WO2007047680A2

WO2007047680A2 - Increasing the activity of radical s-adenosyl methionine (sam) enzymes

Info

Publication number: WO2007047680A2
Application number: PCT/US2006/040563
Authority: WO
Inventors: Ravi R. Gokarn; Steven J. Gort; Holly J. Jessen; Hans H. Liao; Brian J. Brazeau
Original assignee: Cargill, Incorporated
Priority date: 2005-10-14
Filing date: 2006-10-16
Publication date: 2007-04-26
Also published as: WO2007047680A3

Abstract

The present disclosure provides methods of increasing the activity of radical S-adenosyl methionine (SAM) enzymes, for example by increasing expression of ferredoxin, flavodoxin, flavodoxin reductase, or combinations thereof in a cell expressing a radical SAM enzyme. Such methods can be used to increase expression of a radical SAM enzyme product, or a downstream organic chemical. Also provided by the present disclosure are cells that have increased expression of ferredoxin, flavodoxin, flavodoxin reductase, or combination thereof. Such cells can be used to practice the disclosed methods.

Description

BVCREASING THE ACTIVITY OF RADICAL S-ADENOSYL METHIONINE (SAM) ENZYMES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional application No. 60/726,925 filed

October 14, 2005, herein incorporated by reference in its entirety.

FIELD

This application relates to methods of increasing the activity of radical S-adenosyl methionine (SAM) enzymes, as well as cells having such increased activity.

BACKGROUND

Organic chemicals such as organic acids, esters, and polyols can be used to synthesize plastic materials and other products. To meet the increasing demand for organic chemicals, more efficient and cost-effective production methods are being developed which utilize raw materials based on carbohydrates rather than hydrocarbons. For example, certain bacteria have been used to produce large quantities of lactic acid used in the production of polylactic acid.

3-hydroxypropionic acid (3-HP) is an organic acid. Several chemical synthesis routes have been described to produce 3-HP, and biocatalytic routes have also been disclosed (WO 01/16346 to Suthers et al). 3-HP has utility for specialty synthesis and can be converted to commercially important intermediates by known art in the chemical industry, such as acrylic acid by dehydration, malonic acid by oxidation, esters by esterifϊcation reactions with alcohols, and 1,3-propanediol by reduction.

SUMMARY

The inventors have observed that increased expression of flavodoxin, ferredoxin, flavodoxin reductase, or combinations thereof, increases the activity of radical S-adenosyl methionine (SAM) enzymes in cells, such as alanine 2,3-aminomutase activity. This increase in the activity of the radical SAM enzyme led to a corresponding increase in the production of a radical SAM enzyme product, such as beta-alanine.

Based on these observations, methods are provided for increasing the activity of one or more radical SAM enzymes, such as increasing the activity of 1, 2, 3, 4, 5 or more radical SAM enzymes in a cell. In particular examples, the method includes increasing expression of a nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof in a cell, wherein increased expression of the flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof increases the activity of a radical SAM enzyme in the cell. The radical SAM enzyme can be native to the cell, or can be exogenous to the cell, for example expressed by an exogenous nucleic acid encoding a radical SAM enzyme. Particular examples of radical SAM enzymes, and their corresponding radical SAM enzyme products in parenthesis, include, but are not limited to: biotin synthase (biotin), lipoate synthase (lipoic acid), aminomutases that interconvert alpha and beta amino acids (beta amino acid), GcpE protein ((E)-4-hydroxy-3-methylbutly-2-enyl pyrophosphate, HMBPP) and LytB protein (isopentenyl pyrophosphate, IPP, and dimethylallyl pyrophosphate, DMAPP). Non-limiting examples of aminomutases that interconvert alpha and beta amino acids include alanine 2,3-aminomutase (beta-alanine), arginine 2,3- aminomutase (beta-arginine), or lysine 2,3-aminomutase (beta-Iysine). Organic compounds that can be produced from radical SAM enzyme products include but are not limited to: 3- hydroxypropionic acid (3-HP) (and derivatives thereof such as an ester of 3-HP), pantothenate, cryptophycin, and CoA (all downstream compounds of beta-alanine); streptothricin, viomycin, racemomycin, and nourseothricins (all downstream compounds of beta-lysine); blasticidin S (a downstream compound of beta-arginine); and isoprenoids, carotenoids (such as lycopene, lutein, β-carotene, zeazanthin) and terpenoids (such as artemisinin) (downstream compounds of GcpE and LytB). In some examples, increasing the activity of a radical SAM enzyme in the cell (for example by at least 20%) increases the production of one or more the radical SAM enzyme products (or a downstream organic compound thereof), for example by at least 20%. Therefore, the disclosure provides methods of producing radical SAM enzyme products such as beta-alanine, and producing further downstream chemicals such as 3 -HP and pantothenate (and derivatives thereof). In particular examples, when producing a downstream organic chemical, the cell can include additional enzymes activities that permit production of the chemical. Such activities can be endogenous to the cell, or can be supplied by one or more exogenous nucleic acid molecules. For example, for the production of 3 -HP (or a derivative thereof), the cell can include CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity and 3- hydroxypropionyl-CoA hydrolase activity. In another example for the production of 3-HP (or a derivative thereof), the cell can include beta alanine-2-oxoglutarate aminotransferase activity and 3-HP dehydrogenase activity. In an example where pantothenate (or a derivative thereof) is produced, the cell can further include alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity.

Transformed cells having increased radical SAM enzyme activity are also provided by the present disclosure. The cells can be eukaryotic or prokaryotic, such as such as yeast cells, plant cells, fungal cells, or bacterial cells (for example Lactobacillus, Lactococcus, Bacillus, or Escherichia cells). Also provided are transgenic plants that include such cells. A particular example of such cells was deposited with the American Type Culture Collection (Manassas, VA) on July 7, 2005 (Accession No. PTA-6837).

In one example, expression of one or more nucleic acid molecules encoding flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, is increased in the cell. For example, the flavodoxin, ferredoxin, or flavodoxin reductase can be a native sequence that is expressed by an exogenous promoter. In another example, exogenous flavodoxin, ferredoxin, or flavodoxin reductase coding sequences are present in the cell. The radical SAM enzyme can be native to the cell, or can be exogenous to the cell (for example expressed from an exogenous nucleic acid molecule). The disclosed cells can produce one or more radical SAM enzyme products (or a downstream organic chemical thereof), such as cells that have increased production of a radical SAM enzyme product (or a downstream chemical). For example, the disclosed cells can in some examples produce beta-alanine or a downstream chemical thereof, such as 3- HP, pantothenate, CoA, or derivatives thereof. In some examples, cells that produce a downstream organic chemical can include additional enzymes activities that permit production of the chemical. Such activities can be endogenous to the cell, or can be supplied by one or more exogenous nucleic acid molecules. For example, for the production of 3-HP (or a derivative thereof), the cell can include CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity and 3-hydroxypropionyl-CoA hydrolase activity. In another example for the production of 3-HP (or a derivative thereof), the cell can include or beta alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity. In an example where the cell produces pantothenate (or a derivative thereof), the cell can further include alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity.

Also provided by the present disclosure are methods of making radical SAM enzyme products, and downstream organic chemicals thereof. Such methods can be performed in vivo, in vitro, or combinations thereof. For example, radical SAM enzyme products, and downstream organic chemicals thereof, can be generated in vivo by culturing the disclosed cells under conditions that permit generation of the product. For example, the disclosed cells can be cultured to generate a beta amino acid (such as beta-alanine) from an alpha amino acid (such as alpha-alanine) if the cell has the appropriate enzyme activity, such as alanine 2,3-aminomutase activity. In a particular example, such as when a downstream organic chemical is generated, the cell can include additional enzyme activities. Such activities can be endogenous or supplied by an exogenous nucleic acid molecule. For example, a cell that can make pantothenate can include alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity.

In another example, radical SAM enzyme products, and downstream organic chemicals thereof, can be generated by a combination of in vitro and in vivo methods. For example, the disclosed cells can be cultured under conditions that permit generation of a radical SAM enzyme product (such as beta-alanine). The radical SAM enzyme product can be purified or isolated from the cell or culture medium, and the radical SAM enzyme product generated in vivo contacted with the appropriate enzymes or other agents to generate a downstream organic chemical in vitro. For example, 3-HP can be generated by purifying beta-alanine from the cell, and then incubating the beta-alanine with one or more peptides having CoA transferase activity to form beta-alanyl-CoA, contacting the beta- alanyl CoA with a peptide having beta-alanyl-CoA ammonia lyase activity to form acrylyl- CoA, contacting the aery Iy 1-CoA with a peptide having 3HP-CoA dehydratase activity to form 3-HPrCoA, and contacting the 3-HP-CoA with a peptide having CoA transferase activity and 3-hydroxypropionyl-CoA hydrolase activity, to make 3-HP. In another example, 3-HP can be generated by purifying beta-alanine from the cell, and then incubating the beta-alanine with one or more peptides having beta-alanine-2-oxoglutarate aminotransferase activity to form malonic semialdehyde, and contacting the malonic semialdehyde with a peptide having 3-HP dehydrogenase activity, to make 3-HP.

The foregoing and other aspects of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BMEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a pathway for generating 3-HP and derivatives thereof via a beta-alanine intermediate, and for making beta-alanine from alpha-alanine.

FIG. 2 is a diagram of a pathway for generating coenzyme A and pantothenate from beta-alanine. FIG. 3 is a graph showing the effect of flavodoxin (FLDl) and flavodoxin reductase (FNR) on beta-alanine production by alanine 2,3-aminomutase.

FIG. 4 is a graph comparing alanine 2,3-aminomutase activity with E. coli flavodoxin NADP⁺ reductase (FNR) and flavodoxin from E. coli (EcFLDl) or P. gingivalis (PgFLDl).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NOS: 1 and 2 are PCR primers used to PCR amplify an E. coli ferredoxin gene (fdx).

SEQ ID NOS: 3 and 4 are PCR primers used to PCR amplify an E. coli flavodoxin/ferredoxin NADP(H) oxidoreductase gene (fpr).

SEQ ID NOS: 5 and 6 are PCR primers used to remove the Bsal restriction site present in the E. coli fpr coding sequence.

SEQ ID NOS: 7 and 8 are PCR primers used to PCR amplify an E. coli flavodoxin gene (fldA). SEQ ID NOS: 9 and 10 are PCR primers used to PCR amplify a P. gingivalis flavodoxin gene {Pgfldl).

SEQ ID NOS: 11 - 14 are PCR primers used to PCR amplify pKDprom.

SEQ ID NOS: 15 and 16 show exemplary alanine 2,3-aminomutase nucleic acid and protein sequences, respectively. SEQ TD NOS: 17 and 18 show further exemplary alanine 2,3-aminomutase nucleic acid and protein sequences, respectively.

SEQ ID NOS: 19 and 20 show a P. gingivalis flavodoxin nucleic acid and protein sequence, respectively.

DETAILED DESCRIPTION

Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "comprising a cell" includes one or a plurality of such cells, and reference to "comprising the radical SAM enzyme" includes reference to one or more radical SAM enzymes and equivalents thereof known to those skilled in the art, and so forth. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. For example, the phrase "CoA transferase activity or CoA synthetase activity" refers to CoA transferase activity, CoA synthetase activity, or a combination of both CoA transferase activity and CoA synthetase activity.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the disclosure are apparent from the following detailed description and the claims.

Alanine 2,3-aminoniutase: An enzyme that can convert alpha-alanine to beta- alanine, for example in a cell. Includes any alanine 2,3-aminomutase gene, cDNA, RNA, or protein from any organism, such as a prokaryote. In particular examples, an alanine 2,3- aminomutase sequence includes the alanine 2,3-aminomutase sequences disclosed in PCT/US2003/001635 and PCT/US2004/024686 (both herein incorporated by reference as to the alanine 2,3-aminomutase sequences). In particular examples, an alanine 2,3- aminomutase nucleic acid sequence includes the sequences shown in SEQ ID NOS: 15 or 17, as well as fragments, variants, or fusions thereof that retain the ability to encode a protein having alanine 2,3-aminomutase activity. In another example, an alanine 2,3- aminomutase protein includes the amino acid sequence shown in SEQ ID NO: 16 or 18, as well as fragments, fusions, or variants thereof that retain alanine 2,3-aminomutase activity. An alanine 2,3-aminomutase amino acid sequence includes a full-length sequence, such as SEQ ID NO: 16 or 18, as well as shorter sequences which retain the ability to convert alpha-alanine to beta-alanine. Examples of alanine 2,3-aminomutase fragments which can be used include, but are not limited to: amino acids 50-390, 50-350, 60-350, 75- 340, 101-339, 100-339, 1-390, 15-390, 15-340 or 19-331 of SEQ DD NO: 16 or 18.

This description includes alanine 2,3-aminomutase allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to convert alpha-alanine to beta-alanine. In a particular example, alanine 2,3-aminomutase is a mutated B. subtilis lysine 2,3- aminomutase having a substitution at position Ll 03, D339, Ml 36, or combinations thereof. For example, the substitution can include a L103M, L103K, L103R, Ll 03 E, or L103S substitution. In another or additional example, the substitution includes a D339H, D339Q, D339T, or D339N substitution. In yet another example, the substitution can include a L103M, a M136V substitution, a D339H substitution, or any combination thereof.

Alanine 2,3-aminomutase activity: The ability of an alanine 2,3-aminomutase to convert alpha-alanine to beta-alanine. In one example, such activity occurs in a cell. In another example, such activity occurs in vitro. Such activity can be measured using any assay known in the art. For example, alanine 2,3-aminomutase activity can be identified by incubating the enzyme with either alpha-alanine or beta-alanine and determining the reaction products by high-performance liquid chromatography (for example using the method of Abe et al. J. Chromatography B, 712:43-9, 1998).

Examples of substitutions which can be made, while still retaining alanine 2,3- aminomutase activity, include, but are not limited to: T40S; V96I or V96L; Dl 02E; A252V; or L393V of SEQ ID NO: 16 or 18, as well as combinations thereof.

Arginine 2,3-aminomutase: An enzyme which can convert alpha-arginine to beta- arginine. Includes any arginine 2,3-aminomutase nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example Streptomyces. This description includes arginine 2,3-aminomutase allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to convert alpha-lysine to beta-lysine. In one example, includes a peptide encoded by nucleic acid sequence (such as a gene or cDNA) annotated as arginine 2,3-aminomutase in public sequence databases (for example GenBank Accession No. AY196214 discloses an arginine 2,3-aminomutase nucleic acid sequence and GenBank Accession No. AAP03121 discloses an arginine 2,3- aminomutase protein sequence).

Arginine 2,3-aminomutase activity: The ability of an arginine 2,3-aminomutase to convert alpha-arginine to beta-arginine. In one example, such activity occurs in a cell. In another example, such activity occurs in vitro. Such activity can be measured using any assay known in the art. For example, arginine 2,3-aminomutase activity can be identified by incubating the enzyme with either alpha-arginine or beta-arginine and determining the reaction products by reverse phase HPLC (for example using the method of Henrikson and Meredich, Anal. Biochem. 135:55-74, 1984). cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA can be synthesized by reverse transcription from messenger RNA extracted from cells.

Conservative substitution: One or more amino acid substitutions (for example 1, 2, 5 or 10 amino acid residues) for amino acid residues having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting peptide. For example, a conservative substitution is an amino acid substitution in an alanine 2,3-aminomutase peptide that does not substantially affect the ability of the peptide to convert alpha-alanine to beta-alanine. In a particular example, a conservative substitution is an amino acid substitution in an alanine 2,3-aminomutase peptide, such as a conservative substitution in SEQ ID NO: 16 or 18, that does not significantly alter the ability of the protein to convert alpha-alanine to beta-alanine, or other downstream products such as 3-HP.

An alanine scan can be used to identify amino acid residues in a peptide that can tolerate substitution. In one example, activity is not altered (such as decreased) by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid (such as those listed below), is substituted for one or more native amino acids.

In a particular example, alanine "2,3 -aminomutase activity is not substantially altered if the amount of beta-alanine produced is not reduced by more than about 25%, such as not more than about 10%, than an amount of beta-alanine production in the presence of an alanine 2,3-aminomutase containing one or more conservative amino acid substitutions, as compared to an amount of beta-alanine production in the presence of a native alanine 2,3- aminomutase. A peptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that peptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Alternatively, a peptide can be produced to contain one or more conservative substitutions by using standard peptide synthesis methods. Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; GIn or His for Asn; GIu for Asp; Ser for Cys; Asn for GIn; Asp for GIu; Pro for GIy; Asn or GIn for His; Leu or VaI for He; He or VaI for Leu; Arg or GIn for Lys; Leu or Ue for Met; Met, Leu or Tyr for Phe; Tlir for Ser; Ser for Thr; Tyr for Tip; Trp or Phe for Tyr; and Ue or Leu for VaI.

Further information about conservative substitutions can be found in, among other locations in, Ben-Bassat et al, (J. Bacteriol. 169:751-7, 1987), O'Regan et ah, {Gene 77:237-51 , 1989), Sahin-Toth et a , (Protein Sci. 3 :240-7, 1994), Hochuli et al,

(Bio/Technology 6:1321-5, 1988), WO 00/67796 (Curd et al.) and in Standard textbooks of genetics and molecular biology.

Detectable: Capable of having an existence or presence ascertained. For example, production of beta-alanine from alpha-alanine, or the production or 3-HP from beta-alanine, is detectable if the signal generated from beta-alanine or 3-HP, respectively, is strong enough to be measured.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acid in a peptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed. Enhance or increase: To improve the quality, amount, or strength of something.

In one example, an agent enhances the activity of a radical SAM enzyme if the activity of the enzyme is increased in the presence of the agent, as compared to activity in the absence of the agent. In a particular example, an agent enhances the activity of a radical SAM enzyme if the amount of radical SAM enzyme product produced increases in the presence of the agent, such as an increase of at least 10%, at least 20%, at least 50%, or even at least 75%. For example, overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, can increase the production of a radical SAM enzyme product in the presence of a radical SAM enzyme, such as an increase the production of beta-alanine in the presence of alanine 2,3-aminomutase, an increase the production of beta-arginine in the presence of arginine 2,3-aminomutase, an increase the production of beta-lysine in the presence of lysine 2,3-aminomutase, an increase the production of biotin in the presence of biotin synthase, or an increase the production of lipoic acid in the presence of lipoate synthase.

Such enhancement can be measured using the methods disclosed herein, for example determining an amount of beta-alanine produced in the presence of overexpressed flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof using the methods disclosed in the examples below (such as Examples 2 and 5).

Exogenous: The term "exogenous" as used herein with reference to a nucleic acid molecule and a particular cell refers to any nucleic acid molecule that does not originate from that particular cell as found in nature. Thus, a non-naturally-occurring nucleic acid molecule is considered to be exogenous to a cell once introduced into the cell. A nucleic acid molecule that is naturally-occurring also can be exogenous to a particular cell. For example, an entire coding sequence isolated from cell X is an exogenous nucleic acid with respect to cell Y once that coding sequence is introduced into cell Y, even if X and Y are the same cell type.

Expression: The process by which a gene's coded information is converted into the structures and functions of a cell, such as a protein, transfer RNA, or ribosomal RNA. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs).

Ferredoxin (fdx): An iron-containing protein having a high sulfide content and a very low redox potential that can transfer electrons from one enzyme system to another without having enzyme activity itself. Ferredoxins participate in electron transport, for example in photosynthesis, nitrogen fixation, and other biological processes. Includes any ferredoxin nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example Bacillus subtilis, Chlorobium phaeobacteroides, Salmonella, or E. coli. This description includes ferredoxin allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to participate in electron transport. In one example, includes polypeptides encoded by genes annotated as ferredoxin in public sequence databases, such as GenBank or EMBL-EBI.

Ferredoxin nucleic acid and proteins sequences are publicly available (for example Genbank accession nos: X74556, Ml 8003, and D90883 disclose ferredoxin nucleic acid sequences and Genbank accession nos: ZP_00534131, NP_457069 and BAA16419 disclose ferredoxin protein sequences). Flavodoxin (fldA): Electron-transfer proteins that contain the prosthetic group flavin mononucleotide. Flavodoxins serve as electron donors, for example in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase.

Includes any flavodoxin nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example P, gingivalis or E. coll This description includes flavodoxin allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to serve as electron donors. In one example, includes peptides encoded by genes (or other nucleic acid molecules) annotated as flavodoxin in public sequence databases, such as GenBank or EMBL-EBI. Flavodoxin nucleic acid and proteins sequences are publicly available (for example Genbank accession nos: NC_000913, AE016980, and NC_002695 disclose flavodoxin nucleic acid sequences and Genbank accession nos: ZP_00531318, NP__415210 and AAP 16118 disclose flavodoxin protein sequences).

Flavodoxin reductase (fpr): A family of hydrophilic, monomeric flavoenzymes that contain noncovalently bound FAD as a prosthetic group. These enzymes deliver electrons from NADPH to recipients (such as ferredoxin and flavodoxin) and participate in redox-based metabolisms in plastids, mitochondria and bacteria. Also referred to in the art as flavodoxin/ferredoxin NADP(H) oxidoreductase (E.C. 1.18.1.2).

Includes any flavodoxin reductase nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example P. gingivalis, Bacillus or E. coli. This description includes flavodoxin reductase allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to serve as electron donors. In one example, includes peptides encoded by genes (or other nucleic acid molecules) annotated as flavodoxin reductase in public sequence databases, such as GenBank or EMBL-EBI. Flavodoxin reductase nucleic acid and proteins sequences are publicly available (for example Genbank accession nos: NC_000913, NZ_AAIB01000045, and NZ_AAAC02000001 disclose flavodoxin reductase nucleic acid sequences and Genbank accession nos: ZP_00530081, ZP_00542630 and ZP_00391772 disclose flavodoxin reductase protein sequences). Functionally Equivalent: Having a similar function, such as the ability of a sequence variant, fragment or fusion to have a similar function as the native sequence. For example, functionally equivalent molecules of alanine 2,3-aminomutase include those molecules that retain the function of alanine 2,3-aminomutase, that is, the ability to convert alpha- to beta-alanine. For example, functional equivalents can be provided by sequence alterations in an alanine 2,3-aminomutase, wherein the peptide with one or more sequence alterations retains a function of the unaltered peptide, such that it retains its ability to convert alpha-alanine to beta-alanine.

