic acid vector of claim 33, wherein the vector is pMD⁺ and at least one of phlA^~, phlB^~, and phlC.

29. The nucleic acid vector of claim 33, wherein the vector is phlA^~, phlB^~, and phlC.

30. The nucleic acid vector of claim 33, wherein the vector is genetically engineered to increase PhlD expression.

31. The nucleic acid vector of claim 33, further comprising a malonyl-CoA synthesis enzyme gene.

32. A recombinant cell comprising:

a nucleic acid vector comprising an open reading frame encoding the nucleic acid sequence of claim 1 or 2, or corresponding to the amino acid sequence of claim 7 or 8, from which the recombinant cell can express a functioning phloroglucinol synthase.

33. The recombinant cell of claim 32, wherein the vector is phlD and at least one of phlA^~, phlB^~, and phlC.

34. The recombinant cell of claim 32, wherein the vector is phlA^~, phlB^~, and phlC.

35. The recombinant cell of claim 32, wherein the vector is genetically engineered to increase PhlD expression.

36. The recombinant cell of claim 32, wherein the vector comprises a malonyl-CoA synthesis enzyme gene.

37. The recombinant cell of claim 32, wherein the recombinant cell is an E. coli or P. fluorescens cell.

38. A method for producing a phlD⁺ recombinant cell comprising:

transforming a cell with a nucleic acid vector comprising an open reading frame encoding the nucleic acid sequence of claim 1 or 2, or corresponding to the amino acid sequence of claim 7 or 8, from which the recombinant cell can express a functioning phloroglucinol synthase.

39. The method of claim 38, wherein the vector is at least one oiphlA^', phlB^~, and phlC.

40. The method of claim 38, wherein the vector is phlA^~, phlB^~, and phlC.

41. The method of claim 38, wherein the vector is genetically engineered to increase PhlD expression.

42. The method of claim 38, further comprising a malonyl-CoA synthesis enzyme gene.

43. The method of claim 38, wherein the recombinant cell is an E. coli or P. fluorescens cell.

44. A method for producing anabolic O-methyl phloroglucinol comprising: providing a system for converting malonyl-CoA to phloroglucinol comprising a an isolated or recombinant enzyme system or a recombinant cell having a phloroglucinol synthase (PhlD⁺), wherein the enzyme system or cell comprises the nucleic acid sequence of claim 1 or 2, or comprises the amino acid sequence of claim 3 or 4, wherein the system comprises the nucleic acid sequence necessary to express phloroglucinol O-methyl transferase (POMT); and

contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol, the phloroglucinol being further transformed into O-methyl phloroglucinol.

45. The method of claim 44, wherein the enzyme system or cell is at least one of PhlA^", PhlB^", and PhlC^".

46. The method of claim 44, wherein the enzyme system or cell is PhlA^", PhlB^", and PhlC

47. The method of claim 44, wherein the cell is genetically engineered to increase PhlD expression.

48. The method of claim 44, wherein the enzyme system or cell comprises a malonyl- CoA synthesis enzyme.

49. The method of claim 44, wherein the carbon source comprises a saccharide, an aliphatic polyol, or both a saccharide and an aliphatic polyol.

50. The method of claim 44, wherein the contacting step comprises an extractive fermentation.

51. The method of claim 44, wherein the contacting step comprise a dual temperature profile.

52. A method for producing di-O-methyl phloroglucinol and/or tri-O-methyl phloroglucinol comprising: providing a system for converting malonyl-CoA to phloroglucinol comprising an isolated or recombinant enzyme system or a recombinant cell having a phloroglucinol synthase (PhlD⁺), wherein the enzyme system or cell comprises the nucleic acid sequence of claim 1 or 2, or comprises the amino acid sequence of claim 3 or 4,

wherein the system comprises the nucleic acid sequence necessary to express phloroglucinol O-methyl transferase (POMT),

wherein the system comprises the nucleic acid sequence necessary to express orcinol O-methyl transferase (OOMT);

contacting the system and malonyl-CoA or a carbon source that the system can convert into malonyl-CoA, to produce anabolic phloroglucinol, wherein the phloroglucinol is further transformed into O-methyl phloroglucinol, wherein the O-methyl phloroglucinolis further transformed to di-O-methyl phloroglucinol; and

isolating the di-O-methyl phloroglucinol or transforming the di-O-methyl phloroglucinol to tri-O-methyl phloroglucinol.