Examples of sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, and insertions. In one example, a given peptide binds an antibody, and a functional equivalent is a peptide that binds the same antibody. Thus a functional equivalent includes peptides that have the same binding specificity as a peptide, and that can be used as a reagent in place of the peptide (such as in the production of beta-alanine, 3-HP, pantothenate and derivatives thereof). In one example a functional equivalent includes a peptide wherein the binding sequence is discontinuous, wherein the antibody binds a linear epitope. Thus, if the peptide sequence is

MAESRRKYYF (amino acids 1-10 of SEQ ID NO: 18) a functional equivalent includes discontinuous epitopes, that can appear as follows (**=any number of intervening amino acids): NH₂ >*-M**A**E**S**R**R**K**Y**Y**F<!OOH. In this example, the peptide is functionally equivalent to amino acids 1-10 of SEQ ID NO: 18 if the three dimensional structure of the peptide is such that it can bind a monoclonal antibody that binds amino acids 1-10 of SEQ ID NO: 18.

GcpE: Proteins that can reduce 2-C-methyl-D-erythritol cyclopyrophosphate into (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP), for example in the presence of flavodoxin. GcpE proteins include a dioxygen-sensitive [4Fe-4S]²⁺ cluster. Includes any GcpE nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example Mycobacterium avium, Salmonella, or E. coli. This description includes GcpE allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to reduce 2-C-methyl-D- erythritol cyclopyrophosphate into HMBPP. In one example, includes peptides encoded by genes (or other nucleic acid molecules) annotated as GcpE in public sequence databases, such as GenBank or EMBL-EBI. GcpE nucleic acid and proteins sequences are publicly available (for example Genbank accession nos: NC_002944, NC_003198, and D90881 disclose GcpE nucleic acid sequences and Genbank accession nos: NP_961872, NP_457055 and BAA20919 disclose GcpE protein sequences). Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or other molecule) has been substantially separated or purified away from other biological components in the cell in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules, and proteins.

In one example, isolated refers to a naturally-occurring nucleic acid molecule that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid molecule can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid molecule includes, without limitation, a recombinant DNA that exists as a separate molecule (for example, a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (for example, a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

In one example, the term "isolated" as used with reference to a nucleic acid molecule also includes any non-naturally-occurring nucleic acid molecule since non- naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non- naturally-occurring nucleic acid molecules such as an engineered nucleic acid molecule is considered to be an isolated nucleic acid molecule. Engineered nucleic acid molecules can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid molecules can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (such as a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid molecule can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

Lysine 2,3-aminomutase (KAM): An enzyme (EC 5.4.3.2.) which can convert alpha-lysine to beta-lysine. Includes any lysine 2,3-aminomutase nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example Bacillus subtilis, Deinococcus radiodurans, Clostridium subterminale, Porphyromonas gingivalis, Aquifex aeolicus, Haemophilus influenzae, or E. coli. This description includes lysine 2,3-aminomutase allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to convert alpha-lysine to beta-lysine. In one example, includes a peptide encoded by nucleic acid sequence (such as a gene or cDNA) annotated as lysine 2,3-aminomutase in public sequence databases (for example GenBank Accession No. AF 159146 discloses a lysine 2,3-aminomutase nucleic acid sequence and GenBank Accession No. AAD43134 discloses a lysine 2,3-aminomutase protein sequence). Lysine 2,3-aminomutase activity: The ability of a lysine 2,3-aminomutase to convert alpha-lysine to beta-lysine. In one example, such activity occurs in a cell. In another example, such activity occurs in vitro. Such activity can be measured using any assay known in the art. For example, lysine 2,3-aminomutase activity can be identified by incubating the en2yme with either alpha-lysine or beta-lysine and determining the reaction products by reverse phase HPLC (for example using the method of Henrikson and Meredich, Anal. Biochem. 135:55-74, 1984).

LytB: Proteins that can convert (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP) to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), for example in the presence of flavodoxin. In some examples, LytB proteins include a [4Fe-4S] cluster.

Includes any LytB nucleic acid molecule (such as gene, cDNA, RNA, mRNA), or protein from any organism, such as a prokaryote, for example Mycobacterium avium, Neisseria, or E. coli. This description includes LytB allelic variants, as well as any variant, fragment, or fusion sequence which retains the ability to convert HMBPP into IPP and DMAPP. In one example, includes peptides encoded by genes (or other nucleic acid molecules) annotated as LytB in public sequence databases, such as GenBank or EMBL- EBI. LytB nucleic acid and proteins sequences are publicly available (for example Genbank accession nos: NC_003198, AL162753, and NC_002695 disclose LytB nucleic acid sequences and Genbank accession nos: NP_454660, CAB83914 and NP_308059 disclose LytB protein sequences).

Nucleic acid molecule: Encompasses both RNA and DNA including, without limitation, cDNA, genomic DNA, mRNA. Includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced. The nucleic acid molecule can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. In addition, nucleic acid molecule can be circular or linear.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. Configurations of separate genes that are transcribed in tandem as a single messenger RNA are denoted as operons. Thus placing genes in close proximity, for example in a plasmid vector, under the transcriptional regulation of a single promoter, constitutes a synthetic operon.

ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.

Pantothenate or Pantothenic Acid: A vitamin used in cosmetics, medicine, and nourishment. The terms pantothenic acid and pantothenate are used interchangeably herein, and refer not only to the free acid but also to the salts of D-pantothenic acid, such as the calcium salt, sodium salt, ammonium salt or potassium salt. Pantothenate can be produced by chemical synthesis or biotechnologically from beta-alanine using the cells and methods disclosed herein.

Promoter: An array of nucleic acid control sequences that direct transcription of a nucleic acid molecule. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

The term includes endogenous promoter sequences as well as exogenous promoter sequences (such as those introduced into the chromosome to promote expression of a gene, such as ferredoxin, flavodoxin, or flavodoxin reductase). Particular types of promoters that can be used to practice the methods disclosed herein include, but are not limited to, constitutive promoters and inducible promoters (such as a promoter responsive or unresponsive to a particular stimulus, for example such as light, oxygen, or chemical concentration, such as a lactose, IPTG, or tetracycline inducible promoter).

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide in is more enriched than the peptide is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other peptides) that may accompany it. In another example, a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.

In one example, a peptide (such as any of the enzymes shown in FIGS. 1 or 2) is purified when at least about 50% by weight of a sample is composed of the peptide, for example when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of a sample is composed of the peptide. Examples of methods that can be used to purify a peptide, include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 17). Protein purity can be determined by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single peptide band upon staining the polyacrylamide gel; high-pressure liquid chromatography; sequencing; or other conventional methods. Radical S-adenosyl methionine enzyme (Radical SAM): A family of enzymes that juxtapose an iron-sulfur cluster ([4Fe-4S]) and S-adenosyl methionine (SAM) to generate catalytic 5'-deoxyadenosyl radicals in response to reductive activation. This family of over 600 enzymes catalyzes diverse radical chemistry on a vast array of substrates. For an exemplary review, see Layer et al. (Curr. Opin. Chem. Biol. 8:468-76, 2004). Particular examples include, but are not limited to: biotin synthase, lipoate synthase,

GcpE protein, LytB protein, and aminomutases that interconverts alpha and beta amino acids (such as alanine 2,3-aminomutase, arginine 2,3-aminomutase, and lysine 2,3-aminomutase).

Radical S-adenosyl methionine enzyme (radical SAM) product: An agent formed by an enzyme with a radical SAM catalytic mechanism, following reductive activation of the radical SAM enzyme. For example, the radical SAM enzyme product of biotin synthase is biotin, of lipoate synthase is lipoic acid, of alanine 2,3-aminomutase is beta-alanine, of arginine 2,3-aminomutase is beta-arginine, of lysine 2,3-aminomutase is beta-lysine, of GcpE is HMBPP, and of LytB is IPP and DMAPP.

Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring, has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, or both. This artificial combination can be achieved, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules or proteins, such as genetic engineering techniques. Recombinant is also used to describe nucleic acid molecules that have been artificially manipulated, but contain the same regulatory sequences and coding regions that are found in the organism from which the nucleic acid was isolated.

Transformed cell: A cell into which a nucleic acid molecule has been introduced, for example by molecular biology techniques. Transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including, but not limited to, transfection with viral vectors, conjugation, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. In one example, includes culturing cells (such as bacterial cells) in growth medium and a temperature sufficient to allow the desired activity. In particular examples, the desired activity is the production of a radical SAM enzyme product (or downstream product thereof, such as 3 -HP, pantothenate, or derivatives thereof) by the cell. Vector: A nucleic acid molecule as introduced into a cell, thereby producing a transformed cell. A vector can include nucleic acid sequences that permit it to replicate in the cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art.

Methods of Increasing the Activity of a Radical SAM Enzyme

Methods are disclosed for increasing the activity of at least one radical SAM enzyme, such as increasing the activity of 1, 2, 3, 4, 5 or more radical SAM enzymes in a cell. The cell can be a prokaryotic (such as a bacterial cell) or eukaryotic cell (such as a yeast, fungi, or plant cell). Particular examples of radical SAM enzymes include, but are not limited to: biotin synthase, lipoate synthase, pyruvate formate-lyase, benzylsuccinate synthase, spore photoproduct lyase, GcpE, LytB, and aminomutases that interconvert alpha and beta amino acids. Non-limiting examples of aminomutases that interconvert alpha and beta amino acids include alanine 2,3-aminomutase, arginine 2,3-aminomutase, and lysine 2,3-aminomutase. Each radical SAM enzyme catalyzes radical chemistry on a particular substrate, thereby generating a radical SAM enzyme product. For example, biotin synthase catalyzes the reaction of dethiobiotin to biotin, by inserting a sulfur atom into dethiobiotin. Lipoate synthase catalyzes the reaction of octanoic to lipoic acid by inserting two sulfur atoms into octanoic acid. Aminomutases interconvert alpha and beta amino acids. For example, alanine 2,3 aminomutase catalyzes the reaction of alpha-alanine to beta-alanine, arginine 2,3- aminomutase catalyzes the reaction of alpha-arginine to beta-arginine, and lysine 2,3- aminomutase catalyzes the reaction of alpha-lysine to beta-lysine. GcpE catalyzes the reaction of 2-C-methyl-D-erythritol cyclopyrophosphate into HMBPP. LytB catalyzes the reaction of HMBPP to IPP and DMAPP. In particular examples, the method includes increasing the activity of flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, wherein such an increase increases the activity of a radical SAM enzyme in the cell. Methods of increasing the activity flavodoxin, ferredoxin, or flavodoxin reductase include, but are not limited to, increasing expression of a nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof in a cell, wherein such increased expression results in increased production of a flavodoxin, ferredoxin, or flavodoxin reductase protein in the cell, or increasing the activity of a flavodoxin protein, ferredoxin protein, flavodoxin reductase protein, or combination thereof, in the cell. For example, the method can include overexpressing one or more of axιfdx,fldA or fpr gene. Therefore, the method can include increasing expression of one of flavodoxin, ferredoxin, or flavodoxin reductase, or increasing expression of two of these, such as both flavodoxin and ferredoxin, both flavodoxin and flavodoxin reductase, or both ferredoxin and flavodoxin reductase, or increasing expression of all three of these.

The radical SAM en2yme in the cell can be an endogenous or exogenous radical SAM enzyme. For example, an exogenous radical SAM enzyme can be expressed in the cell by an exogenous nucleic acid encoding the radical SAM enzyme, such as a plasmid or vector containing a promoter operably linked to the coding sequence for one or more radical SAM enzymes. In a particular example, the radical SAM enzyme coding sequence (such as alanine 2,3-aminomutase, biotin synthase (bioB) or lipoate synthase (HpA) gene or cDNA) is part of an operon that also includes one or more of a flavodoxin, ferredoxin, or flavodoxin reductase cDNA or other coding sequence (such as an fdx,fldA, or fpr gene). The expression of the radical SAM enzyme sequences and the one or more of flavodoxin, ferredoxin, or flavodoxin reductase sequences, can be induced simultaneously, for example from a single promoter. In particular examples, increasing the activity (such as the expression) of flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, results in an increase of radical SAM enzyme activity in the cell by at least 20%, such as at least 30%, at least 40%, at least 50%, or at least 75%. The increase in radical SAM enzyme activity can be relative to a cell of the same type that does not have increased flavodoxin, ferredoxin, or flavodoxin reductase expression, such as a cell with native flavodoxin, ferredoxin, or flavodoxin reductase expression. For example, the increase can be relative to a reference value of radical SAM enzyme activity expected when native expression of one or more of flavodoxin, ferredoxin, flavodoxin reductase is present. In another example, the increase can be relative to an experimental sample containing native expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase. Methods for measuring the activity of a radical SAM enzyme are known in the art, and particular non-limiting examples are disclosed herein. In one particular example, the activity of a radical SAM enzyme is determined by measuring an amount of radical SAM enzyme product produced (for example using HPLC or an immunoassay, such as ELISA). Increasing expression of flavodoxin, ferredoxin, orflavodoxin reductase

Methods of increasing expression of a nucleic acid molecule and its corresponding protein are known in the art. Although particular methods are disclosed, the disclosure is not limited to such methods. In one example, overexpression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase is achieved by manipulating the growth conditions of the cells, thereby inducing expression of endogenous flavodoxin, ferredoxin, or flavodoxin reductase. For example, steady state levels of flavodoxin reductase in E. coli cells increased 20-fold upon exposure to methyl viologen, such as 0.01 mM - 1 mM viologen, for example 0.1 mM viologen (Liochev et al, Proc. Natl. Acad. Sci. USA 91: 1328-31, 1994). In another example, the method includes expressing one or more of flavodoxin, ferredoxin, and flavodoxin reductase from an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter. Methods of replacing an endogenous promoter with an exogenous promoter are known, and a particular example is provided in Example 4. Alternatively or in addition, the method can include expressing one or more of flavodoxin, ferredoxin, and flavodoxin reductase from an exogenous nucleic acid molecule, such as a plasmid or vector. In one example, the method includes introducing into the cell one or more plasmids or vectors that include the coding sequence flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, using standard transformation or transfection techniques. The vector can include a promoter operably linked to the flavodoxin, ferredoxin, or flavodoxin reductase sequence, to allow expression of flavodoxin, ferredoxin, or flavodoxin reductase. Such a vector could be co-transformed into a cell (such as E. coli) with another vector that includes a radical SAM enzyme coding sequence, such as an aminomutase (for example alanine 2,3-aminomutase), biotin synthase, pyruvate formate- lyase activator, benzylsuccinate synthase, spore photoproduct lyase, GcpE, LytB, or lipoate synthase.

In particular examples, expression of flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, is increased by at least 50%, such as at least 60%, at least 75%, or at least 90%. Such expression can be relative to an amount of expression in the same cell type in the absence of the conditions used to overexpress flavodoxin, ferredoxin, or flavodoxin reductase (such as a reference or experimental value). Levels of expression activity can be measured by methods known in the art, such as by RNA hybridization (Northern blot) or immunological analytical methods (such as ELISA). Alanine 2,3-aminomutase and 3-HP pathway

One enzyme in the beta-alanine pathway for 3-HP production is alanine 2,3- aminomutase, which interconverts alpha and beta amino acids. This aminomutase was derived from lysine 2,3-aminomutase by mutagenesis (for example see PCT/US2003/001635 and PCT/US2004/024686), and belongs to the class of enzymes known as "radical SAM" enzymes because the catalytic cycle utilizes an organic radical derived from enzyme-bound S-adenosyl methionine (SAM). The adenosyl radical is generated by a 4Fe-4S cluster within the enzyme, which in turn is activated by reduction from the nominally +2 state to +1. The redox potential for this reduction is approximately - 420 raV. Thus, the first step towards generating the catalytic radical likely requires an interaction between the enzyme and a highly electronegative reductant.

Application to other enzymes and biosynthetic pathways

Biotin synthase (encoded by the bioB gene) and lipoate synthase (lipA) are enzymes found in E. coli, as well as many other organisms, that are involved in the biosynthesis of biotin and lipoic acid, respectively. Biotin synthase and lipoate synthase are also members of the radical SAM superfamily of enzymes that share the requirements of SAM and iron- sulfur cluster(s) for catalytic activity. The 4Fe-4S cluster is reduced and then reductively cleaves SAM yielding L-methionine and a 5 '-deoxy adenosyl radical which subsequently initiates the reaction.

(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (encoded by the GcpE gene) and LytB are enzymes found in E. coli, as well as many other organisms, that are involved in the biosynthesis of isoprenoids. GcpE and LytB are also members of the radical SAM superfamily of enzymes that share the requirements of SAM and iron-sulfur cluster(s) for catalytic activity.

Based on the results presented herein, it is proposed that flavodoxin, ferredoxin, or flavodoxin reductase (or combinations thereof) are involved in the reductive activation of these enzymes, and that overexpression of one or more of these enzymes, congruently with bioB, HpA, GcpE, or LytB encoding sequences (for example in E. coli) will increase the yield of the respective products biotin, lipoic acid, or isoprenoids in vivo.

Increasing Production of a Radical SAM Enzyme Product

The present disclosure also provides methods of increasing the production of one or more radical SAM enzyme products. Such methods can be performed in vivo, in vitro, or combinations thereof. For example, the method can include culturing a cell having increased flavodoxin, ferredoxin, or flavodoxin reductase expression and increased radical SAM enzyme activity under conditions sufficient for the cell to make the radical SAM enzyme product, such as beta-alanine from alpha-alanine via alanine 2,3-aminomutase. For methods involving in vivo steps, the cells can be isolated cultured cells or whole organisms such as transgenic plants, or single-celled organisms such as yeast and bacteria (for example Lactobacillus, Lactococcus, Bacillus, and Escherichia cells). Such cells are referred to as production cells. Products produced by these production cells can be organic products such as beta-alanine, biotin, lipoic acid, carotenoids, or terpenoids.

For example, increasing the activity of a radical SAM enzyme in the cell can increase the production or yield of a radical SAM enzyme product, such as an increase of at least 10%, such as at least 15%, at least 20%, at least 30%, or at least 50%. The increased production of a radical SAM enzyme product can be relative to a cell of the same type that does not have increased flavodoxin, ferredoxin, or flavodoxin reductase expression, such as a cell with native flavodoxin, ferredoxin, or flavodoxin reductase expression. For example, the increase can be relative to a reference value of radical SAM enzyme product expected when native expression of one or more of flavodoxin, ferredoxin, flavodoxin reductase is present. In another example, the increase can be relative to an experimental sample containing native expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase. Methods for measuring a radical SAM enzyme product are known in the art, and particular non-limiting examples are disclosed herein.

Particular examples of radical SAM enzyme products include, but are not limited to, a beta amino acid (such as beta-alanine from alanine 2,3-aminomutase, beta-arginine from arginine 2,3-aminomutase, and beta-lysine from lysine 2,3-aminomutase), biotin (from biotin synthase), lipoic acid (from lipoate synthase), and isoprenoids, carotenoids and terpenoids (from GcpE and LytB).

In one example, a cell having increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase can increase alanine 2,3-aminomutase activity (or other aminomutase), thereby increasing production of beta-alanine (or the appropriate beta amino acid) by at least 10%, such as at least 20%, at least 25%, or at least 30%, as compared to an amount of beta-alanine (or the appropriate beta amino acid) produced in the absence of increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase. For example, cultures of a cell having increased flavodoxin expression (thereby increasing alanine 2,3-aminomutase activity) produced 11.4 μM/OD₆oo beta-alanine as compared to a cell not having increased alanine 2,3-aminomutase activity that produced 8.1 μM/OD^oo beta-alanine in 25 hours under anaerobic conditions, while in aerobic conditions the cell having increased alanine 2,3-aminomutase activity produced 4.1 μM/ODβoo beta-alanine as compared to a cell not having increased alanine 2,3-aminomutase activity that produced 2.7 μM/OD₆oo (see Table 2). OD₆oo is the optical density (lcm pathlength) of the cultures at 600 nra, In another example, a cell having increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase can increase biotin synthase or lipoate synthase activity, thereby increasing production of biotin or lipoic acid, respectively, by at least 10%, such as at least 20%, at least 25%, or at least 30% , as compared to an amount of biotin or lipoic acid, respectively, produced in the absence of increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase.

In one example, a cell having increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase can increase isoprenoid biosynthesis, thereby increasing production of isoprenoid, carotenoids, or terpenoids by at least 10%, such as at least 20%, at least 25%, or at least 30%, as compared to such an amount produced in the absence of increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase.

In some examples where the method includes increasing the production of a radical SAM enzyme product, the cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, further includes one or more exogenous nucleic acid molecules (such as a vector or operon) that includes the coding sequence of one or more radical SAM enzyme sequences (such as a cDNA or gene sequence). For example, the cell can include one or more of the following radical SAM enzyme activities, thereby permitting production of the appropriate radical SAM enzyme product: alanine 2,3-aminomutase, arginine 2,3-aminomutase, lysine 2,3-aminomutase, biotin synthase, lipoate synthase, GcpE or LytB.

Such enzymes can be native to the cell, or supplied to the cell using exogenous nucleic acid molecules. For example, the cell can include at least one exogenous nucleic acid molecule that encodes an alanine 2,3-aminomutase capable of producing beta-alanine from alpha-alanine. In another example, the cell includes at least one exogenous nucleic acid molecule that encodes an arginine 2,3-aminomutase capable of producing beta-arginine from alpha-arginine. In yet another example, the cell includes at least one exogenous nucleic acid molecule that encodes a lysine 2,3-aminomutase capable of producing beta- lysine from alpha-lysine. In yet another example, the cell includes at least one exogenous nucleic acid molecule that encodes a biotin synthase that can produce biotin from dethiobiotin. In yet another example, the cell includes at least one exogenous nucleic acid molecule that encodes a lipoate synthase that can produce lipoic acid from octanoic acid. In yet another example, the cell includes at least one exogenous nucleic acid molecule that encodes a GcpE that can produce HMBPP from 2-C-methyl-D-erythritol cyclopyrophosphate. In yet another example, the cell includes at least one exogenous nucleic acid molecule that encodes a LytB that can produce DPP and DMAPP from HMBPP.

Increasing Production of a Radical SAM Enzyme Downstream Organic Chemical

The present disclosure also provides methods of increasing the production of one or more radical SAM enzyme downstream organic chemicals. Such methods can be performed in vivo (within a cell), in vitro (outside of a cell), or combinations thereof. For example, the disclosed methods can also be used to increase expression of a radical SAM enzyme downstream organic chemical. For example, 3-hydroxypropionic acid (3-HP), pantothenate, cryptophycin, and derivatives thereof can be produced in a cell from the radical SAM enzyme product beta-alanine; streptothricin, viomycin, racemomycin, and nourseothricins can be produced in a cell from the radical SAM enzyme product beta-lysine; blasticidin S can be produced in a cell from the radical SAM enzyme product beta-arginine; and carotenoids (such as lycopene, lutein, beta-carotene, zeazanthin) or terpenoids (such as artemisinin) can be produced in a cell from the radical SAM enzyme product HMBPP, IPP and DMAPP. The compound 3 -HP can be produced by biocatalysis from beta-alanine. The resulting 3 -HP can be used in the nutritional industry as a food, feed additive or preservative. In addition, 3-HP can be used to produce derivatives thereof, such as those disclosed herein. Several methods of producing 3-HP from beta-alanine using the disclosed cells are disclosed. In one example, the cell is transfected with one or more enzymes needed to convert 3-HP from beta-alanine. In another example, the method includes purifying beta- alanine (or other intermediate) from the cell, then contacting the beta-alanine (or other intermediate) with peptides needed to convert 3-HP from beta-alanine.

In some examples where the method includes increasing the production of a radical SAM enzyme downstream organic chemical, the cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, further includes one or more exogenous nucleic acid molecules (such as a vector or operon) that include the coding sequence of one or more enzyme sequences (such as a cDNA or gene sequence) needed to produce the radical SAM enzyme downstream chemical. The sequences of such enzymes are publicly available. Although particular methods are provided for producing beta-alanine downstream chemicals, one skilled in the art will appreciate that similar methods (with the appropriate enzymes), can be used to produce downstream organic chemicals from other radical SAM enzymes. For example, using the methods disclosed herein, the production of beta-alanine downstream chemicals (such as 3-HP, pantothenate, and derivatives thereof) is enabled (see FIGS. 1 and 2). Each step provided in the pathways depicted in FIGS. 1 and 2 can be performed within a cell (in vivo) or outside a cell (in vitro, such as in a container or column). Additionally, the organic compound products can be generated through a combination of in vivo synthesis and in vitro synthesis. Moreover, the in vitro synthesis step, or steps, can be via chemical reaction or enzymatic reaction.

For example, the cells disclosed herein can be used to perform the steps provided in FIGS. 1 and 2, or an extract containing peptides having the indicated enzymatic activities can be used to perform the steps provided in FIGS. 1 and 2. In addition, chemical treatments can be used to perform the conversions provided in FIGS. 1 and 2. For example, acrylyl-CoA can be converted into acrylate by hydrolysis. Other chemical treatments include, without limitation, trans esterification to convert acrylate into an acrylate ester.

In addition, the compounds produced from any of the steps provided in FIGS. 1 and 2 can be chemically converted into other organic compounds. For example, 3-HP can be hydrogenated to form 1,3-propanediol, a polyester monomer. Hydrogenating an organic acid such as 3-HP can be performed using any method such as those used to hydrogenate succinic acid or lactic acid. For example, 3-HP can be hydrogenated using a metal catalyst. In another example, 3-HP can be dehydrated to form acrylic acid. Any method can be used to perform a dehydration reaction. For example, 3-HP can be heated in the presence of a catalyst (such as a metal or mineral acid catalyst) to form acrylic acid. 1,3-propanediol also can be created using polypeptides having oxidoreductase activity (such as enzymes in the 1.1.1.- class of enzymes) in vitro or in vivo.

Producing 3-HP and derivatives thereof

Methods and materials related to producing 3-HP from beta-alanine, as well as derivatives thereof such as 1,3-propanediol, esters of 3-HP, acrylate or acrylic acid, polymerized acrylate, esters of acrylate, polymerized 3-HP, co-polymers of 3-HP and other compounds such as butyrates, valerates, are disclosed. Several metabolic pathways can be used to produce organic compounds from beta-alanine that has been produced from alpha- alanine (FIG. 1). As shown in FIG. 1, beta-alanine can be converted into beta-alanyl-CoA by a peptide having CoA transferase activity (EC 2.8.3.1) or CoA synthase activity (E.G. 6.2.1.-). Beta-alanine can be produced from alpha-alanine by using a cell transformed with recombinant alanine 2,3-aminomutase or from aspartate by aspartate decarboxylase. Beta- alanyl-CoA can then be converted into acrylyl-CoA by a peptide having beta-alanyl-CoA ammonia lyase activity (EC 4.3.1.6). Acrylyl-CoA can then be converted into 3- hydroxypropionyl-CoA (3 -HP-CoA) by a peptide having 3 -HP-CoA dehydratase activity (EC 4.2.1.-). 3-HP-CoA can then be converted into 3-HP through several enzymes, including, but not limited to: a peptide having CoA transferase activity (EC 2.8.3.1), or a peptide having 3-hydroxypropionyl-CoA hydrolase activity (EC 3.1.2.-) which may be an additional activity of a peptide having 3-hydroxyisobutryl-CoA hydrolase activity (EC 3.1.2.4).

In addition, as shown in FIG. 1, 3-HP can be made from beta-alanine by a peptide having beta-aIanine-2-oxoglutarate aminotransferase activity which generates malonic semialdehyde from beta-alanine, and which may be an additional activity of a peptide characterized in databases as having 4-aminobutyrate aminotransferase activity. The malonic semialdehyde can be converted into 3-HP with a peptide having 3-HP dehydrogenase activity (EC 1.1.1.59) which may be an additional activity of a peptide characterized in databases as having 3 -hydroxy isobutyrate dehydrogenase activity (EC 1.1.1.31).

Derivatives of 3-HP can be made from beta-alanine as shown in FIG. 1. The resulting 3 -HP-CoA can be converted into polymerized 3-HP by a peptide having poly hydroxyacid synthase activity (EC 2.3.1.-). Alternatively or in addition, 3-HP-CoA can be converted into 1,3 -propanediol by a peptide having acetylating oxidoreductase activity. The resulting acrylyl-CoA can be converted into polymerized acrylate by a peptide having poly hydroxyacid synthase activity (EC 2.3.1.-). Alternatively or in addition, acrylyl-CoA can be converted into acrylate by a peptide having CoA transferase activity or CoA hydrolase activity; and the resulting acrylate can be converted into an ester of acrylate by a peptide having lipase or esterase activity. The resulting 3-HP can be converted into an ester of 3-HP by a peptide having lipase or esterase activity (EC 3.1.1.-). Alternatively or in addition, 1,3 -propanediol can be created from 3-HP, by a combination of a peptide having aldehyde dehydrogenase activity and a polypeptide having alcohol dehydrogenase activity.

Therefore, 3-HP or a derivative thereof can be produced in the presence of the appropriate enzymes. For example, for producing 3-HP (or a derivative thereof) from beta- alanine, the cell having increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, and increased radical SAM enzyme activity (specifically increased alanine 2,3-aminomutase activity), can further include CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and 3- hydroxypropionyl-CoA hydrolase activity. In another example, the cell can further include beta alanine-2-oxoglutarate aminotransferase activity and 3 -HP dehydrogenase activity. To produce a derivative of 3-HP from 3-HP, the cell can further include these enzyme activities, in addition to others. For example, for producing an ester of 3-HP (such as methyl acrylate, ethyl acrylate, propyl acrylate, or butyl acrylate, for example methyl 3- hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3- hydroxypropionate, or 2-ethylhexyl 3-hydroxypropionate) from 3-HP, the cell can further include lipase or esterase activity. For producing polymerized 3-HP from 3-HP, the cell can further include esterase activity. For producing 1,3 propanediol from 3-HP, the cell can further include aldehyde dehydrogenase activity (EC 1.2.1.3) and alcohol dehydrogenase activity (EC 1.1.1.1).

Polymerized 3-HP and 1,3 propanediol can also be produced from 3-HP-CoA, as an alternative (or in addition to) producing them from 3-HP. For producing polymerized 3-HP from 3 -HP-CoA, the cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, can further include alanine 2,3- aminomutase activity, CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, and poly hydroxyacid synthase activity. For producing 1,3 propanediol from 3-HP-CoA, the cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, can further include alanine 2,3-aminomutase activity, CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and acetylating aldehyde oxidoreductase and alcohol oxidoreductase activities (such as enzymes from the 1.1.1.1 or 1.2.1.10 class of enzymes).

Producing pantothenate and derivatives thereof Methods and materials related to producing pantothenate from beta-alanine, as well as derivatives thereof such as CoA, are disclosed. Metabolic pathways that can be used to produce pantothenate and derivatives thereof are shown in FIG. 2.

As shown in FIG. 2, pantothenate can be made from beta-alanine by one or more peptides having alpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.11), alpha- ketopantoate reductase (E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1) activities, which converts beta-alanine to pantothenate.

Derivatives of pantothenate can be made from beta-alanine as follows. The resulting pantothenate can be converted into CoA by polypeptides having pantothenate kinase (E.C. 2.7.1.33), 4'-phosphopantethenoyl-l -cysteine synthetase (E.C. 6.3.2.5), 4'- phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36), ATP:4'-phosphopantetheine adenyltransferase (E.C. 2.7.7.3), and dephospho-CoA kinase (E.C. 2.7.1.24) activities.

Pantothenate, a vitamin essential to many animals for growth and health, is involved in fatty acid synthesis and degradation. Deficiency of the vitamin results in generalized malaise clinically. Therefore, pantothenate produced using the methods disclosed herein can be administered to a subject having a pantothenic deficiency, at a therapeutically effective dose. Cells that produce pantothenate, and methods of producing pantothenate from beta-alanine using the disclosed cells, are disclosed.

For example, for producing pantothenate (or a derivative thereof) from beta-alanine, the cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, can further include increased alanine 2,3- aminomutase activity, as well as alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity.

Producing radical SAM enzyme downstream organic chemicals using both in vivo and in vitro methods

In particular examples, methods for producing a radical SAM enzyme downstream organic chemical are performed using a combination of in vivo and in vitro methods. For example, to generate organic chemicals from a radical SAM enzyme product, the radical SAM enzyme product can be generated in vivo in a cell (which in some examples is subsequently isolated or purified from the cell or culture medium), and the radical SAM enzyme product contacted with other enzymes or chemicals in vitro to generate the desired downstream chemical. Although particular examples are provided for particular radical SAM enzyme products and downstream chemicals, the disclosure is not limited to such examples, as one skilled in the art can determine how to achieve similar results for other such products and chemicals, using the appropriate enzymes.

For example, beta-alanine can be generated in vivo in a cell that has increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, and has alanine 2,3-aminomutase activity, and subsequently purified using standard methods known in the art. The beta-alanine is then contacted or incubated with the appropriate enzymes to generate the desired radical SAM enzyme downstream organic chemical in vitro. For example, to generate 3-HP from beta-alanine, beta-alanine can be contacted with a peptide having CoA transferase activity to form beta-alanyl-CoA, and the beta-alanine CoA contacted with a peptide having beta-alanyl-CoA ammonia lyase activity to form acrylyl-CoA, contacting the acrylyl-CoA with a peptide having 3HP-CoA dehydratase activity to form 3 -HP-CoA, and contacting 3 -HP-CoA with a peptide having CoA transferase activity or 3-hydroxypropionyl-CoA hydrolase activity to make 3-HP. In another example, 3-HP is generated from beta-alanine, by contacting beta-alanine with a peptide having beta-alanine - 2-oxoglutarate aminotransferase activity to form malonic semialdehyde, and contacting the malonic semialdehyde with a peptide having 3-HP dehydrogenase activity to make 3-HP. Derivatives of 3-HP, such as an ester of 3-HP, polymerized 3-HP, or 1,3 propanediol, can be generated from 3-HP in vitro by incubating 3- HP in the presence of the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, all of these enzymes are incubated with beta-alanine and the reactions allowed to proceed in vitro.

For example, to generate 3-HP-CoA from beta-alanine, beta-alanine can be contacted with a peptide having CoA transferase activity to form beta-alanyl-CoA, and the beta-alanyl CoA contacted with a peptide having beta-alanyl-CoA ammonia lyase activity to form acrylyl-CoA, and contacting the acrylyl-CoA with a peptide having 3HP-CoA dehydratase activity to form 3-HP-CoA. Derivatives of 3-HP-CoA, such as polymerized 3- HP or 1,3 propanediol can be generated from 3-HP -CoA in vitro by incubating 3-HP-CoA in the presence of the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, all of these enzymes are incubated with beta-alanine and the reactions allowed to proceed in vitro. For example, to generate acrylyl-CoA from beta-alanine, beta-alanine can be contacted with a peptide having CoA transferase activity to form beta-alanyl-CoA, and the beta-alanyl CoA contacted with a peptide having beta-alanyl-CoA ammonia lyase activity to form acrylyl-CoA. Derivatives of acrylyl-CoA, such as polymerized acrylate, or acrylate esters can be generated from acrylyl-CoA in vitro by incubating acrylyl-CoA in the presence of the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, all of these enzymes are incubated with beta-alanine and the reactions allowed to proceed in vitro. For example, to generate pantothenate from beta-alanine, beta-alanine can be contacted with a peptide having alpha-ketopantoate hydroxymethyltransferase activity, with a peptide having alpha-ketopantoate reductase activity, and with a peptide having pantothenate synthase activity to make pantothenate. To generate CoA from the pantothenate, the method can further include contacting the pantothenate with a peptide having pantothenate kinase (E.C. 2.7.1.33) activity, a peptide having 4'- phosphopantethenoyl-1 -cysteine synthetase (E.C. 6.3.2.5) activity, a peptide having 4'- phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36) activity, a peptide having ATP:4'-phosphopantetheine adenyltransferase (E.C. 2.7.7.3) activity, and a peptide having dephospho-CoA kinase (E.C. 2.7.1.24) activity. In one example, all of these enzymes are incubated with beta-alanine and the reactions allowed to proceed in vitro.

In one example, to generate DPP and DMAPP from HMBPP, 2-C-methyl-D- erythritol cyclopyrophosphate can be contacted with a peptide having GcpE activity to make HMBPP. To generate DPP and DMAPP from the HMBPP, the method can further include contacting the HMBPP with a peptide having LytB activity. In one example, all of these enzymes are incubated with one or more of flavodoxin, ferredoxin, or flavodoxin reductase and the reactions allowed to proceed in vitro.

Production of organic chemicals in vitro Purified peptides having the desired enzymatic activity can be used to produce pantothenate, 3 -HP, or derivatives thereof such as CoA, and organic compounds such as 1,3 -propanediol, acrylic acid, polymerized aery late, esters of acrylate, esters of 3 -HP, and polymerized 3 -HP. For example, a preparation including a substantially pure peptide having 3-hydroxypropionyl-CoA dehydratase activity can be used to catalyze the formation of 3- HP-CoA, a precursor to 3-HP.

Further, cell-free extracts containing a peptide having the desired enzymatic activity can be used alone or in combination with purified peptides or cells to produce pantothenate, 3-HP, or derivatives thereof. For example, a cell-free extract that includes a peptide having CoA transferase activity can be used to form beta-alanyl-CoA from beta-alanine, and peptides having the enzymatic activities needed to catalyze the reactions needed to form 3- HP from beta-alanyl-CoA can be used to produce 3-HP. In another example, a cell-free extract which includes alpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.11), alpha- ketopantoate reductase (E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1) can be used to form pantothenate from beta-alanine. Any method can be used to produce a cell- free extract. For example, osmotic shock, sonication, or a repeated freeze-thaw cycle followed by filtration or centrifugation can be used to produce a cell-free extract from intact cells.

A purified peptide or cell-free extract can be used to produce 3-HP that is, in turn, treated chemically to produce another compound. For example, a chemical process can be used to modify 3-HP into a derivative such as 1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate, esters of 3-HP, and polymerized 3-HP. In one example, 3-HP can be converted into a 3-HP ester by trans esterification, or into 1,3-propanediol by hydrogenation. Hydrogenating an organic acid such as 3-HP can be performed using any method such as those used to hydrogenate succinic acid or lactic acid. For example, 3-HP can be hydrogenated using a metal catalyst. In another example, 3-HP can be dehydrated to form acrylic acid. Any method can be used to perform a dehydration reaction. For example, 3-HP can be heated in the presence of a catalyst (such as a metal or mineral acid catalyst) to form acrylic acid.

Similarly, a purified peptide or cell-free extract can be used to produce pantothenate that is, in turn, treated chemically to produce another compound. For example, following the production of pantothenate in vitro, a chemical process can be used to modify pantothenate into a derivative such as CoA.

Producing radical SAM enzyme downstream chemicals using in vivo methods The cells disclosed herein can be used to produce beta-alanine, pantothenate and 3-

HP, as well as derivatives thereof such as CoA, and organic compounds such as 1,3- propanediol, acrylic acid, polymerized acrylate, esters of acrylate, esters of 3-HP, and polymerized 3-HP. In addition, methods are provided for producing a radical SAM enzyme downstream chemical in vivo. For example, the radical SAM enzyme product and the radical SAM enzyme downstream chemical can be produced in a cell. The cells can be isolated cultured cells or whole organisms such as transgenic plants, or single-celled organisms such as yeast and bacteria (for example Lactobacillus, Lactococcus, Bacillus, and Escherichia cells). Such cells are referred to as production cells. Products produced by these production cells can be organic products such as 3-HP, pantothenate, and derivatives thereof such as organic acids, polyols (such as 1,3-propanediol), and coenzyme A (CoA). In particular examples, the method includes culturing the cell under conditions sufficient for the desired product to be produced. The desired product can be extracted from the cells, or can be recovered from the extracellular medium if the product is secreted by the cell. Although particular examples are provided for particular radical SAM enzyme products and downstream organic chemicals, the disclosure is not limited to such examples, as one skilled in the art can determine how to achieve similar results for other products and chemicals, using the appropriate enzymes. In addition, one skilled in the art will appreciate that the exogenous nucleic acid molecules encoding the appropriate enzymes can be part of one or more vectors, and can include other nucleic acid sequences, such as ferredoxin, flavodoxin, or flavodoxin reductase.

For example, a cell having increased activity (such as increased expression) of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, and has increased alanine 2,3- aminomutase activity, can be transfected with one or more nucleic acid molecules that can express the appropriate enzymes to generate the desired radical SAM enzyme downstream chemical. For example, to generate 3 -HP from beta-alanine, the cell can be transfected with one or more nucleic acid molecules that can express a protein having CoA transferase activity, one or more nucleic acid molecules that can express a protein having beta-alanyl- CoA ammonia lyase activity, one or more nucleic acid molecules that can express a protein having 3HP-CoA dehydratase activity, and one or more nucleic acid molecules that can express a protein having CoA transferase activity or 3-hydroxypropionyl-CoA hydrolase activity. The cell is cultured under conditions sufficient to make 3-HP. In another example, 3 -HP is generated from beta-alanine by transfecting the cell with one or more nucleic acid . molecules that can express a protein having peptide having beta-alanine-2-oxoglutarate aminotransferase activity, and with one or more nucleic acid molecules that can express a protein having 3-HP dehydrogenase activity. The cell is cultured under conditions sufficient to make 3-HP. Derivatives of 3-HP, such as an ester of 3-HP, polymerized 3-HP, or 1,3 propanediol, can be generated from 3-HP in vivo by using cells that express the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, these enzymes are supplied to the cell by transfecting the cell with one or more nucleic acid molecules that can express a protein having the necessary enzyme activity.

For example, to generate 3 -HP-CoA from beta-alanine, the cell can be transfected with one or more nucleic acid molecules that can express a protein having CoA transferase activity or CoA synthetase activity, with one or more nucleic acid molecules that can express a protein having beta-alanyl-CoA ammonia lyase activity, and with one or more nucleic acid molecules that can express a protein having 3HP-CoA dehydratase activity. The cell is cultured under conditions sufficient to make 3-HP-CoA. Derivatives of 3-HP- CoA, such as polymerized 3-HP or 1,3 propanediol can be generated from 3-HP-CoA in vivo by using cells that express the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, these enzymes are supplied to the cell by transfecting the cell with one or more nucleic acid molecules that can express a protein having the necessary enzyme activity.

For example, to generate acrylyl-CoA from beta-alaπine, the cell can be transfected with one or more nucleic acid molecules that can express a protein having CoA transferase activity, and with one or more nucleic acid molecules that can express a protein having beta- alanyl-CoA ammonia lyase activity. The cell is cultured under conditions sufficient to make acrylyl-CoA. Derivatives of acrylyl-CoA, such as polymerized acrylate, or acrylate esters can be generated from acrylyl-CoA in vivo by using cells that express the appropriate enzymes (see FIG. 1 and the present disclosure). In one example, these enzymes are supplied to the cell by transfecting the cell with one or more nucleic acid molecules that can express a protein having the necessary enzyme activity.

For example, to generate pantothenate from beta-alanine, the cell can be transfected with one or more nucleic acid molecules that can express a protein having alpha- ketopantoate hydroxymethyltransferase activity, with one or more nucleic acid molecules that can express a protein having alpha-ketopantoate reductase activity, and with one or more nucleic acid molecules that can express a protein having pantothenate synthase activity to make pantothenate. To generate CoA from the pantothenate, the method can further include transfecting the cell with one or more nucleic acid molecules that can express a protein having pantothenate kinase (E.G. 2.7.1.33) activity, a peptide having 4'- phosphopantethenoyl-1 -cysteine synthetase (E.C. 6.3.2.5) activity, one or more nucleic acid molecules that can express a protein having 4'-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36) activity, a peptide having ATP:4'-phosphopantetheine adenyltransferase (E.C. 2.7.7.3) activity, and one or more nucleic acid molecules that can express a protein having dephospho-CoA kinase (E.C. 2.7.1.24) activity. The cell is cultured under conditions sufficient to make pantothenate or CoA. In one example, to generate BPP and DMAPP from HMBPP, the cell can be transfected with one or more nucleic acid molecules that can express a protein having GcpE activity, and with one or more nucleic acid molecules that can express a protein having LytB activity. The cell is cultured under conditions sufficient to make IPP and DMAPP, or products thereof such as carotenoids or terpenoids. In one example, these proteins are supplied to the cell by transfecting the cell with one or more nucleic acid molecules that can express a protein having the necessary activity.

Fermentation of Cells to Produce Organic Acids

As disclosed above, methods are provided for producing a radical SAM enzyme product, or a downstream chemical thereof such as carotenoids, terpenoids, pantothenate, 3- HP, or derivatives thereof, that include in vivo methods (for example alone or in combination with in viti'o methods). In vivo methods include culturing the cell (such as a microorganism) having the appropriate enzyme activities and increased expression of one or more of flavodoxin, flavodoxin reductase, or ferredoxin, in culture medium such that desired product is produced, In general, the culture media or culture conditions can be such that the cells grow to an adequate density and produce the product efficiently. For large- scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2^nd Edition, Editors: Demain and Davies, ASM Press; and Principles of Fermentation Technology, Stanbury and Whitaker, Pergamon).

Briefly, a tank (such as a 1 gallon, 5 gallon, 10 gallon, 50 gallon, 100 gallon, 200 gallon, 500 gallon, or more tank) containing appropriate culture medium with, for example, a glucose carbon source is inoculated with a particular production cell (such as microorganism). After inoculation, the cells are incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the cells can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank. For example, the first tank can contain medium with xylose, while the second tank contains medium with glucose.

Once transferred, the cells can be incubated to allow for the production of a radical SAM enzyme product, or downstream chemical thereof such as pantothenate, 3 -HP, or derivatives thereof. Once produced, any method can be used to isolate the formed product. For example, common separation techniques can be used to remove the biomass from the broth, and common isolation procedures (such as extraction, distillation, and ion-exchange procedures) can be used to obtain the radical SAM enzyme product, or downstream chemical thereof from the cell-free broth. Alternatively, the product can be isolated while it is being produced, or it can be isolated from the broth after the product production phase has been terminated. In some examples, the cells are isolated and the radical SAM enzyme product, or downstream chemical thereof, extracted from the cells.

Cells Having Increased Radical SAM Enzyme Activity

Transformed cells having increased radical SAM enzyme activity are provided. In one example, such cells have increased flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof. For example, the cell can have increased expression of one or more nucleic acid molecules that encode flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof. Such cells can produce a radical SAM enzyme product, such as a beta amino acid, and in particular examples can produce a downstream chemical thereof, such as 3 -HP.

Transformed cells having increased radical SAM enzyme activity can be eukaryotic or prokaryotic. A particular example of the disclosed cells were deposited as American Type Culture Collection No. PTA-6837 on July 6, 2005. For example, transformed production cells can be mammalian cells (such as human, murine, or bovine cells), plant cells (such as corn, wheat, rice, or soybean cells), fungal cells (such as Aspergillus or

Rhizopus cells), yeast cells, or bacterial cells (such as Lactobacillus, Lactococcus, Bacillus, Escherichia, Geobacillus, Corynebacterium, or Clostridium cells). In one example, a cell is a microorganism. The term "microorganism" refers to any microscopic organism including, but not limited to, bacteria, algae, fungi, and protozoa. Thus, E. coli, B. subtilis, B. licheniforrnis, S. cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, and Pichia pastoris are microorganisms and can be used as described herein. In another example, the cell is part of a larger organism, such as a plant, such as a transgenic plant. Examples of plants that can be used to make 3-HP, pantothenate, or other organic compounds from beta-alanine include, but are not limited to, genetically engineered plant crops such as corn, rice, wheat, and soybean. Therefore, also provided by the present disclosure are transgenic plants that include a cell having increased radical SAM enzyme activity. hi one example, the cell includes an exogenous promoter that controls expression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof. Examples of such promoters include the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter. Methods of introducing an exogenous promoter into a chromosome in the cell, for example by recombination, are known in the art. In another example, flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof is expressed from an exogenous plasmid or vector introduced into the cell. Flavodoxin, ferredoxin, and flavodoxin reductase nucleic acid sequences are publicly available, and methods of introducing such sequences into a cell are routine.

The increase in activity of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof can be any amount that increases the activity of a radical SAM enzyme. In particular examples, the activity of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, such as the expression or protein levels, is increased by at least 50% in the cell, such as at least 75%, at least 100% or at least 200%. Methods of determining such activities are known. For example, expression of fiavodoxin, ferredoxin, flavodoxin reductase nucleic acid molecules can be detected by Southern blotting, northern blotting, and RT-PCR, and flavodoxin, ferredoxin, flavodoxin reductase proteins can be detected by polyacrylamide gel electrophoresis, western blotting or flow cytometry. In some example, the activity of flavodoxin, ferredoxin, or flavodoxin reductase, is compared to a reference value, such as a control (for example a native amount of flavodoxin, ferredoxin, or flavodoxin reductase activity).

In particular examples, cells have an increase in radical SAM enzyme activity of at least 20%, such as at least 40%, or at least 50%. Such activity can be determined by measuring the production of a radical SAM enzyme product using methods known in the art. In some example, the radical SAM enzyme activity is compared to a reference value, such as a control (for example a native amount of radical SAM enzyme activity, such as in the absence of increased flavodoxin, ferredoxin, or flavodoxin reductase activity). The radical SAM enzyme can be endogenous to the cell, or can be expressed by an exogenous nucleic acid molecule that encodes the radical SAM enzyme. For example, the cell can be transformed with a radical SAM enzyme nucleic acid sequence that confers to the transformed cells radical SAM enzyme activity. Such an exogenous nucleic acid molecule can further include a promoter to drive expression of the radical SAM enzyme sequence. In particular examples, the exogenous nucleic acid molecule further includes one or more flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, nucleic acid molecules (such as a gene or cDNA sequence).

In addition, the cell can include one or more endogenous or exogenous nucleic acid molecules that permit production of a radical SAM enzyme products, as well as chemicals derived from those products. For example, radical SAM enzyme products can be used to catalyze the formation of organic compounds. In particular examples, the disclosed cells can be used to produce one or more of beta-alanine, beta-arginine, beta-lysine, biotin, lipoic acid, 3-HP, pantothenate, cryptophycin, CoA, polyols such as 1,3-propanediol, acrylic acid, polymerized acrylate, esters of acrylate, polymerized 3-HP, co-polymers of 3-HP, butyrates, valerates, esters of 3-HP, as well as streptothricin, viomycin, racemomycin, and nourseothricins (downstream compounds of beta-lysine), IPP, DMAPP, HMBPP, one or more carotenoids, one or more terpenoids (downstream compounds of GcpE and LytB), and blasticidin S (a downstream compound of beta-arginine). Methods for detecting specific enzymatic activities or the presence of particular radical SAM enzyme products (or a downstream chemical thereof) are well known, for example, the presence of an organic compound such as 3-HP can be determined as described in Sullivan and Clarke (J. Assoc. Offic. Agr. Chemists, 38:514-8, 1955).

In some examples, the radical SAM enzyme product (or downstream organic chemical thereof) is secreted from the cell, reducing or eliminating the need to disrupt cell membranes to retrieve the desired compound. In one example, the cell produces a radical SAM enzyme product (or downstream organic chemical thereof) with the concentration of the product(s) being at least 1 mg per L (such as at least 1 mg/L, at least 5 mg/L, at least 10 mg/L, at least 25 mg/L, at least 50 mg/L, at least 75 mg/L, at least 100 mg/L, or at least 200 mg/L). When determining the yield of a radical SAM enzyme product (or downstream organic chemical thereof) such as 3-HP, pantothenate, or derivatives thereof, for a particular cell, any method can be used. See, for example, Applied Environmental Microbiology 59(12):4261-5 (1993). A cell within the scope of the disclosure can utilize a variety of carbon sources. In another example, the radical SAM enzyme product (or downstream organic chemical thereof) is not secreted from the cell. In such examples, the cell membrane can be disrupted using methods known in the art to retrieve the organic compound.

The disclosed transformed cells can have increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, and increased radical SAM enzyme activity, and can therefore produce a radical SAM enzyme product. In particular examples, the activity of the radical SAM enzyme in the cell is increased by at least 20%, thereby increasing the production of a radical SAM enzyme product by the cell (for example by at least 20%, at least 40%, or at least 50%). In one example, the cell having increased expression of one or more of flavodoxin, ferredoxin, and flavodoxin reductase, includes increased biotin synthase activity, increased lipoate synthase activity, increased GcpE activity, increased LytB activity, or increased aminomutase activity that interconverts alpha and beta amino acids (such as increased alanine 2,3-aminomutase activity, increased arginine 2,3-aminomutase activity, or increased lysine 2,3-aminomutase activity). The radical SAM enzyme can be native to the cell, or can be supplied to the cell via one or more exogenous nucleic acid molecules using standard recombinant methods. In one example, the disclosed transformed cell includes increased lipoate synthase activity. In particular examples, such cells produce the radical SAM enzyme product lipoic acid. In another example, the disclosed transformed cell includes increased GcpE activity. In particular examples, such cells produce the radical SAM enzyme product HMBPP. In another example, the disclosed transformed cell includes increased LytB activity. In particular examples, such cells produce one or more of the radical SAM enzyme products IPP and DMAPP. In another example, the disclosed transformed cell includes increased biotin synthase. In particular examples, such cells produce the radical SAM enzyme product biotin. In another example, the disclosed transformed cell includes increased lysine 2,3-aminomutase. In particular examples, such cells produce the radical SAM enzyme product beta-lysine. In another example, the disclosed transformed cell includes increased alanine 2,3-aminomutase. In particular examples, such cells produce the radical SAM enzyme product beta-alanine. In another example, the disclosed transformed cell includes increased arginine 2,3-aminomutase. In particular examples, such cells produce the radical SAM enzyme product beta-arginine. The disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities. These other activities can permit production of radical SAM enzyme downstream chemicals, such as carotenoids, terpenoids, acrylyl-CoA, 3-HP-CoA, 3-HP, pantothenate, and derivates of these. For example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities such as one or more of CoA transferase activity, CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-hydroxypropionyl-CoA dehydratase activity, 3- hydroxypropionate dehydrogenase activity (EC 1.1.1.59), lipase activity or esterase activity (EC 3.1.1.-), aldehyde dehydrogenase activity (EC 1.2.1.3), alcohol dehydrogenase activity (EC 1.1.1.1), glutamate dehydrogenase activity, beta-alanine-2-oxoglutarate aminotransferase, acetylating aldehyde oxidoreductase and alcohol oxidoreductase activities (such as enzymes from the 1.1.1.1 and/or 1.2.1.10 class of enzymes), poly hydroxyacid synthase activity, alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, pantothenate synthase activity, pantothenate kinase activity (E.C. 2.7.1.33), 4'-phosphopantethenoyl-l -cysteine synthetase activity (E.C. 6.3.2.5), 4'- phosphopantothenoylcysteine decarboxylase activity (E.C. 4.1.1.36), ATP:4'- phosphopantetheine adenyltransferase activity (E.C. 2.7.7.3), or dephospho-CoA kinase (E.C. 2.7.1.24) activity. Such enzyme activities can be endogenous to the cell, or can be supplied to the cell via one or more exogenous nucleic acid molecules that encode one or more peptides having the desired enzyme activity.

In a particular example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities. For example, a cell having increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and arginine 2,3-aminomutase activity, can include additional enzyme activities needed to produce blasticidin S from beta-arginine , such as the activities encoded by the blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes (Cone et at, Chembiochem 4: 821-8, 2003). In another example, cell having increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and lysine 2,3-aminomutase activity, can include additional enzyme activities needed to produce streptothricin, viomycin, racemomycin, or nourseothricins from beta-lysine, such as the activities encoded by the nourserothricin biosynthesis genes of Streptomyces noursei (Grammel et al, Eur. J. Biochem. 269:347-57, 2002). In another example, cell having increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and GcpE and LytB activity, can include additional enzyme activities needed to produce one or more terpenoids or carotenoids.

For example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities that permit the production of acrylyl- CoA or derivatives thereof. In particular examples, such cells have alanine 2,3 aminomutase activity, CoA transferase activity or CoA synthetase activity, and beta-alanyl- CoA ammonia lyase activity, wherein the cell produces acrylyl-CoA. In one example, the cells can further include poly hydroxyacid synthase activity, wherein the cell produces polymerized acrylate. In another example, the cells further include lipase or esterase activity, wherein the cell produces an ester of acrylate (such as methyl acrylate, ethyl acrylate, propyl acrylate, and butyl acrylate).

For example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities that permit the production of 3-HP- CoA or derivatives thereof. In particular examples, such cells have alanine 2,3 aminomutase activity, CoA transferase activity or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and 3 -HP-CoA dehydratase activity, wherein the cell produces 3- HP-CoA. In one example, the cells can further include poly hydroxyacid synthase activity, wherein the cell produces polymerized 3-HP, co-polymers of 3-HP or other compounds such as butyrates and valerates. In another example, the cells further include acerylating aldehyde oxidoreductase or alcohol oxidoreductase activities (such as enzymes from the 1.1.1.1 or 1.2.1.10 class of enzymes), wherein the cell produces 1,3 propanediol.

For example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities that permit the production of 3-HP or derivatives thereof. In particular examples, such cells have alanine 2,3 aminomutase activity, CoA transferase activity or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, and 3-hydroxypropionyl-CoA activity, wherein the cell produces 3-HP. In another particular examples, cells that produce 3-HP or a derivative thereof have alanine 2,3 aminomutase activity, beta-alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity. In one example, the cells that produce 3-HP can further include lipase or esterase activity, wherein the cell produces an ester of 3-HP, such as methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3- hydroxypropionate, or butyl 3-hydroxypropionate. In another example, the cells that produce 3-HP can further include aldehyde dehydrogenase activity (such as an enzyme from the EC 1.2.1.3 class) and alcohol dehydrogenase activity (such as an enzyme from the EC 1.1.1.- class) activity, wherein the cell produces 1 ,3 propanediol. In yet another example, the cells that produce 3-HP can further include esterase activity, wherein the cell produces polymerized 3-HP, co-polymers of 3-HP or other compounds such as butyrates and valerates.

For example, the disclosed transformed cells, which in addition to increased flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, activity, and radical SAM enzyme activity, include other enzyme activities that permit the production of pantothenate or derivatives thereof. In particular examples, such cells have alanine 2,3 aminomutase activity, alpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.11) activity, alpha-ketopantoate reductase (E.C. 1.1.1.169) activity, and pantothenate synthase (E.C. 6.3.2.1) activity, wherein the cell produces pantothenate. In one example, the cells can further include pantothenate kinase (E.C. 2.7.1.33) activity, 4'-phosphopantethenoyl-l- cysteine synthetase (E.C. 6.3.2.5) activity, 4'-phosphopantothenoylcysteine decarboxylase (E.C. 4.1.1.36) activity, ATP:4'-phosphopantetheine adenyltransferase (E.C. 2.7.7.3) activity, and dephospho-CoA kinase (E.C. 2.7 ^'.1. ,24) activity, wherein the cell produces coenzyme A (CoA).

The disclosed cells can include other enzyme activities, such as one or more of pyruvate-alanine aminotransferase activity and beta-alanine-pyruvate aminotransferase activity.

Enzymes

The disclosed cells can include one or more of the following enzymes. Such enzymes can be endogenous to the cell, exogenous to the cell, or combinations thereof. In addition, these enzymes can be used to catalyze the desired reaction in vitro. The term "peptide having enzymatic activity" refers to any peptide that catalyzes a chemical reaction of other substances without itself being destroyed or altered upon completion of the reaction. Typically, a peptide having enzymatic activity catalyzes the formation of one or more products from one or more substrates. Such peptides can have any type of enzymatic activity including, without limitation, the enzymatic activity or enzymatic activities associated with enzymes such as those described below.

Peptides having lysine 2,3-aminomutase activity as well as nucleic acid encoding such polypeptides are publicly available and can be obtained from various species including, but not limited to: Clostridium subterminale (Genbank Accession number AF159146), E. coli, B. subtilis, Deinococcus radiodurans, Porphyromonas gingivalis (Genbank Accession number NC_002950), Aquifex aeolicus, or Haemophilus influenza. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains lysine 2,3-aminomutase activity. Peptides having alanine 2,3-aminomutase activity as well as nucleic acid encoding such peptides can be obtained by mutagenesis of genes encoding lysine 2,3-aminomutase activity from various species including, but not limited to: B. subtilis and P. gingivalis. Exemplary sequences are provided in PCT/US2003/001635 and PCT/US2004/024686 and are shown in SEQ DD NO: 15 for a variant of the lysine 2,3-aminomutase from P. gingivalis with alanine 2,3-aminomutase activity (the corresponding amino acid sequence is shown in SEQ ID NO: 16), and in SEQ ID NO: 17 another variant of the lysine 2,3-aminomutase from P. gingivalis with alanine 2,3-aminomutase activity (the corresponding amino acid sequence is shown in SEQ ID NO: 18). In addition, other polypeptides having alanine 2,3- aminomutase activity can be generated (for example see PCT publication WO 03/062173 and PCT/US2004/024686). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains alanine 2,3-aminomutase activity.

Peptides having arginine 2,3-aminomutase activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to: Streptomyces griseochromogenes. Such sequences are publicly available, for example Genbank accession no. AYl 96214 discloses an arginine 2,3-aminomutase nucleic acid sequences and Genbank accession no. AAP03121 discloses an arginine 2,3-aminomutase protein sequences. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains arginine 2,3-aminomutase activity.

Peptides having biotin synthase activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to: E. coli and P. gingivalis. Such sequences are publicly available, for example Genbank accession no. NC_000913 and E00893 disclose biotin synthase nucleic acid sequences and Genbank accession no. P12996 and P12678 disclose biotin synthase protein sequences. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains biotin synthase activity.

Peptides having lipoate synthase activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to: E. coli and P. gingivalis. Such sequences are publicly available, for example Genbank accession no. NC_000913 and NC_002950 disclose lipoate synthase nucleic acid sequences and Genbank accession no. NP_415161 and NP_904799 disclose lipoate synthase protein sequences. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains lipoate synthase activity.

Peptides having GcpE activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to: E. coli, Mycobacterium avium, and Salmonella. Such sequences are publicly available, for example Genbank accession nos. NC__002944, NC_003198, and D90881 disclose GcpE nucleic acid sequences and Genbank accession nos. NP_961872, NP_457055 and BAA20919 disclose GcpE protein sequences. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains GcpE activity. Peptides having LytB activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to: Mycobacterium avium, Neisseria, and E. coli. Such sequences are publicly available, for example Genbank accession nos. NC_003198, AL162753, and NC_002695 disclose LytB nucleic acid sequences and Genbank accession nos. NP_454660, CAB83914 and NP_308059 disclose LytB protein sequences. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains LytB activity.

Peptides having CoA transferase activity as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to, Megasphaera elsdenii, Clostridium propionicum, Clostridium kluyveri, and E. coli. For example, CoA transferase nucleic acids and proteins are disclosed in WO 03/062173 for M elsdenii. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains CoA transferase activity. CoA transferase activity refers to the ability to catalyze the reversible transfer of CoA (coenzyme A) from one CoA-thioester to a free acid. For example, the following variations can be made to the CoA transferase nucleic acid sequence disclosed in WO 03/062173: the "a" at position 49 can be substituted with an "c"; the "a" at position 590 can be substituted with a "atgg"; an "aaac" can be inserted before the "g" at position 393; or the "gaa" at position 736 can be deleted. It will be appreciated that the sequences set forth in the sequence listing can contain any number of variations as well as any combination of types of variations, as long as the peptide retains CoA transferase activity. In addition, the following variations can be made to the CoA transferase amino acid sequence disclosed in WO 03/062173: the "k" at position 17 of can be substituted with a "p" or "h"; and the "v" at position 125 can be substituted with an "i" or "f."

Peptides having CoA synthetase or ligase activity (E.C. 6.2. Ln) as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to, Rhodococcus sp., Nocardiafarcinica, Rhodococcus Sp. RHA, and Ralstonia metallidurans CH34. For example, Co-A synthetase nucleic acids sequences are disclosed in GenBank Accession Nos: CP000431, BAD59539, ABH00669 and NC_007973, and Co- A synthetase proteins are disclosed in GenBank Accession Nos: ABG97803, YP_120903, YP_708827, and YP_584671. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains Co-A synthetase activity. Co-A synthetase activity refers to the ability to ligate a fatty acid with Coenzyme A.

Peptides having beta-alanyl-CoA ammonia lyase activity (EC 4.3.1.6) as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, C. propionicum. For example, nucleic acid encoding a peptide complex having beta-alanyl-CoA ammonia lyase activity can be obtained from C. propionicum. Beta- alanyl-CoA ammonia lyase nucleic acid and peptide sequences are disclosed in WO 03/062173 (as well as GenBank Accession Nos. AJ715482, AJ715481, and CAG29275, CAG29274, respectively). It will be appreciated that publicly available beta-alanyl-CoA ammonia lyase sequences can contain variations as long as the peptide retains beta-alanyl- CoA ammonia lyase activity. Beta-alanyl-CoA ammonia lyase activity refers to the ability to convert beta-alanyl-CoA to acrylyl-CoA.

Peptides having 3-hydroxypropionyl-CoA dehydratase activity (also referred to as acrylyl-CoA hydratase activity) as well as nucleic acid encoding such peptides can be obtained from various species including, but not limited to, Chloroflexus aurantiacus, Candida rugosa, RhodosprilHum rubrum, and Rhodobacter capsulates. For example, a nucleic acid that encodes a peptide having 3-hydroxypropionyl-CoA dehydratase activity is disclosed in WO 02/42418. It will be appreciated that publicly available 3- hydroxypropionyl-CoA dehydratase sequences can contain variations as long as the peptide retains 3-hydroxypropionyl-CoA dehydratase activity. 3-hydroxypropionyl-CoA dehydratase activity refers to the ability to convert acrylyl-CoA to 3HP-CoA. Peptides having glutamate dehydrogenase activity (EC 1.4.1.3) as well as nucleic acid encoding such peptides can be obtained from various species, such as E. coli, Pseudomonas putia, and Bacillus subtilis. Such sequences are publicly available, for example GenBank Accession Nos J01615, AEOl 5451, and L47648 (nucleic acid sequences) and AAA87979, AAN66300 and AAC83953 (proteins). It will be appreciated that publicly available glutamate dehydrogenase sequences can contain variations as long as the peptide retains glutamate dehydrogenase activity. Glutamate dehydrogenase activity refers to the ability to convert glutamate to α-ketoglutarate (or 2-oxoglutarate), and vice versa.

Peptides having 3-hydroxypropionyl-CoA hydrolase activity (also referred to as 3- hydroxyisobutyryl-CoA hydrolase), as well as nucleic acid encoding such peptides, can be obtained from various species including, without limitation, Plasmodium falciparum, Graniilobacter bethesdensis, and Homo sapiens. Such sequences are publicly available, for example GenBank Accession Nos: NC_004316, NC_008343, and U66669 (nucleic acid sequences) and NP_701750, YP_745636, and AAC52114 (proteins). It will be appreciated that publicly available 3-hydroxypropionyl-CoA hydrolase sequences can contain variations as long as the peptide retains 3-hydroxypropionyl-CoA hydrolase activity. 3- hydroxypropionyl-CoA hydrolase activity refers to the ability to hydrolyze 3- hydroxypropionyl-CoA to 3-HP and CoA.

Peptides having alanine dehydrogenase activity (EC 1.4.1.1), as well as nucleic acid encoding such peptides can be obtained from various species such as Bacillus, Thermus caldophilus, and Rhizobium leguminosarwn. Such sequences are publicly available, for example GenBank Accession Nos: AJ238118, AY293734, and NZ_AAOX01000067 (nucleic acid sequences) and CAB60094, AAP44334 and ZP_01174047 (proteins). It will be appreciated that publicly available alanine dehydrogenase sequences can contain variations as long as the peptide retains alanine dehydrogenase activity. Alanine dehydrogenase activity refers to the ability to catalyzes the NAD(P)H-dependent reversible reductive amination of pyruvate into alanine.

Peptides having pyruvate/glutamate transaminase activity (EC 2.6.1.2), as well as nucleic acid encoding such peptides can be obtained from various species such as Bacillus and yeast. Such sequences are publicly available, for example GenBank Accession Nos

Z99120 and CAA88665 (nucleic acid sequences) and CAB 15129 and P52892 (proteins). It will be appreciated that publicly available pyruvate/glutamate transaminase sequences can contain variations as long as the peptide retains pyruvate/glutamate transaminase activity. Pyruvate/glutamate transaminase activity refers to the ability to convert L-alanine and 2- oxoglutarate to pyruvate and L-glutamate. Peptides having beta-alanine-2-oxoglutarate aminotransferase activity, as well as nucleic acid encoding such peptides can be obtained from various species such as rat Pseudomonas aeruginosa, and Rhizobium leguminosarum. Such sequences are publicly available, for example GenBank Accession Nos D87839, AE004091, and AF335502 (nucleic acid sequences) and BAA25570, AAG08698, and AAK21246 (proteins). It will be appreciated that publicly available beta-alanine-2-oxoglutarate aminotransferase sequences can contain variations as long as the peptide retains beta-alanine-2-oxoglutarate aminotransferase activity. Beta-alanine-2-oxoglutarate aminotransferase activity refers to the ability to convert beta alanine to malonic semialdehyde. Peptides having 3-HP dehydrogenase activity as well as nucleic acid encoding such peptides can be obtained from various species, such as Pseudomonas aeruginosa. For example, nucleic acid that encodes a peptide having 3-HP dehydrogenase activity can be obtained from the 3 -hydroxy isobutyrate dehydrogenase (mmsB) gene of Pseudomonas aeruginosa and can have a sequence as set forth in GenBank accession number M84911 (with a corresponding protein sequence shown in GenBank accession number

AAA25892.1). Peptides having 3-HP dehydrogenase activity as well as nucleic acid encoding such peptides can also be obtained from Rhodobacter sphaeroides, having nucleic acid sequence as set forth in GenBank accession number AF316325 (with a corresponding protein sequence shown in GenBank accession number AAL26884). It will be appreciated that publicly available 3-HP dehydrogenase sequences can contain variations as long as the peptide retains 3-HP dehydrogenase activity. 3-HP dehydrogenase activity refers to the ability to convert malonic semialdehyde to 3-HP.

Peptides having acetylating aldehyde oxidoreductase activity (EC 1.2.1.10) as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, E. coli. Such sequences are publicly available. For example, nucleic acid that encodes a peptide having acetylating aldehyde oxidoreductase activity can be obtained from E. coli and can have a sequence as set forth in GenBank accession number NC_000913 (protein sequence NP_415757), from Photorhabdus luminescens with a nucleic acid sequence as set forth in GenBank accession number NC_005126 (protein sequence NP_929732), or from Clostridium perfiingens with a nucleic acid sequence as set forth in GenBank accession number NC_003366 (protein sequence NP_563447). It will be appreciated that publicly available acetylating aldehyde oxidoreductase sequences can contain variations as long as the peptide retains acetylating aldehyde oxidoreductase activity. Acetylating aldehyde oxidoreductase activity refers to the ability to convert acetaldehyde and CoA to acetyl-CoA. Peptides having alcohol oxidoreductase activity (EC 1.1.1.1) as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Saccharomyces cerevisiae, Pseudomonas fluorescens, or Rhodobacter sphaeroides. Such sequences are publicly available, for example GenBank Accession Nos: NC_001134, NC__004129, or NC_007493 (nucleic acid sequences) and NP_009703, YP_258528, or YP_351873 (protein sequences). It will be appreciated that publicly available alcohol oxidoreductase sequences can contain variations as long as the peptide retains alcohol oxidoreductase activity. Alcohol oxidoreductase activity refers to the ability to convert an alcohol to an aldehyde. Aldehyde oxidoreductase activity and alcohol oxidoreductase activities can be carried out by two different peptides as described above, or carried out by a single peptide, such as a multi-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No. M33504, protein sequence set forth as Accession No. AAA23420). Peptides having poly hydroxyacid synthase activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Rhodobacter sphaeroides, Comamonas acidororans, and Rhodospirillum rubrum. Such sequences are publicly available, for example GenBank Accession Nos X97200, AF061446 and AFl 78117 (nucleic acid sequences) and CAA65833, AAC69615, and AAD53179 (proteins). Addition information about poly hydroxyacid synthase can be found in Song et al. {Biomacromolecules 1:433-9, 2000). It will be appreciated that publicly available poly hydroxyacid synthase sequences can contain variations as long as the peptide retains poly hydroxyacid synthase activity. Poly hydroxyacid synthase activity refers to the ability to synthesize polymers of hydroxyacid. Peptides having lipase activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Candida rugosa, Candida tropicalis, and Candida albicans. For example, lipase nucleic acids and proteins are disclosed in GenBank Accession Nos: A81171, Z30945, AF188894 (nucleic acids) and Z30945 and AFl 88894 (proteins). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains lipase activity. Lipase activity refers to the ability to catalyze the hydrolysis or formation of ester bonds, in particular, between 3- FIP and an alcohol.

Peptides having esterase activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Candida rugosa, Candida tropicalis, and Candida albicans. For example, esterase nucleic acids and proteins are disclosed in GenBank Accession Nos: Z30945 and AFl 88894 (nucleic acids) and CAA83122 and AAF35171 (proteins). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains esterase activity. Esterase activity refers to the ability to catalyze the hydrolysis or formation of ester bonds, in particular, to form an ester linkage between two molecules of 3 -HP or between one molecule of 3-HP and a polymer of 3-HP, or between two polymers of 3-HP.

Peptides having aldehyde dehydrogenase (EC 1.2.1.-) activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Pseudomonas putida, E. coli, and S. cerevisiae. For example, aldehyde dehydrogenase nucleic acids and proteins are disclosed in GenBank Accession Nos: AB100375, L40742, and Z17314 (nucleic acids) and BAD07372, AAC36938, and CAA78962 (proteins). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains aldehyde dehydrogenase activity. Aldehyde dehydrogenase activity refers to the ability to reduce a carboxylic group to an aldehyde group, using NADH or NADPH as the reductant.

Peptides having alcohol dehydrogenase activity (EC 1.1.1.1) as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, Pseudomonas putida, Z. mobilis, and S. cerevisiae. For example, alcohol dehydrogenase nucleic acids and proteins are disclosed in GenBank Accession Nos: AB100375, M32100, and M38457 (nucleic acids) and BAD07371, AAA27682, and AAA3441 1 (proteins). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains alcohol dehydrogenase activity. Alcohol dehydrogenase activity refers to the ability to reduce an aldehyde group to an alcohol group, using NADH or NADPH as the reductant. Peptides having alpha-ketopantoate hydroxymethyltransferase (EC 2.1.2.11) and pantothenate synthase (EC:6.3.2.1) activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, E. coli. For example, nucleic acids that encode peptides having alpha-ketopantoate hydroxymethyltransferase and pantothenate synthase activity are provided in GenBank accession number L 17086 (corresponding peptides disclosed in GenBank Accession Nos: AAA24271 and

AAA24272). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains alpha-ketopantoate hydroxymethyltransferase or pantothenate synthase activity. Alpha-ketopantoate hydroxymethyltransferase activity refers to the ability to form 2-dehydropantoate and pantothenate synthase activity refers to the ability to catalyze the formation of pantothenate from pantoate and alanine. Peptides having alpha-ketopantoate reductase (EC 1.1.1.169) activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, E. coli. For example, nucleic acids that encode peptides having alpha- ketopantoate reductase activity are provided in GenBank accession No. AAC73528 (corresponding peptide disclosed in GenBank Accession No. NP_414959). It will be appreciated that publicly available sequences can contain variations as long as the peptide retains alpha-ketopantoate reductase activity. Alpha-ketopantoate reductase activity refers to the ability to catalyze the NADPH-dependent reduction of alpha-ketopantoate to D-(-)- pantoate. Peptides having pantothenate kinase, 4 -phosphopantethenoyl-l -cysteine synthetase,

4'-phosphopantothenoylcysteine decarboxylase, ATP:4'-phosphopantetheine adenyltransferase, and dephospho-CoA kinase activity as well as nucleic acid encoding such peptides can be obtained from various species including, without limitation, E. coli. For example, nucleic acids that encodes peptides having alpha-ketopantoate reductase pantothenate kinase, 4'-phosphopantethenoyl-l -cysteine synthetase, 4'- phosphopantothenoylcysteine decarboxylase, ATP:4'-phosphopantetheine adenyltransferase, and dephospho-CoA kinase activity can be obtained from E. coli and can have a sequence as set forth in GenBank accession number NC_000913, with protein sequences set forth in accession numbers NP_418405, NP_418096, NP_418096, NP_418091, and NP_414645. It will be appreciated that publicly available sequences can contain variations as long as the peptide retains the desired enzyme activity. Pantothenate kinase activity refers to the ability to phosphorylate pantothenate using ATP, 4'-phosphopantethenoyl-l -cysteine synthetase activity refers to the ability to condense 4'-phosphopantothenate with cysteine to foπn 4'- phosphopantenoylcysteine, 4'-phosphopantothenoylcysteine decarboxylase activity refers to the ability to decarboxylate 4'-phosphopantenoylcysteine to form 4'-phosphopantetheine, ATP:4'-phosphoρantetheine adenyltransferase activity refers to the ability to transfer the AMP moiety of ATP to 4'-phosphopantetheine to form dephospho-CoA, and dephospho- CoA kinase activity refers to the ability to phosphorylate dephospho-CoA to form CoA.

Although particular examples of enzymes that can be used are disclosed, one skilled in the art will appreciate that a nucleic acid molecule encoding a peptide having the desired enzymatic activity can be identified and obtained using methods known in the art. For example, nucleic acid molecules that encode a peptide having the desired enzymatic activity can be identified and obtained using common molecular cloning or chemical nucleic acid synthesis procedures and techniques, including PCR. In addition, standard nucleic acid sequencing techniques and software programs that translate nucleic acid sequences into amino acid sequences based on the genetic code can be used to determine whether or not a particular nucleic acid has any sequence homology with known enzymatic peptides. Sequence alignment software such as MEGALIGN (DNASTAR, Madison, WI₅ 1997) can be used to compare various sequences. Further, nucleic acid and amino acid databases (such as GenBank and EMBL) can be used to identify a nucleic acid sequence that encodes a peptide having the desired enzymatic activity. Briefly, any amino acid sequence having at least 80% homology to a peptide having the desired enzymatic activity (such as a lipase), or any nucleic acid sequence having at least 50% homology to a sequence encoding a peptide having the desired enzymatic activity can be used as a query to search GenBank. The identified peptides then can be analyzed to determine whether or not they exhibit the desired enzymatic activity.

Nucleic acid hybridization techniques can also be used to identify and obtain a nucleic acid molecule that encodes a peptide having the desired enzymatic activity. Briefly, a nucleic acid molecule that encodes a known enzymatic peptide, or fragment thereof, can be used as a probe to identify similar nucleic acid molecules by hybridization under conditions of moderate to high stringency. Such similar nucleic acid molecules then can be isolated, sequenced, and analyzed to determine whether the encoded peptide has the desired enzymatic activity.

Expression cloning techniques also can be used to identify and obtain a nucleic acid molecule that encodes a peptide having the desired enzymatic activity. For example, a substrate known to interact with a particular enzyme can be used to screen a phage display library containing that enzyme. Phage display libraries can be generated as described (Burritt et ah, Anal. Biochem. 238: 1-13, 1990), or can be obtained from commercial suppliers such as Novagen (Madison, WI). Peptide sequencing techniques can also be used to identify and obtain a nucleic acid molecule that encodes a peptide having the desired enzymatic activity. For example, a purified peptide can be separated by gel electrophoresis, and its amino acid sequence determined by, for example, amino acid microsequencing techniques. Once determined, the amino acid sequence can be used to design degenerate oligonucleotide primers. Degenerate oligonucleotide primers can be used to obtain the nucleic acid encoding the polypeptide by PCR. Once obtained, the nucleic acid can be sequenced, cloned into an appropriate expression vector, and introduced into a microorganism. Recombinant Expression of Proteins

The enzymes described herein, as well as flavodoxin, ferredoxin, and flavodoxin reductase, can be produced individually or in combination in a cell. For example, recombinant nucleic acid molecules can be used to generate the cells and practice the methods disclosed herein. In particular examples, the disclosed cells (or methods that use such cells) include at least one exogenous nucleic acid molecule.

For example, nucleic acid molecules encoding the enzymes described herein, as well as flavodoxin, ferredoxin, and flavodoxin reductase, can be introduced into a cell using standard molecular biology methods. A single nucleic acid molecule can encode more than one enzyme or other desired molecule. For example, operons including two or more nucleic acid coding sequences, such as two, three, four, five, six, or even seven coding sequences, can be used. For example, each nucleic acid sequence can encode a radical SAM enzyme, flavodoxin, ferredoxin, flavodoxin reductase, or an enzyme needed for the production of a radical SAM enzyme downstream chemical (such as 3 -FIP or pantothenate). In a particular example, the recombinant nucleic acid sequence includes a sequence encoding one or more of flavodoxin, ferredoxin, and flavodoxin reductase and a nucleic acid sequence encoding a radical SAM enzyme, such as alanine 2,3-aminomutase. Such recombinant nucleic acid sequences can further include a nucleic acid sequence that encodes one or more enzymes shown in FIG. 1 or 2. In addition, recombinant nucleic acid sequences can additionally include one or more promoter sequences to drive expression of the coding sequence. The disclosed nucleic acids can be incorporated into a vector, which can be used to transform a cell, or be incorporated into the genome of the cell, or both.

Transformed cells disclosed can be used to produce a radical SAM enzyme product or downstream chemical thereof, and can therefore be used to perform one or more steps of the steps in the pathways described herein. For example, an individual microorganism can contain exogenous nucleic acid(s) encoding each of the peptides needed to perform the steps depicted in FIGS. 1 and 2. Such cells can contain any number of exogenous nucleic acid molecules. For example, a particular cell can contain one, two, three, or four different exogenous nucleic acid molecules with each one encoding the peptide(s) needed to convert pyruvate into beta-alanine (or a later product such as acrylyl-CoA, 3-HP, or pantothenate) as shown in FIG. 1 or 2, or a particular cell can endogenously produce peptides needed to convert pyruvate into alpha-alanine while containing an exogenous nucleic acid molecule that encodes peptides needed to convert alpha-alanine into beta-alanine.

In addition, a single exogenous nucleic acid molecule can encode one, or more than one, peptide. For example, a single exogenous nucleic acid molecule can contain sequences that encode two, three, or even four different peptides. Further, the cells described herein can contain a single copy, or multiple copies (such as at least 5, 10, 20, 35, 50, 75, 100 or 150 copies), of a particular exogenous nucleic acid molecule, such as a particular enzyme. The cells described herein can contain more than one particular exogenous nucleic acid. For example, a particular cell can contain about 15 copies of exogenous nucleic acid molecule X as well as about 25 copies of exogenous nucleic acid molecule Y.

A nucleic acid molecule encoding a peptide having enzymatic activity can be identified and obtained using any method known in the art. For example, nucleic acid molecules that encode a peptide having enzymatic activity can be identified and obtained using common molecular cloning or chemical nucleic acid synthesis procedures and techniques, including PCR. In addition, standard nucleic acid sequencing techniques and software programs that translate nucleic acid sequences into amino acid sequences based on the genetic code can be used to determine whether or not a particular nucleic acid has any sequence homology with known enzymatic peptides. Sequence alignment software such as MEGALIGN (DNASTAR, Madison, WI, 1997) can be used to compare various sequences. Further, nucleic acid and amino acid databases (such as GenBank and EMBL) can be used to identify a nucleic acid sequence that encodes a peptide having the desired enzymatic activity. Briefly, any amino acid sequence having at least 50% homology (such as at least 80% or at least 90% sequence homology) to a peptide having enzymatic activity, or any nucleic acid sequence having at least 50% homology (such as at least 80% or at least 90% sequence homology) to a sequence encoding a peptide having enzymatic activity can be used as a query to search GenBank. The identified peptides then can be analyzed to determine whether or not they exhibit the desired enzymatic activity using routine methods known in the art. Any method known to those skilled in the art can be used to introduce an exogenous nucleic acid molecule into a cell. For example, heat shock, lipofection, electroporation, conjugation, fusion of protoplasts, and biolistic delivery are common methods for introducing nucleic acid into bacteria and yeast cells. (For example see Ito et al., J. Bacterol. 153: 163-8, 1983; Durrens et al, Curr. Genet. 18:7-12, 1990; Sambrook et al., Molecular cloning: A laboratory manual, Cold Spring Harbour Laboratory Press, New York, USA, second edition, 1989; and Becker and Guarente, Methods in Enzymology 194: 182-7, 1991). Other methods for expressing an amino acid sequence from an exogenous nucleic acid molecule include, but are not limited to, constructing a nucleic acid such that a regulatory element promotes the expression of a nucleic acid sequence that encodes a peptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. Any type of promoter can be used to express an amino acid sequence from an exogenous nucleic acid molecule. Examples of promoters include, without limitation, constitutive promoters, and promoters responsive or unresponsive to a particular stimulus (such as light, oxygen, chemical concentration). An exogenous nucleic acid molecule contained within a particular cell of the disclosure can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state. That is, a cell can be a stable or transient transformant.

Example 1

Cloning, expression, and purification of proteins

This example describes methods used to generate and obtain purified proteins. One skilled in the art will appreciate that similar methods can be used to generate and obtain other purified proteins (for example, see the methods disclosed in Scopes, R.K., Protein Purification, Principles and Practice, 3^rd Ed. Springer-Verlag, 1993) or to generate and obtain the same proteins disclosed below from other organisms. For example, similar methods can be used to clone, express or purify any of the enzymes listed in FIGS. 1 and 2, or other enzymes needed to produce a radical SAM enzyme product or downstream organic chemical thereof.

Cloning of E, colifdx (ferredoxin)

The sequence of the E. coli ferredoxin gene (fdx) can be found in public databases (Genbank accession number D90883). The gene was synthesized from oligonucleotides and assembled as described by Stemmer et a {Gene 164:49-53, 1995). The assembled gene was PCR-amplified with the following primers: SEQ ID NO 1 : 5'- ggccccggtctccaatgccaatgccaaagattgttattttg-3', and SEQ ID NO 2: 5'- gataatggtctctgcgctatgctcacgcgcatggttgatag-3'. The PCR product containing the fdx gene was then cloned into pASK IBA3 (DBA, Goettingen, Germany) as described by the manufacturer. DNA sequence of the clone was confirmed by standard sequencing methods.

Cloning of E. colifpr (flavodoxin/ferredoxin NADP(H) oxidoreductase; EC 1.18.1.2)

The sequence of E. colifpr gene can be found in public databases (Genbank accession number NC_000913). The following primers were designed using this sequence to PCR-amplify the gene from genomic DNA prepared from the E. coli strain ATCC 11303 (American Type Culture Collection, Manassas, VA): SEQ ID NO 3: 5'- gtgcatggtctcgaatggctgattgggtaacagg-3', and SEQ ID NO 4: 5'- gtctaaggtctcagcgctccagtaatgctccgctgtc-3'. The resulting PCR product was cloned into pCR Blunt TOPO using the Zero Blunt TOPO cloning kit from Invitrogen (Carlsbad, CA). To enable cloning of the fpr gene into pASK IB A3 using Bsal restriction endonuclease cleavage as described by the manufacturer of this plasmid, the Bsal restriction site present in the coding sequence was removed using the QuikChange II kit from Stratagene (La Jolla, CA) and the following primers: SEQ ED NO 5: 5'- cagttgctgaaagaaacgcggcagatgacgaaacatttacgtcg-3', and SEQ ID NO 6: 5'- cgacgtaaatgtttcgtcatctgccgcgtttctttcagcaactg-3 '. The DNA sequence was altered to remove the Bsal site but preserves the native amino acid sequence. The fpr gene was cloned into pASK IBA3 as described by the manufacturer using Bsal restriction sites engineered into SEQ ID NOS: 3 and 4. When the clone in pASK D3A3 was sequenced, 24 base changes were found leading to three amino acid changes: L29H, H105D, and T197A.

Cloning of E. colifldΛ (flavodoxin)

The E. colifldA gene (GenBank M59426) was PCR-amplified from chromosomal DNA prepared from E. coli strain ATCCl 1303 (American Type Culture Collection, Manassas, VA) with the following primers: SEQ ED NO 7: 5'- gtctagggtctcgaatggctgtcactggcatctttttc-3 ', and SEQ ID NO 8: 5 '- gtcaatggtctcagcgctggcattgagaatttcgtcgag-3'. The PCR product containing the fldA gene was cloned into pASK IBA3 using Bsal sites engineered into the primers. Sequences were confirmed by standard DNA sequencing techniques. A single base change at position 79 of the coding sequence (relative to the sequence in GenBank Accession number M59426) was found. The change detected (G to an A) at this position is a silent mutation.

Cloning of P. gingivalis flavodoxin

The P. gingivalis genome (Genbank accession no. NC_002950) has two sequences with a high degree of nucleic acid sequence homology to the E. colifldA gene. However, translation of the DNA sequences yields only one protein with similar characteristics to the protein encoded by the E. colifldA gene; therefore, this P. gingivalis gene is designated as Pgfldl. Pgfldl was PCR-amplified from chromosomal DNA prepared from P. gingivalis strain ATCC BAA-308D (American Type Culture Collection, Manassas, VA) with the primers: SEQ ID NO 9: 5'-ccggaattccatatgaaatcaatcggaatcttctacgg-3\ and SEQ ID NO 10: 5 '-cccaagcttctcgagcaagcccatggcagcg-3 ' . The PCR product containing the Pgfldl gene was cloned into the pCR Blunt II vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen Corporation) according to the manufacturer's instructions. The Pgfldl insert was then excised using Ndel and Xhol sites engineered into the primers and inserted into those same sites in pET23 (Novagen, Madison WI). The nucleic acid sequence of the cloned Pgfldl gene is provided as SEQ E⁾ NO: 19 and the corresponding amino acid sequence is provided as SEQ ID NO: 20.

Cloning of P. gingivalis lysine 2,3-aminomutase

A P. gingivalis lysine 2,3-aminomutase was cloned as described in WO03/062173. A P. gingivalis alanine 2,3-aminomutase was previously obtained by mutagenesis of the lysine 2,3-aminomutase gene. The sequence is provided in SEQ ID NOS: 17 and 18.

Proteins cloned into the pASK IBA3 vector carry a C-terminal extension of 10 amino acids that provide binding to biotin (the "strep-tag"). These proteins were thus purified using biotin affinity chromatography as described by the supplier (IBA, Goettingen, Germany).

Example 2 in vitro Assay to Measure Alanine 2,3 aminomutase Activity

This example describes methods used to demonstrate that flavodoxin reductase and flavodoxin can increase alanine 2,3 aminomutase activity in vitro. One skilled in the art will appreciate that similar assays can be used to determine the amount of alanine 2,3 aminomutase activity in the presence of other combinations of ferredoxin, flavodoxin reductase, and flavodoxin.

Briefly, purified flavodoxin reductase and flavodoxin proteins were combined with purified alanine 2,3-aminomutase (SEQ ID NO: 18), NADPH, and glucose dehydrogenase plus glucose (to regenerate NADPH) in an anaerobic in vitro reaction, and the production of beta-alanine was monitored over time. All in vitro assays were done in a Coy anaerobic chamber at 37⁰C. The concentration of alanine 2,3-aminomutase monomer varied between 0.005 and 0.025 mM. The reaction mixture (final volume = 0.5 ml) contained 50 mM HEPPS pH 8, 0.4 mM SAM, 50 mM L-alanine, 1 mM NADPH, 1 mM DTT, 0.2 mM PLP, 10 mM glucose, and 13.5 U of glucose dehydrogenase (Sigma Catalog Number 19369, St. Louis, MO). The concentration of E. coli flavodoxin reductase, E. coli flavodoxin, and P. gingivalis flavodoxin (see Example 1) varied between 0.001 and 0.1 mM.

At each time-point 0.1 mL of the reaction was added to 0.1 mL of the reaction quench buffer (50% 50 mM HEPPS pH 8 and 50% formic acid). The quenched samples were vortexed for fifteen seconds and then centrifuged at 10,000 rpm for one minute, and the supernatant was removed for analysis of the beta-alanine concentration by HPLC (AMINOSep-511 column, Transgenomic, gradient development with Pickering pH 3.28 and 7.48 buffers, and post-column development using O-phthaladehyde). The results of an assay comparing the effect of the biological reductant system containing flavodoxin and flavodoxin reductase versus the chemical reductant dithionite in the in vitro production of beta-alanine by alanine 2,3-aminomutase is summarized in Table 1, and shows that the flavodoxin system is at least as efficient as chemical reduction.

Table 1. Effect of reductants on alanine 2,3-aminomutase activity

Reaction components* beta-alanine CmM)

FNR, FLDl, NADPH 0.123

Dithionite, preincubation 0.102

Dithionite 0.096

FNR, NADPH 0.024

No reduction 0

*FNR = E. coli flavodoxin reductase; FLDl = E. coli flavodoxin

FIG. 3 shows that alanine 2,3-aminomutase activity is increased by increasing levels of flavodoxin in the presence of flavodoxin reductase. The results shown in Table 1 and FIG. 3 demonstrate that this biologically-relevant reduction system is capable of generating active alanine 2,3-aminomutase in vitro without the use of chemical reductants such as dithionite or a period of reductive preincubation.

To determine if the beneficial effect of flavodoxin on the activity of alanine 2,3- aminomutase could be enhanced by using a cognate flavodoxin from Porphyromonas gingivalis instead of (or in addition to) the E, coli flavodoxin, the fldA homolog was cloned from P. gingivalis (Example 1). This protein, designated as Pgfldl, was expressed as a His- tag fusion and purified as described as above. As shown in FIG. 2 flavodoxin from P. gingivalis has the same effect as the flavodoxin from E, coli in augmenting the activity of alanine 2,3-aminomutase.

Example 3 Isolation of flavodoxin gene by selection for increased alanine 2,3-aminomutase activity

This example describes methods used to identify genes that contribute to the reduction of the alanine 2,3-aminomutase 4Fe-4S cluster in vivo. It was proposed that increased activity of the alanine 2,3-aminomutase could be achieved by increased cluster reduction. One skilled in the art will appreciate that similar methods can be used to identify additional examples of such genes.

To identify the genes that contribute to the reduction of the alanine 2,3- aminomutase 4Fe-4S cluster in vivo, a random library of genomic E. coli DNA in a multicopy plasmid was constructed to select for "helper" genes that augment the in vivo function of alanine 2,3-aminomutase. The selection is based on the complementation of cells carrying a bφanD deletion, which results in pantothenate auxotrophy, by the activity of alanine 2,3-aminomutase to supply beta-alanine for pantothenate synthesis. A genomic library of BW251 13 bφanD ApanF was constructed by ligation of 2-5 Kb Sau3k fragments into a pPROLar-based plasmid also bearing a weak alanine 2,3-aminomutase (SEQ DD NO: 15; the corresponding protein sequence is shown in SEQ ID NO: 16). A weak alanine 2,3- aminomutase with low activity was used so that the effects of a positive helper gene would be more easily visualized on selective media, and a bφanD bφanF mutant was used as the source of genomic DNA to avoid cloning genes that bypass the genetic complementation selection.

The ligation reaction was transformed into the BW25113 ApanD ApanF strain, the transformation recovery was washed with 500 μL 0.85% NaCl, and 20 μL was plated on rich media containing beta-alanine to obtain a colony count. The remaining transformation mix, saved at 4⁰C, was then plated on selective media (M9 salts, 4 g/L glucose, 100 mM MOPS pH 7, 0.5 g/ L-alanine, 20 μM ferric citrate, 100 μM DPTG, 40 μg/mL kanamycin, and trace elements) to obtain approximately 375 colonies/plate. A total of approximately 5,600 colonies were plated and plates were placed in an anaerobic chamber. After 3-4 days, eight colonies that were visibly larger than the plate average were streaked to new selective media for purification. Five of the eight colonies grew well in the restreaks.

A liquid growth test in selective media was conducted for two single colonies from each of these five clones, along with controls containing only a alanine 2,3-aminomutase gene. Growth was done in 1.8 mL glass screw-capped tubes containing 1.4 mL of media as described above, with occasional mixing, and compared to growth in non-selective media (with beta-alanine).

Two of the clones performed approximately 2.3-2.7 times better than the control. Sequencing of plasmid DNA from these two clones showed that their genomic inserts both contained the E. colifldA gene encoding flavodoxin. This result indicates that the increased availability of flavodoxin in turn increases the activity of alanine 2,3-aminomutase, and demonstrates that flavodoxin interacts with alanine 2,3-aminomutase in vivo. This observed in vivo effect of the flavodoxin gene on alanine 2,3-aminomutase agrees with the direct measurement of the effect of the protein in vitro, accomplished by cloning and expressing the genes for flavodoxin (fldA) and flavodoxin reductase φr) from E. coli, and purification of the proteins using affinity chromatography (see Example 2).

Example 4

Cells with flavodoxin or flavodoxin reductase promoter replacements This example describes methods used to generate cells that included flavodoxin or flavodoxin reductase with a non-native promoter, to increase expression. One skilled in the art will appreciate that similar methods can be used to operably link any non-native promoter, such as an inducible promoter, to a flavodoxin, ferredoxin, or flavodoxin reductase gene.

Based on the in vivo and in vitro demonstrations in Examples 2 and 3 that flavodoxin participates in the reductive activation of the alanine 2,3-aminomutase 4Fe-4S cluster, a strain in which the artificial Pi_ac/ara hybrid promoter was placed immediately upstream of th& fldA gene was constructed, using a modification of the Datsenko and Wanner method for the insertion of PCR-generated fragments in specific sites in the E. coli genome (Datsenko and Wanner, Proc. Natl. Acad. Soc. USA 97:6640-5, 2000). A novel plasmid, pKDprom, was constructed by insertion of the Pi_ac/ara promoter region of pPRO-Nde into plasmid pKD3 of Datsenko and Wanner. Plasmid pKDprom carries the chloramphenicol-resistance marker between two FRT regions (to allow excision of the marker by the FLP protein) immediately adjacent to the Pi_ac/ara promoter. This segment comprising the marker and promoter on pKDprom was amplified using the following primers that carried 42-basepair 5' extensions homologous to regions immediately upstream of the fldA gene: SEQ ID NO 11 : 5'- gtgggcaattttccacccccatttcaataagtttcaagaggtgtgtaggctggagctgcttc, and SEQ ID NO 12: 5'- attaccggtgtcgctgccgaaaaagatgccagtgatagccatatgtacctttctcctctttaa.

The PCR product was recombined into this site in the E. coli genome by the lambda phage Red recombinase functions as described by Datsenko and Wanner. Insertion was followed by selection for chloramphenicol resistance, and confirmed by PCR. A strain carrying the artificial promoter and chloramphenicol resistance cassette in front of the fldA gene is designated as KIfldA::cam, and was deposited with the American Type Culture Collection (Manassas, VA) on July 7, 2005 (Accession No. PTA-6837). Strains carrying the artificial promoter before the fldA gene, from which the chloramphenicol-resistance marker was excised as described by Datsenko and Wanner, are designated KlfldA.

A strain in which the artificial promoter was located just upstream of the fpr gene in the E. coli chromosome was constructed in a similar manner. Primers: SEQ ID NO 13: 5'- tcggagaacgaagataaggcaagtcaatcaaaacaggagaaaaacgtgtaggctggagctgcttc and SEQ ID NO 14: 5'-gtccagttctgcactttagtgactttgcctgttacccaatcagccatatgtacctttctcctctttaa were used to amplify pKDprom and recombined into E, coli as carried out for the fldA gene above. Strains carrying the artificial promoter before fpr are designated Klfpr.

Example 5 Increased production of beta-alanine in cells with increased expression of flavodoxin

This example describes methods used to demonstrate that increased expression of flavodoxin using the cells generated in Example 4, increase the activity of alanine 2,3 aminomutase, thereby increasing the production of beta-alanine. One skilled in the art will appreciate that similar methods can be used to increase the activity of other radical SAM enzymes, such as other aminomutases.

The bφanD KlfldA strain of E. coli was transformed with a plasmid bearing variant of the P. gingivalis lysine 2,3 -aminomutase with alanine 2,3-aminomutase activity (SEQ ID NO: 17), and the in vivo production of beta-alanine compared with a control strain in which expression of fldA was under the native regulation. As shown in Table 2, the KlfldA strain (Example 4) produced almost twice as much beta-alanine under all conditions tested.

Table 2. Enhancement of beta-alanine production by fldA overexpression

Condition Host beta-alanine (uM/OD^nn)

Hours: 0 3 6 25

Anaerobic control 0 1.9 4.5 8.1

KlfldA 0 4.3 7.8 11.4

02/AnO control 0 1.2 1.4 1.3

KlfldA 0.3 4.2 4.1 4.1

Aerobic control 0 0.1 1.4 2.7

KψdA 0 0.2 1.2 4.1 Similar methods can be used to increase expression of fpr using the Yλfpr strain (Example 4).

Example 6 Increased production of biotin or lipoic acid by increasing expression of flavodoxin and flavodoxin reductase

This example describes methods that can be used to demonstrate that increased expression of ferredoxin, flavodoxin, flavodoxin reductase, or a combination thereof, increases the activity of other radical SAM enzymes, such as biotin synthase and lipoate synthase. Specifically, this example describes methods of producing biotin or lipoic acid, and cells that can produce biotin or lipoic acid.

Biotin can be produced from dethiobiotin, for example via biotin synthase, and lipoic acid can be generated from octanoic acid, in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing dethiobiotin can be engineered to make biotin by expressing one or more nucleic acid molecules that encode an enzyme having biotin synthase activity in the cell, for example using recombinant technologies, while cells can be engineered to make lipoic acid by expressing one or more nucleic acid molecules that encode an enzyme having lipoate synthase activity in the cell. The cell that produces biotin or lipoic acid overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase biotin synthase activity, lipoate synthase activity, or both, thereby increasing production of biotin or lipoic acid in the cell. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes biotin synthase activity. Such biotin synthase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for a biotin synthase. Likewise, in another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes lipoate synthase activity. Such lipoate synthase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for a lipoate synthase.

Methods of producing biotin or lipoic acid in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has biotin synthase or lipoate synthase activity, under conditions sufficient for producing biotin from dethiobiotin or lipoic acid from octanoic acid. In a particular example, such methods of making biotin or lipoic acid include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having biotin synthase or lipoate synthase activity, and culturing the transfected cell to allow the transfected cell to make biotin or lipoic acid. The resulting products can be isolated from the cell culture medium or extracted from the cells. The formation of biotin and/or lipoic acid can be analyzed using methods described in WO 02/085293.

Example 7

Production of Beta-Alanine

This example describes methods of producing beta-alanine, and cells that can produce beta-alanine. One skilled in the art will appreciate that similar methods can be used to produce other beta amino acids from alpha amino acids, using the appropriate aminomutase. For example lysine 2,3-aminomutase can be substituted for alanine 2,3- aminomutase to convert alpha-lysine to beta-lysine, and arginine 2,3-aminomutase can be substituted for alanine 2,3-aminomutase to convert alpha-arginine to beta-arginine (see Examples 18-19, respectively).

Beta-alanine can be generated from alpha-alanine, for example via alanine 2,3- aminomutase (FIG. 1) in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing alpha-alanine can be engineered to make beta-alanine by expressing nucleic acid molecules that encode for an enzyme having alanine 2,3-aminomutase activity in the cell, for example using recombinant technologies.

The cell that produces beta-alanine overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of beta-alanine in the cell. Therefore, alpha-alanine can be converted to beta- alanine in the presence of alanine 2,3-aminomutase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity. Such alanine 2,3-aminomutase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for an alanine 2,3-aminomutase.

Genes (or cDNAs) encoding for alanine 2,3-aminomutase are disclosed herein (for example see SEQ ID NOS: 15 and 17), and can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of alanine 2,3-aminomutase in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert alpha-alanine to beta-alanine.

Methods of producing beta-alanine in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity, under conditions sufficient for producing beta-alanine from alpha-alanine. In a particular example, such methods of making beta-alanine include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, and culturing the transfected cell to allow the transfected cell to make beta-alanine. The resulting beta-alanine can be isolated from the cell culture medium or extracted from the cells. Methods of producing beta-alanine from alpha-alanine in vitro are also provided. Conversion of alpha-alanine to beta-alanine can be achieved by contacting alpha-alanine with an enzyme having alanine 2,3-aminomutase activity, and with enzymes having flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof, to enhance the activity of the alanine 2,3-aminomutase. Such conversion can also be performed by a combination of in vivo and in vitro methods.

The formation of beta-alanine during fermentation in vivo or in an in vitro assay can be analyzed using HPLC (see Example 2).

Example 8

Production of 3HP-CoA from Beta-Alanine

This example describes methods of producing 3 -HP-CoA, and cells that can produce 3-HP-CoA. In particular examples, such methods and cells can serve as a starting point for making derivatives of 3-HP-CoA, such as 3-HP, 1,3-propanediol, and polymerized 3-HP (see Examples 9 and 11-12, respectively).

3 -HP-CoA can be generated from beta-alanine (FIG. 1) in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing beta-alanine (see Example 7) can be engineered to make 3-HP-CoA by expressing one or more nucleic acid molecules that encode for an enzyme having CoA transferase or CoA synthetase activity, one or more nucleic acid molecules that encode for an enzyme having beta-alanyl-CoA ammonia lyase activity, and one or more nucleic acid molecules that encode for an enzyme having 3-HP-CoA dehydratase activity, in the cell, for example using recombinant technologies. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3-aminomutase. The cell that produces 3-HP-CoA overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of 3 -HP-CoA in the cell. Therefore, beta-alanine can be converted to 3 -HP-CoA in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and 3-HP-CoA dehydratase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and 3 -HP-CoA dehydratase activity.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert beta-alanine to 3-HP-CoA.

Methods of producing 3 -HP-CoA in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and 3-HP-CoA dehydratase activity, under conditions sufficient for producing 3 -HP-CoA from beta-alanine.

In a particular example, such methods of making 3 -HP-CoA include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, and with a nucleic acid encoding a peptide having 3 -HP-CoA dehydratase activity, and culturing the transfected cell to allow the transfected cell to make 3-HP-CoA. The resulting 3-HP-CoA can be isolated from the cell culture medium or extracted from the cells. Methods of producing 3-HP-CoA from beta-alanine in vitro are also provided. Conversion of beta-alanine to 3-HP-CoA can be achieved by contacting beta-alanine with an enzyme having CoA transferase activity to form beta-alanyl-CoA, contacting the beta- alanine CoA with a peptide having beta-alanyl-CoA ammonia lyase activity to form acrylyl- CoA, and contacting the acrylyl-CoA with a peptide having 3HP-CoA dehydratase activity to form 3 -HP-CoA.

The formation of 3 -HP-CoA during fermentation in vivo or in an in vitro assay can be analyzed using methods described in US 20040076982 Al (herein incorporated by reference).

Example 9

Production of 3-HP from Beta- Alanine

This example describes methods of producing 3-HP, and cells that can produce 3- HP. In particular examples, such methods and cells can serve as a starting point for making derivatives of 3-HP, such as 3-HP esters, 1,3 -propanediol, and polymerized 3-HP (see Examples 10-12, respectively).

3-HP can be generated from beta-alanine via two different pathways (FIG. 1) in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing beta-alanine (see Example 7) can be engineered to make 3-HP by expressing one or more nucleic acid molecules that encode for an enzyme having CoA transferase or CoA synthetase activity, one or more nucleic acid molecules that encode for an enzyme having beta-alanyl-CoA ammonia lyase activity, one or more nucleic acid molecules that encode for an enzyme having 3-HP-CoA dehydratase activity, and one or more nucleic acid molecules that encode for an enzyme having 3-hydroxypropionyl-CoA hydrolase activity, in the cell, for example using recombinant technologies. For example, cells producing 3-HP-CoA (see Example 8) can be engineered to make 3-HP by expressing one or more nucleic acid molecules that encode 3-HP-CoA dehydratase activity. In another example, cells producing beta-alanine can be engineered to make 3-HP by expressing one or more nucleic acid molecules that encode for an enzyme having beta alanine-2-oxoglutarate aminotransferase activity, and one or more nucleic acid molecules that encode for an enzyme having 3-HP dehydrogenase activity. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3-aminomutase.

The cell that produces 3-HP overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of 3-HP in the cell. Therefore, beta-alanine can be converted to 3-HP in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and 3-hydroxypropionyl-CoA hydrolase activity. In another example, beta-alanine can be converted to 3-HP in the presence of alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity.

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, and 3-hydroxypropionyl-CoA hydrolase activity. In another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert beta-alanine to 3-HP.

Methods of producing 3-HP in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and 3-hydroxypropionyl-CoA hydrolase activity, under conditions sufficient for producing 3-HP from beta-alanine. In another example, the method includes culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity.

In a particular example, such methods of making 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta- alanyl-CoA ammonia lyase activity, with a nucleic acid encoding a peptide having 3 -HP- Co A dehydratase activity, and with a nucleic acid encoding a peptide having 3- hydroxypropionyl-CoA hydrolase activity and culturing the transfected cell to allow the transfected cell to make 3-HP. In another particular example, such methods of making 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having beta alanine-2-oxoglutarate aminotransferase activity, and with a nucleic acid encoding a peptide having 3-HP dehydrogenase activity, and culturing the transfected cell to allow the transfected cell to make 3-HP. The resulting 3-HP can be isolated from the cell culture medium or extracted from the cells.

Methods of producing 3-HP from beta-alanine in vitro are also provided. Conversion of beta-alanine to 3-HP can be achieved by contacting beta-alanine with an enzyme having CoA transferase activity to form beta-alanyl-CoA, contacting the beta- alanine CoA with a peptide having beta-alanyl-CoA ammonia lyase activity to form aery IyI- CoA, contacting the acrylyl-CoA with a peptide having 3HP-CoA dehydratase activity to form 3-HP-CoA, and contacting 3-HP-CoA with a peptide having CoA transferase activity or 3-hydroxypropionyl-CoA hydrolase activity to make 3-HP.

In another example, conversion of beta-alanine to 3-HP is achieved by contacting beta-alanine with an enzyme having beta-alanine-2-oxoglutarate aminotransferase activity to form malonic semialdehyde, and contacting the malonic semialdehyde with a peptide having 3-HP dehydrogenase activity to make 3-HP. Such conversions can also be performed by a combination of in vivo and in vitro methods.

The formation of 3-HP during fermentation in vivo or in an in vitro assay can be analyzed using methods described in US 20040076982A1 (herein incorporated by reference).

Example 10 Producing an Ester of 3-HP This example describes methods of producing an ester of 3-HP, and cells that can produce an ester of 3-HP. Particular examples of 3-HP esters include, but are not limited to: methyl 3-hydroxypropionate, ethyl 3-hydroxypropionate, propyl 3-hydroxypropionate, butyl 3-hydroxypropionate, or 2-ethylhexyl 3-hydroxypropionate.

An ester of 3-HP can be generated from 3-HP. For example, cells or microorganisms producing 3-HP (such as those disclosed in Example 9) can be engineered to make an ester of 3-HP by expressing genes that encode for enzymes having lipase or esterase activity (EC 3.1.1.-).

For example, cells (such as a microorganism) producing 3-HP can be engineered to make an ester of 3-HP by expressing genes that encode for enzymes having lipase or esterase activity. In a particular example, a transformed cell that produces an ester of 3-HP can include one or more nucleic acid sequences (such as an exogenous nucleic acid sequence) that encode a protein having lipase or esterase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and 3-hydroxypropionyl-CoA hydrolase activity, or can further include one or more nucleic acid sequences that encode a protein having beta alanine-2-oxoglutarate aminotransferase activity, and 3-HP dehydrogenase activity. In particular examples, the cell also includes a nucleic acid that encodes an alanine 2,3-aminomutase.

A cell that produces an ester of 3-HP overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of an ester of 3-HP in the cell. Therefore, 3-HP can be converted to an ester of 3-HP in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and lipase or esterase activity. In another example, 3-HP be converted to an ester of 3-HP in the presence of alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity and lipase or esterase activity.

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, 3-HP-CoA hydrolase activity and lipase or esterase activity. In another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity, and lipase or esterase activity. Such en∑yme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert 3-HP to an ester of 3-HP.

Methods of producing an ester of 3-HP in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and lipase or esterase activity under conditions sufficient for producing an ester of 3 -HP from 3 -HP. In another example, the method includes culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity, and lipase or esterase activity.

In a particular example, such methods of making an ester of 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, with a nucleic acid encoding a peptide having 3 -HP-CoA dehydratase activity, with a nucleic acid encoding a peptide having 3 -HP- CoA hydrolase activity, and with a nucleic acid encoding a peptide having lipase or esterase activity and culturing the transfected cell to allow the transfected cell to make an ester of 3- HP. In another particular example, such methods of making an ester of 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having beta alanine-2-oxoglutarate aminotransferase activity, with a nucleic acid encoding a peptide having 3-HP dehydrogenase activity, and with a nucleic acid encoding a peptide having lipase or esterase activity, and culturing the transfected cell to allow the transfected cell to make an ester of 3-HP. The resulting ester of 3-HP can be isolated from the cell culture medium or extracted from the cells.

Methods of producing an ester of 3-HP from 3-HP in vitro are also provided. One skilled in the art will appreciate that the in vitro synthesis step or steps can be via chemical reaction or enzymatic reaction. For example, 3-HP can be converted into a 3-HP ester by trans esterification. In another example, conversion of 3-HP to an ester of 3-HP can be achieved by contacting 3-HP with an enzyme having lipase or esterase activity to form an ester of 3-HP. These conversions can also be performed using a combination of in vivo and in vitro methods.

The formation of an ester of 3-HP during fermentation in vivo or in an in vitro assay can be analyzed using methods described in US 20040076982A1. Example 11 Production of 1,3-propanediol

This example describes methods of producing 1,3-propanediol, and cells that can produce 1,3-propanediol. 1,3-propanediol can be generated from 3-HP-CoA or from 3-HP (FIG. 1). For example, cells (such as a microorganism) producing 3 -FEP-CoA or 3 -FTP (such as those disclosed in Examples 8 and 9, respectively) can be engineered to make 1,3-propanediol by expressing nucleic acid molecules that encode for enzymes having an acetylating combined aldehyde/alcohol oxidoreductase (E.C.1.2.1.10) activity, or having aldehyde dehydrogenase (EC 1.2.1.-) and alcohol dehydrogenase (EC 1.1.1.1) activities.

For example, cells (such as a microorganism) producing 3-HP-CoA can be engineered to make 1,3-propanediol by expressing nucleic acid molecules that encode for enzymes having acetylating aldehyde/alcohol oxidoreductase (E.C.1.2.1.10) activity. These activities can be carried out by a single peptide or by two different peptides. Single enzymes include the multi-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No. M33504). Enzymes having a singular activity of aldehyde oxidoreductase (EC 1.2.1.3) or alcohol oxidoreductase (EC 1.1.1.1) are known. Nucleic acid molecules encoding for acylating aldehyde dehydrogenase from E. coli (GenBank Accession No. Y09555) and alcohol dehydrogenase from Z. mobilis (GenBank Accession No. M32100) have been isolated and sequenced. The coding sequences can be cloned into a 3-HP-CoA producing organism or cell by well-known molecular biology techniques. Expression of these enzymes in 3 -HP- CoA producing cells will impart it the ability to convert 3-HP-CoA to 1,3-propanediol. The substrate specificity of these enzymes for 3 -HP-CoA can be changed or improved using well-known techniques such as error prone PCR or mutator E. coli strains.

In a particular example, a transformed cell that produces 1,3-propanediol can include one or more nucleic acid sequences (such as an exogenous nucleic acid molecule) that encode a protein having acetylating combined aldehyde/alcohol oxidoreductase activity (EC 1.2.1.10), and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase or CoA synthetase activity, beta- alanyl-CoA ammonia lyase activity, and 3-HP-CoA dehydratase activity. In another particular example, a transformed cell that produces 1,3-propanediol can include one or more nucleic acid sequences that encode a protein having aldehyde dehydrogenase activity (EC 1.2.1.3) and one or more nucleic acid sequences that encode a protein having alcohol dehydrogenase activity (EC 1.1.1.1), and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase activity, one or more nucleic acid sequences that encode a protein having beta-alanyl-CoA ammonia lyase activity, one or more nucleic acid sequences that encode a protein having 3 -HP-CoA dehydratase activity, and one or more nucleic acid sequences that encode a protein having 3- hydroxypropionyl-CoA hydrolase activity. In particular examples, the cell also comprises a nucleic acid that encodes alanine 2,3-aminomutase.

A cell that produces 1,3 propanediol overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or EPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of 1,3 propanediol in the cell. Therefore, 3-HP or 3-HP-CoA can be converted to 1,3 propanediol in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP- CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, aldehyde dehydrogenase activity (EC 1.2.1.3), alcohol dehydrogenase activity (EC 1.1.1.1), and acetylating combined aldehyde/alcohol oxidoreductase activity (EC 1.2.1.10).

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, aldehyde dehydrogenase activity (EC 1.2.1.3), alcohol dehydrogenase activity (EC 1.1.1.1), and acetylating combined aldehyde/alcohol oxidoreductase activity (EC 1.2.1.10). In another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity and aldehyde dehydrogenase activity (EC 1.2.1.3) and alcohol dehydrogenase activity (EC 1.1.1.1). Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert 3-HP or 3-HP-CoA to 1,3- propanediol.

Methods of producing 1,3-propanediol in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and acetylating combined aldehyde/alcohol oxidoreductase activity (EC 1.2.1.10) or aldehyde dehydrogenase activity (EC 1.2.1.3) and alcohol dehydrogenase activity (EC 1.1.1.1), under conditions sufficient for producing 1,3 propanediol from 3-HP or 3-HP-CoA. In another example, the method includes culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity, aldehyde dehydrogenase activity (EC 1.2.1.3), and alcohol dehydrogenase activity (EC 1.1.1.1).

In a particular example, such methods of making 1,3-propanediol include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, with a nucleic acid encoding a peptide having 3-HP-CoA dehydratase activity, with a nucleic acid encoding a peptide having 3- hydroxypropionyl-CoA hydrolase activity, and with a nucleic acid encoding a peptide having acetylating combined aldehyde/alcohol oxidoreductase activity (EC 1.2.1.10) and culturing the transfected cell to allow the transfected cell to make 1,3-propanediol. In another particular example, such methods of making 1,3-propanediol include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having beta alanine-2-oxoglutarate aminotransferase activity, with a nucleic acid encoding a peptide having 3 -HP dehydrogenase activity, with a nucleic acid encoding a peptide having aldehyde dehydrogenase activity (EC 1.2.1.3), and with a nucleic acid encoding a peptide having alcohol dehydrogenase activity (EC 1.1.1.1), and culturing the transfected cell to allow the transfected cell to make 1,3-propanediol. The resulting 1,3-propanediol can be isolated from the cell culture medium or extracted from the cells.

Methods of producing 1,3-propanediol from 3-HP-CoA in vitro are also provided. Conversion of 3-HP-CoA to 1,3-propanediol can be achieved by contacting 3-HP-CoA with an enzyme having acetylating aldehyde oxidoreductase activity and an enzyme having alcohol oxidoreductase activity to form 1,3-propanediol. In addition, 3-HP can be converted to 1,3-propanediol by contacting 3-HP with an enzyme having aldehyde dehydrogenase activity (EC 1.2.1.-) and an enzyme having alcohol dehydrogenase activity (EC 1.1.1.1). Alternatively, chemical methods of producing 1,3-propanediol from 3-HP in vitro can be used, such as the method disclosed in US 2005/0283029 by Meng, Tsobanakis, and Abraham. In one example, 3-HP is converted into 1,3-propanediol by hydrogenation. Hydrogenating an organic acid such as 3-HP can be performed using any method such as those used to hydrogenate succinic acid or lactic acid. For example, 3-HP can be hydrogenated using a metal catalyst.

The formation of 1,3-propanediol during fermentation or in an in vitro assay can be analyzed using a High Performance Liquid Chromatography (HPLC). The chromatographic separation can be achieved by using a Bio-Rad 87H ion-exchange column. A mobile phase of 0.01N sulfuric acid is passed at a flow rate of 0.6 ml/min and the column maintained at a temperature of 45-65°C. The presence of 1,3-propanediol in the sample can be detected using a refractive index detector (Skraly et al, Appl. Environ. Microbiol. 64:98-105, 1998).

Example 12

Producing Polymerized 3-HP

This example describes methods of producing polymerized 3-HP, and cells that can produce polymerized 3-HP. Polymerized 3-HP can be generated from 3-HP-CoA or 3-HP (FIG. 1). For example, cells (such as a microorganism) producing 3 -HP-CoA (such as those described in Example 8) can be engineered to make polymerized 3-HP by expressing nucleic acid molecules that encode for enzymes having poly hydroxyacid synthase activity, and cells producing 3-HP (such as those described in Example 9) can be engineered to make polymerized 3-HP by expressing nucleic acid molecules that encode for enzymes having esterase activity. For example, cells producing 3 -HP-CoA (such as those described in Example 8) can be engineered to make polymerized 3-HP by expressing nucleic acid molecules that encode for enzymes having poly hydroxyacid synthase activity. In a particular example, a transformed cell that produces polymerized 3-HP can include one or more nucleic acid sequences (such as an exogenous nucleic acid molecule) that encode a protein having poly hydroxyacid synthase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase activity, one or more nucleic acid sequences that encode a protein having beta-alanyl-CoA ammonia lyase activity, and one or more nucleic acid sequences that encode a protein having 3 -HP-CoA dehydratase activity. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3-aminomutase.

In another example, cells producing 3-HP (such as those described in Example 9) can be engineered to make polymerized 3-HP by expressing nucleic acid molecules that encode for enzymes having esterase activity. In a particular example, a transformed cell that produces polymerized 3-HP can include one or more nucleic acid sequences that encode a protein having esterase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase or CoA synthetase activity, one or more nucleic acid molecules that encode for an enzyme having beta-alanyl- CoA ammonia lyase activity, one or more nucleic acid molecules that encode for an enzyme having 3 -HP-CoA dehydratase activity, and one or more nucleic acid molecules that encode for an enzyme having 3-hydroxypropionyl-CoA hydrolase activity. In another particular example, a transformed cell that produces polymerized 3-HP can include one or more nucleic acid sequences that encode a protein having poly hydroxyacid synthase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having beta alanine-2-oxoglutarate aminotransferase activity, and one or more nucleic acid molecules that encode for an enzyme having 3-HP dehydrogenase activity. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3- aminomutase.

A cell that produces polymerized 3-HP overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxiπ, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of polymerized 3 -HP in the cell. Therefore, 3 -HP can be converted to polymerized 3 -HP in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP- CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and esterase activity, or in the presence of alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3 -HP dehydrogenase activity, and esterase activity. Similarly, 3-HP-CoA can be converted to polymerized 3-HP in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and poly hydroxyacid synthase activity.

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP- CoA dehydratase activity, and poly hydroxyacid synthase activity. In another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3-HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and esterase activity. In yet another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity, and esterase activity. In another example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and poly hydroxyacid synthase activity. Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert 3 -HP-CoA or 3 -HP to polymerized 3 -HP.

Methods of producing polymerized 3-HP in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, and poly hydroxyacid synthase activity under conditions sufficient for producing polymerized 3-HP from 3-HP-CoA. Similarly, polymerized 3-HP can be produced in vivo from 3-HP by culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, 3 -HP-CoA dehydratase activity, 3-hydroxypropionyl-CoA hydrolase activity, and esterase activity. In addition, polymerized 3-HP can be produced in vivo from 3-HP by culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with beta alanine-2-oxoglutarate aminotransferase activity, 3-HP dehydrogenase activity, and esterase activity.

In a particular example, such methods of making polymerized 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase activity, with a nucleic acid encoding a peptide having beta-alanyl- CoA ammonia lyase activity, with a nucleic acid encoding a peptide having 3-HP-CoA dehydratase activity, and with a nucleic acid encoding a peptide having poly hydroxyacid synthase activity and culturing the transfected cell to allow the transfected cell to make polymerized 3-HP.

In another example, methods of making polymerized 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, with a nucleic acid encoding a peptide having 3- HP-CoA dehydratase activity, with a nucleic acid encoding a peptide having 3- hydroxypropionyl-CoA hydrolase activity, and with a nucleic acid encoding a peptide having esterase activity, and culturing the transfected cell to allow the transfected cell to make polymerized 3-HP.

In yet another example, methods of making polymerized 3-HP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having beta alanine-2-oxoglutarate aminotransferase activity, with a nucleic acid encoding a peptide having 3-HP dehydrogenase activity, and esterase activity, and with a nucleic acid encoding a peptide having poly hydroxyacid synthase activity, and culturing the transfected cell to allow the transfected cell to make polymerized 3-HP. The resulting polymerized 3-HP can be isolated from the cell culture medium or extracted from the cells.

Methods of producing polymerized 3-HP from 3-HP-CoA in vitro are also provided. Conversion of 3-HP-CoA to polymerized 3-HP can be achieved by contacting 3-HP-CoA with an enzyme having poly hydroxyacid synthase activity to form polymerized 3-HP. Methods of producing polymerized 3-HP from 3-HP in vitro are also provided. Conversion of 3-HP to polymerized 3-HP can be achieved by contacting 3-HP with an enzyme having esterase activity to form polymerized 3-HP. These conversions can also be performed using a combination of in vivo and in vitro methods.

The formation of polymerized 3-HP during fermentation in vivo or in an in vitro assay can be analyzed using size-exclusion HPLC.

Example 13 Producing Acrylic acid or Acrylate

This example describes methods of producing acrylate, and cells that can produce acrylate. Acrylate can be generated from acrylyl-CoA (FIG. 1). For example, cells (such as a microorganism) producing acrylyl-CoA from beta-alanine can be engineered to make acrylate by expressing nucleic acid molecules that encode for enzymes having acrylyl-CoA transferase or acrylyl-CoA hydrolase activity.

In a particular example, a transformed cell that produces acrylate can include one or more nucleic acid sequences (such as an exogenous nucleic acid sequence) that encode a protein having acrylyl-CoA transferase or acrylyl-CoA hydrolase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase activity, and one or more nucleic acid sequences that encode a protein having beta-alanyl-CoA ammonia lyase activity. In particular examples, the cell also includes a nucleic acid sequence that encodes alanine 2,3-aminomutase.

A cell that produces acrylate overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or LPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods. Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of acrylate in the cell. Therefore, aery Iy 1-CoA can be converted to acrylate in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and acrylyl-CoA hydrolase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and acrylyl-CoA transferase or acrylyl-CoA hydrolase activity.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these en:zymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert acrylyl-CoA to acrylate.

Methods of producing acrylate in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta- alanyl-CoA ammonia lyase activity, and acrylyl-CoA hydrolase activity under conditions sufficient for producing acrylate from acrylyl-CoA.

In a particular example, such methods of making acrylate include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, and with a nucleic acid encoding a peptide having acrylyl-CoA transferase or acrylyl-CoA hydrolase activity and culturing the transfected cell to allow the transfected cell to make acrylate. The resulting acrylate can be isolated from the cell. Methods of producing acrylate from acrylyl-CoA in vitro are also provided.

Conversion of acrylyl-CoA to acrylate can be achieved by contacting acrylyl-CoA with an enzyme having acrylyl-CoA transferase or acrylyl-CoA hydrolase activity to form acrylate, or by non-enzymatic hydrolysis methods. These conversions can also be performed using a combination of in vivo and in vitro methods. In another example, 3 -HP can be dehydrated to form acrylic acid. Any method can be used to perform a dehydration reaction. For example, 3 -HP can be heated in the presence of a catalyst (such as a metal or mineral acid catalyst) to form acrylic acid.

The formation of acrylate during fermentation in vivo or in an in vitro conversion can be analyzed using the method described in Schweiger and Buckel, FEBS Letts 185:253- 6, 1985. Example 14 Producing Polymerized Acrylate

This example describes methods of producing polymerized acrylate, and cells that can produce polymerized acrylate. Polymerized acrylate can be generated from acrylyl- CoA (FIG. 1). For example, cells (such as a microorganism) producing acrylyl-CoA from beta-alanine can be engineered to make polymerized acrylate by expressing nucleic acid molecules that encode for enzymes having poly hydroxyacid synthase activity.

In a particular example, a transformed cell that produces polymerized acrylate can include one or more nucleic acid sequences (such as an exogenous nucleic acid sequence) that encode a protein having poly hydroxyacid synthase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase activity, and one or more nucleic acid sequences that encode a protein having beta-alanyl-CoA ammonia lyase activity. In particular examples, the cell also includes a nucleic acid sequence that encodes alanine 2,3-aminomutase. A cell that produces polymerized acrylate overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods. Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of polymerized acrylate in the cell. Therefore, acrylyl-CoA can be converted to polymerized acrylate in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and poly hydroxyacid synthase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and poly hydroxyacid synthase activity.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert aery Iy 1-CoA to polymerized aery late.

Methods of producing polymerized acrylate in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase activity, beta-alanyl-CoA ammonia lyase activity, and poly hydroxyacid synthase activity under conditions sufficient for producing polymerized acrylate from acrylyl-CoA.

In a particular example, such methods of making polymerized acrylate include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase activity, with a nucleic acid encoding a peptide having beta-alanyl- CoA ammonia lyase activity, and with a nucleic acid encoding a peptide having poly hydroxyacid synthase activity and culturing the transfected cell to allow the transfected cell to make polymerized acrylate. The resulting polymerized acrylate can be isolated from the cell culture medium or extracted from the cells.

Methods of producing polymerized acrylate from acrylyl-CoA in vitro are also provided. Conversion of acrylyl-CoA to polymerized acrylate can be achieved by contacting acrylyl-CoA with an enzyme having poly hydroxyacid synthase activity to form polymerized acrylate. These conversions can also be performed using a combination of in vivo and in vitro methods.

The formation of polymerized acrylate during fermentation in vivo or in an in vitro assay can be analyzed using size-exclusion HPLC. Example 15 Producing an Ester of Acrylate

This example describes methods of producing an ester of acrylate and cells that can produce an ester of acrylate. Particular examples of esters of acrylate include, but are not limited to: methyl acrylate, ethyl acrylate, propyl acrylate, and butyl acrylate. An ester of acrylate can be generated from acrylyl-CoA through an acrylate intermediate (FIG. 1). For example, cells (such as a microorganism) producing acrylyl-CoA from beta-alanine can be engineered to make an ester of acrylate by expressing nucleic acid molecules that encode for enzymes having Co-A transferase or CoA synthetase activity, and lipase or esterase activity. In a particular example, a transformed cell that produces an ester of acrylate can include one or more nucleic acid sequences (such as an exogenous nucleic acid sequence) that encode a protein having lipase or esterase activity, and in some examples the cell further include one or more nucleic acid sequences that encode a protein having CoA transferase or CoA synthetase activity, and one or more nucleic acid sequences that encode a protein having beta-alanyl-CoA ammonia lyase activity, hi particular examples, the cell also includes a nucleic acid sequence that encodes alanine 2,3-aminomutase.

A cell that produces an ester of acrylate overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of an ester of acrylate in the cell. Therefore, acrylyl-CoA can be converted to an ester of acrylate in the presence of alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and lipase or esterase activity.

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and lipase or esterase activity.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert acrylyl-CoA to an ester of acrylate. Methods of producing an ester of acrylate in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with CoA transferase or CoA synthetase activity, beta-alanyl-CoA ammonia lyase activity, and lipase or esterase activity under conditions sufficient for producing an ester of acrylate from acrylyl-CoA. In a particular example, such methods of making an ester of acrylate include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having CoA transferase or CoA synthetase activity, with a nucleic acid encoding a peptide having beta-alanyl-CoA ammonia lyase activity, and with a nucleic acid encoding a peptide having lipase or esterase activity and culturing the transfected cell to allow the transfected cell to make an ester of acrylate. The resulting ester of acrylate can be isolated from the cell culture medium or extracted from the cell.

Methods of producing an ester of acrylate from acrylyl-CoA in vitro are also provided. Conversion of acrylyl-CoA to polymerized acrylate can be achieved by contacting acrylyl-CoA with an enzyme having CoA transferase or CoA synthetase activity to generate acrylate, and contacting acrylate with an enzyme having lipase or esterase activity to form polymerized acrylate. These conversions can also be performed using a combination of in vivo and in vitro methods. The formation of an ester of acrylate during fermentation in vivo or in an in vitro assay can be analyzed using HPLC or GC methods.

EXAMPLE 16 Production of Pantothenate from Beta- Alanine

This example describes methods of producing pantothenate and cells that can produce pantothenate. Pantothenate can be generated from beta-alanine (FIG. 2). For example, cells (such as a microorganism) producing beta-alanine (for example from alpha alanine) can be engineered to make pantothenate by expressing nucleic acid molecules that encode for enzymes having alpha-ketopantoate hydroxymethyltransferase (E.C. 2.1.2.1 1), alpha-ketopantoate reductase (E.C. 1.1.1.169), and pantothenate synthase (E.C. 6.3.2.1) activity.

In a particular example, a transformed cell that produces pantothenate can include one or more exogenous nucleic acid sequences that encode a protein having alpha- ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3-aminomutase.

A cell that produces pantothenate overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of pantothenate in the cell. Therefore, beta-alanine can be converted to pantothenate in the presence of alanine 2,3-aminomutase activity in combination with alpha- ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity. In some examples, exogenous pantothenic acid is also included.

In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity. In some examples, the cell culture medium further includes pantothenic acid.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available or are disclosed herein. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert beta-alanine to pantothenate.

Methods of producing pantothenate in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, and pantothenate synthase activity under conditions sufficient for producing pantothenate from beta-alanine. In some examples, the cell culture medium further includes pantothenic acid.

In a particular example, such methods of making pantothenate include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having alpha-ketopantoate hydroxymethyltransferase activity, with a nucleic acid encoding a peptide having alpha-ketopantoate reductase activity, and with a nucleic acid encoding a peptide having pantothenate synthase activity, and culturing the transfected cell to allow the transfected cell to make pantothenate. In some examples, the cell culture medium further includes pantothenic acid. The resulting pantothenate can be isolated from the cell medium or extracted from the cell.

Methods of producing pantothenate from beta-alanine in vitro are also provided. Conversion of beta-alanine to pantothenate can be achieved by contacting beta-alanine with a peptide having alpha-ketopantoate hydroxymethyltransferase activity, with a peptide having alpha-ketopantoate reductase activity, and with a peptide having pantothenate synthase activity, to form pantothenate. In some examples, the in vitro reaction further includes pantothenic acid. These conversions can also be performed using a combination of in vivo and in vitro methods. The formation of pantothenate during fennentation in vivo or in an in vitro assay can be analyzed using known methods (for example see U.S. Patent No. 6,184,006 to Rieping et al. and U.S. Patent No. 6,177,264 to Eggeling et al). For example, a quantitative determination of D-pantothenate can be made by using the Lactobacillus plantarum pantothenate assay (test strain: Lactobacillus plantarum ATCC 8014, Cat. No. 3211-30-3; culture medium: Bacto pantothenate assay medium (DIFCO Laboratories, Michigan, USA), cat. No. 0604-15-3). This indicator strain can grow only in the presence of pantothenate in the indicated culture medium and displays a photometrically measurable, linear dependency of the growth on the concentration of pantothenate in the medium. The hemicalcium salt of pantothenate can be used for calibration (Sigma Catalog Number P 2250). The optical density can be determined at a wavelength of 580 nm.

Example 17 Production of CoA

This example describes methods of producing coenzyme A (CoA) and cells that can produce CoA. CoA can be generated from pantothenate (FIG. 2). For example, cells (such as a microorganism) producing pantothenate (for example from beta alanine) can be engineered to make CoA by expressing nucleic acid molecules that encode for enzymes having pantothenate kinase activity, 4'-phosphopantethenoyl-l -cysteine synthetase activity, 4'-phosphopantothenoylcysteine decarboxylase activity, ATP:4'-phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity.

In a particular example, a transformed cell that produces CoA can include one or more exogenous nucleic acid sequences that encode a protein having pantothenate kinase activity, 4'-phosphopantethenoyl-l -cysteine synthetase activity, 4'- phosphopantothenoylcysteine decarboxylase activity, ATP:4'-phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity. In particular examples, the cell also includes a nucleic acid that encodes alanine 2,3-aminomutase. For example, a cell that produces pantothenate (for example those described in Example 16), can be transformed with one or more exogenous nucleic acid sequences that encode a protein having pantothenate kinase activity, 4'-phosphopantethenoyl-l -cysteine synthetase activity, 4'-phosphopantothenoylcysteine decarboxylase activity, ATP:4'-phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity, thereby allowing the cell to produce CoA. In some examples, the cell culture medium further includes pantothenic acid.

A cell that produces CoA overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transfonned or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase alanine 2,3-aminomutase activity in the cell, thereby increasing production of CoA in the cell. Therefore pantothenate an be converted to CoA in the presence of alanine 2,3-aminomutase activity in combination with alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, pantothenate synthase activity, pantothenate kinase activity, 4'-phosphopantethenoyl-l -cysteine synthetase activity, 4'-phosphopantothenoylcysteine decarboxylase activity, ATP:4'- phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes alanine 2,3-aminomutase activity in combination with alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, pantothenate synthase activity, pantothenate kinase activity, 4'- phosphopantethenoyl-1 -cysteine synthetase activity, 4'-phosphopantothenoylcysteine decarboxylase activity, ATP:4'-phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity. In some examples, the cell culture medium further includes pantothenic acid.

Such enzyme activities can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for the enzyme (such as introducing an exogenous nucleic acid into the cell). Genes and cDNAs encoding for these enzymes are publicly available. Such nucleic acid molecules can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of these enzymes in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert pantothenate to CoA.

Methods of producing CoA in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has alanine 2,3-aminomutase activity in combination with alpha-ketopantoate hydroxymethyltransferase activity, alpha-ketopantoate reductase activity, pantothenate synthase activity, pantothenate kinase activity, 4'-phosphopantethenoyl-l -cysteine synthetase activity, 4'-phosphopantothenoylcysteine decarboxylase activity, ATP:4'- phosphopantetheine adenyltransferase activity, and dephospho-CoA kinase activity, under conditions sufficient for producing CoA from pantothenate. In some examples, the cell culture medium further includes pantothenic acid. In a particular example, such methods of making CoA include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having alanine 2,3-aminomutase activity, with a nucleic acid encoding a peptide having alpha- ketopantoate hydroxymethyltransferase activity, with a nucleic acid encoding a peptide having alpha-ketopantoate reductase activity, with a nucleic acid encoding a peptide having pantothenate synthase activity, with a nucleic acid encoding a peptide having pantothenate kinase activity, with a nucleic acid encoding a peptide having 4'-phosphopantethenoyl-l- cysteine synthetase activity, with a nucleic acid encoding a peptide having 4'- phosphopantothenoylcysteine decarboxylase activity, with a nucleic acid encoding a peptide having ATP:4'-phosphopantetheine adenyltransferase activity, and with a nucleic acid encoding a peptide having dephospho-CoA kinase activity, and culturing the transfected cell to allow the transfected cell to make pantothenate. The resulting CoA can be isolated from the cell.

Methods of producing CoA from pantothenate in vitro are also provided. Conversion of pantothenate to CoA can be achieved by contacting pantothenate with a peptide having pantothenate kinase activity, with a peptide having 4'-phosphopantethenoyl- 1 -cysteine synthetase activity, with a peptide having 4'-phosphopantothenoylcysteine decarboxylase activity, with a peptide having ATP:4'-phosphopantetheine adenyltransferase activity, and with a peptide having dephospho-CoA kinase activity. In some examples, the in vitro reaction further includes pantothenic acid. These conversions can also be performed using a combination of in vivo and in vitro methods.

The formation of CoA during fermentation in vivo or in an in vitro assay can be analyzed using methods described in Jackowski and Rock, J. Bacteriol. 148:926-32, 1981.

Example 18

Production of Beta-Lysine

This example describes methods of producing beta-lysine, and cells that can produce beta-lysine. Beta-lysine can be generated from alpha-lysine, for example via lysine 2,3-aminomutase in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing alpha-lysine can be engineered to make beta- lysine by expressing nucleic acid molecules that encode for an enzyme having lysine 2,3- aminomutase activity in the cell, for example using recombinant technologies.

The cell that produces beta-lysine overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase lysine 2,3-aminomutase activity in the cell, thereby increasing production of beta-lysine in the cell. Therefore, alpha-lysine can be converted to beta-lysine in the presence of lysine 2,3-aminomutase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes lysine 2,3-aminomutase activity. Such lysine 2,3-aminomutase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for a lysine 2,3-aminomutase. Genes (or cDNAs) encoding for lysine 2,3-arainomutase are publicly available, and can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of lysine 2,3-aminomutase in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert alpha-lysine to beta-lysine.

Methods of producing beta-lysine in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has lysine 2,3-aminomutase activity, under conditions sufficient for producing beta-lysine from alpha-lysine. In a particular example, such methods of making beta-lysine include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having lysine 2,3-aminomutase activity, and culturing the transfected cell to allow the transfected cell to make beta-lysine. The resulting beta-lysine can be isolated from the cell medium, or extracted from the cell (for example using the methods described in Maharjan and Ferenci, Anal. Biochem. 313:145-54, 2003).

Methods of producing beta-lysine from alpha-lysine in vitro are also provided. Conversion of alpha-lysine to beta-lysine can be achieved by contacting alpha-lysine with an enzyme having lysine 2,3-aminomutase activity, and with enzymes having flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof, to enhance the activity of the lysine 2,3-aminomutase. Such conversion can also be performed by a combination of in vivo and in vitro methods.

The formation of beta-lysine during fermentation in vivo or in an in vitro assay can be analyzed using HPLC (see Example 2).

Example 19

Production of Beta-Arginine

This example describes methods of producing beta-alanine, and cells that can produce beta-arginine. Beta-arginine can be generated from alpha-arginine, for example via arginine 2,3-aminomutase in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing alpha-arginine can be engineered to make beta-arginine by expressing nucleic acid molecules that encode for an enzyme having arginine 2,3-aminomutase activity in the cell, for example using recombinant technologies.

The cell that produces beta-arginine overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase arginine 2,3-aminomutase activity in the cell, thereby increasing production of beta-arginine in the cell. Therefore, alpha-arginine can be converted to beta- arginine in the presence of arginine 2,3-aminomutase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes arginine 2,3-aminomutase activity. Such arginine 2,3-aminomutase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for an arginine 2,3-aminomutase. Genes (or cDNAs) encoding for arginine 2,3-aminomutase are publicly available, and can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of arginine 2,3-aminomutase in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert alpha-arginine to beta-arginine.

Methods of producing beta-arginine in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has arginine 2,3-aminomutase activity, under conditions sufficient for producing beta- arginine from alpha-arginine. In a particular example, such methods of making beta- arginine include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having arginine 2,3-aminomutase activity, and culturing the transfected cell to allow the transfected cell to make beta-arginine. The resulting beta- arginine can be isolated from the cell medium or extracted from the cell. Methods of producing beta-arginine from alpha-arginine in vifro are also provided. Conversion of alpha-arginine to beta-arginine can be achieved by contacting alpha-arginine with an enzyme having arginine 2,3-aminomutase activity, and with enzymes having flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof, to enhance the activity of the arginine 2,3-aminomutase. Such conversion can also be performed by a combination of in vivo and in vitro methods.

The formation of beta-arginine during fermentation in vivo or in an in vitro assay can be analyzed using HPLC (see Example 2).

Example 20

Production of EPP and DMAPP

This example describes methods of producing IPP and DMAPP, and cells that can produce IPP and DMAPP. Such methods and cells can be used to produce downstream products of IPP and DMAPP, such as one or more carotenoids or terpenoids. IPP and DMAPP can be generated from HMBPP, for example via LytB, in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing 2- C-methyl-D-erythritol cyclopyrophosphate can be engineered to make HMBPP by expressing nucleic acid molecules that encode for an enzyme having GcpE activity in the cell, and cells making HMBPP can be engineered to make IPP and DMAPP by expressing nucleic acid molecules that encode for an enzyme having LytB activity in the cell, for example using recombinant technologies.

The cell that produces IPP, DMAPP, or both, overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods. Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase GcpE activity, LytB activity, or both activities in the cell, thereby increasing production of IPP and DMAPP in the cell. Therefore, 2-C-methyl-D-erythritol cyclopyrophosphate can be converted to HMBPP in the presence of GcpE activity, and HMBPP can be converted to IPP and DMAPP in the presence of LytB activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes GcpE activity, LytB activity, or both GcpE and LytB activity. The GcpE and LytB can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for GcpE and LytB. Genes (or cDNAs) encoding for GcpE and LytB are publicly available, and can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of GcpE and LytB in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert HMBPP to IPP and DMAPP. Methods of producing IPP and DMAPP in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, and has LytB activity (and in some examples also GcpE activity), under conditions sufficient for producing IPP and DMAPP from HMBPP. In a particular example, such methods of making IPP and DMAPP include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having LytB activity (and in some examples also with a nucleic acid encoding a peptide having GcpE activity), and culturing the transfected cell to allow the transfected cell to make IPP and DMAPP. The resulting IPP and DMAPP can be isolated from the cell medium or extracted from the cell. Methods of producing IPP and DMAPP from HMBPP in vitro are also provided.

Conversion of HMBPP to EPP and DMAPP can be achieved by contacting HMBPP with an enzyme having LytB activity, and with enzymes having flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof, to enhance the activity of the LytB. Such conversion can also be performed by a combination of in vivo and in vitro methods.

The formation of IPP and DMAPP during fermentation in vivo or in an in vitro assay can be analyzed using HPLC. Example 21 Production of Lipoic Acid

This example describes methods of producing lipoic acid, and cells that can produce lipoic acid. Lipoic acid can be generated from octanoic acid, for example via lipoate synthase in vitro, in vivo (such as in a cell), or combinations thereof. For example, cells (such as a microorganism) producing or supplied with octanoic acid can be engineered to make lipoic acid by expressing nucleic acid molecules that encode for an enzyme having lipoate synthase activity in the cell, for example using recombinant technologies.

The cell that produces lipoic acid overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof. In one example, flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by at least 50%, such as at least 100%, or at least 200%, for example as compared to an amount of flavodoxin, ferredoxin, or flavodoxin reductase expression in a non-transformed or normal cell of the same type. Methods to achieve overexpression are known. In one example flavodoxin, ferredoxin, or flavodoxin reductase is overexpressed by expressing flavodoxin, ferredoxin, or flavodoxin reductase from an exogenous promoter. For example, the native flavodoxin, ferredoxin, or flavodoxin reductase promoter can be replaced with an exogenous promoter, such as the lac promoter inducible with lactose or IPTG, or the tet promoter inducible with tetracycline, or a constitutive promoter, using standard molecular biology methods (for example see Example 4). In another example, overexpression of flavodoxin, ferredoxin, or flavodoxin reductase (or combination thereof) is achieved by expressing a nucleic acid that encodes flavodoxin, ferredoxin, or flavodoxin reductase on a plasmid using standard molecular biology methods.

Overexpression of flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will increase lipoate synthase activity in the cell, thereby increasing production of lipoic acid in the cell. Therefore, octanoic acid can be converted to lipoic acid in the presence of lipoate synthase activity. In one example, a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, also includes lipoate synthase activity. Such lipoate synthase can be native to the cell, or can be supplied to the cell by expressing one or more nucleic acid molecules that encode for a lipoate synthase. Genes (or cDNAs) encoding for lipoate synthase are publicly available, and can be cloned into a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, by well-known molecular biology techniques. Expression of lipoate synthase in cells that overexpress flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof, will impart the cell an ability to convert octanoic acid to lipoic acid. Methods of producing lipoic acid in vivo can therefore include culturing a cell that overexpresses flavodoxin, ferredoxin, fJavodoxin reductase, or a combination thereof, and has lipoate synthase activity, under conditions sufficient for producing lipoic acid from octanoic acid. In a particular example, such methods of making lipoic acid include transfecting a cell that overexpresses flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof (such as those disclosed in Example 4), with a nucleic acid encoding a peptide having lipoate synthase activity, and culturing the transfected cell to allow the transfected cell to make lipoic acid. The resulting lipoic acid can be isolated from the cell medium or extracted from the cell. Methods of producing lipoic acid from octanoic acid in vitro are also provided.

Conversion of octanoic acid to lipoic acid can be achieved by contacting octanoic acid with an enzyme having lipoate synthase activity, and with enzymes having flavodoxin activity, ferredoxin activity, flavodoxin reductase activity, or a combination thereof, to enhance the activity of the lipoate synthase. Such conversion can also be performed by a combination of in vivo and in vitro methods.

The formation of lipoic acid during fermentation in vivo or in an in vitro assay can be analyzed using methods described in WO 02/085293 A2.

Example 22 Recombinant Expression

With the disclosed and publicly available enzyme nucleic acid and amino acid sequences, such as lipoate synthase, biotin synthase, GcpE, LytB, aminomutases that interconvert alpha and beta amino acids (such as alanine 2,3-aminomutase), CoA transferase, CoA synthetase, beta-alanyl-CoA ammonia lyase, 3 -HP-CoA dehydratase, beta- alanine-2-oxo-glutarate aminotransferase, 3-hydroxypropionate dehydrogenase, glutamate dehydrogenase, 3 -HP-CoA hydrolase, poly hydroxyacid synthase, lipase, esterase, CoA hydrolase, alpha-ketopantoate hydroxymethyltransferase, alpha-ketopantoate reductase, pantothenate synthase, pantothenate kinase, 4^r-phosphopantethenoyl-l -cysteine synthetase, 4'-phosphopantothenoylcysteine decarboxylase, ATP:4'-ρhosphopantetheine adenyltransferase, dephospho-CoA kinase, acetylating aldehyde/alcohol oxidoreductase, aldehyde dehydrogenase, and alcohol dehydrogenase, as well as variants, fragments and fusions thereof, the expression or purification of such proteins by standard laboratory techniques is enabled. Similarly, with the disclosed and publicly available ferredoxin, flavodoxin, and flavodoxin reductase nucleic acid and amino acid sequences, as well as variants, fragments and fusions thereof, the expression or purification of such proteins by standard laboratory techniques is enabled. One skilled in the art will understand that peptides can be produced recombinantly in any cell or organism of interest, and purified prior to use, for example prior to production of 3-HP, pantothenate, and derivatives thereof. Methods for producing recombinant proteins are well known in the art. Therefore, the scope of this disclosure includes recombinant expression of any protein or fragment thereof, such as an enzyme. For example, see U.S. Patent No: 5,342,764 to Johnson et al; U.S. Patent No: 5,846,819 to Pausch e/ α/.; U.S. Patent No: 5,876,969 to Fleer et al. and Sambrook et al {Molecular Cloning: A Laboratoiγ Manual, Cold Spring Harbor, New York, 1989, Ch. 17). Briefly, partial, full-length, or variant cDNA sequences, which encode for a functional enzyme protein (such as those listed in FIGS. 1 and 2), can be ligated into an expression vector, such as a bacterial expression vector. Proteins can be produced by placing a promoter upstream of the cDNA sequence. Examples of promoters include, but are not limited to lac, trp, tac, trc, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.

Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, 1981, Nature 292:128), pKK177-3 (Amann and Brosius, 1985, Gene 40: 183) and pET-3 (Studier and Moffatt, 1986, J. MoI. Biol. 189:113). A DNA sequence can be transferred to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al, 1987, Science 236:806-12). These vectors can be introduced into a variety of hosts including cells and simple or complex organisms, such as bacteria, yeast, fungi (Timberlake and Marshall, 1989, Science 244:1313-7), invertebrates, and plants (Gasser and Fraley, 1989, Science 244:1293), which are rendered transgenic by the introduction of the heterologous cDNA.

The transfer of DNA into eukaryotic cells is a conventional technique. For example, vectors can be introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52:466) strontium phosphate (Brash et al, 1987, MoI. Cell Biol. 7:2013), electroporation (Neumann et al, 1982, EMBO J. 1:841), lipofection (Feigner et al, 1987, Proc. Natl. Acad. Sci USA 84:7413), DEAE dextran (McCuthan e/ α/., 1968, J. Natl. Cancer lnst. 41:351), microinjection (Mueller et al , 1978, Cell 15 : 579), protoplast fusion (Schafner, 1980, Proc. Natl Acad. Sci. USA 77:2163-7), or pellet guns (Klein et al, 1987, Nature 327:70). Alternatively, the cDNA can be introduced by infection with virus vectors.

Example 23 Peptide Synthesis and Purification

The enzymes disclosed herein, such as lipoate synthase, biotin synthase, GcpE, LytB, aminomutases that interconvert alpha and beta amino acids (such as alanine 2,3- aminomutase), CoA transferase, CoA synthetase, beta-alanyl-CoA ammonia lyase, 3 -HP- Co A dehydratase, beta-alanine-2-oxo-glutarate aminotransferase, 3-hydroxypropionate dehydrogenase, glutamate dehydrogenase, 3-HP-CoA hydrolase, poly hydroxyacid synthase, lipase, esterase , CoA hydrolase, alpha-ketopantoate hydroxymethyltransferase, alpha-ketopantoate reductase, pantothenate synthase, pantothenate kinase, 4'- phosphopantethenoyl-1 -cysteine synthetase, 4'-phosphopantothenoylcysteine decarboxylase, ATP:4'-phosphopantetheine adenyltransferase, dephospho-CoA kinase, acetylating aldehyde/alcohol oxidoreductase, aldehyde dehydrogenase and alcohol dehydrogenase (and variants, fusions, polymorphisms, fragments, and mutants thereof) can be chemically synthesized by any of a number of manual or automated methods of synthesis known in the art. For example, solid phase peptide synthesis (SPPS) is carried out on a 0.25 millimole (mmole) scale using an Applied Biosystems Model 43 IA Peptide Synthesizer and using 9- fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection, coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or 2-( 1 H-benzo-triazol- 1 -yl)- 1 , 1 ,3,3- tetramethyluronium hexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and using /7-hydroxymethylphenoxymethylpolystyrene (HMP) or Sasrin resin for carboxyl-terminus acids or Rink amide resin for carboxyl-terminus amides. Fmoc-derivatized amino acids are prepared from the appropriate precursor amino acids by tritylation and triphenylmethanol in trifluoroacetic acid, followed by Fmoc derivitization as described by Atherton et a {Solid Phase Peptide Synthesis, IRL Press: Oxford, 1989).

Sasrin resin-bound peptides are cleaved using a solution of 1% TFA in dichloromethane to yield the protected peptide. Where appropriate, protected peptide precursors are cyclized between the amino- and carboxyl-termini by reaction of the amino- terminal free amine and carboxyl-terminal free acid using diphenylphosphorylazide in nascent peptides wherein the amino acid sidechains are protected.

HMP or Rink amide resin-bound products are routinely cleaved and protected sidechain-containing cyclized peptides deprotected using a solution comprised of trifluoroacetic acid (TFA), optionally also comprising water, thioanisole, and ethanedithiol, in ratios of 100 : 5 : 5 : 2.5, for 0.5 - 3 hours at RT.

Crude peptides are purified by preparative high pressure liquid chromatography (HPLC), for example using a Waters Delta-Pak Cl 8 column and gradient elution with 0.1% TFA in water modified with acetonitrile. After column elution, acetonitrile is evaporated from the eluted fractions, which are then lyophilized. The identity of each product so produced and purified may be confirmed by fast atom bombardment mass spectroscopy (FABMS) or electrospray mass spectroscopy (ESMS).

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of increasing the activity of a radical S-adenosyl methionine (SAM) enzyme, comprising: increasing expression of a nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof in a cell, wherein increased expression of the flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof increases the activity of a radical SAM enzyme in the cell.

2. The method of claim 1, wherein increasing expression of the nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof comprises expressing the flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, from an exogenous promoter.

3. The method of claim 1, wherein the nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof comprises an exogenous nucleic acid molecule.

4. The method of claim 1, wherein the expression of a nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, is increased by at least

50%.

5. The method of claim 1, wherein the radical SAM enzyme is expressed by an exogenous nucleic acid encoding the radical SAM enzyme.

6. The method of claim 5, wherein the radical SAM enzyme comprises biotin synthase, lipoate synthase, GcpE, LytB, or an aminomutase that interconverts alpha and beta amino acids.

7. The method of claim 6, wherein the aminomutase that interconverts alpha and beta amino acids comprises alanine 2,3-aminomutase, arginine 2,3 -aminomutase, or lysine 2,3- aminomutase

8. The method of claim 3, further comprising introducing the exogenous nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof into the cell.

9. The method of claim 1, wherein the activity of the radical SAM enzyme in the cell increased by at least 20%.

10. The method of claim 1, wherein increasing the activity of the radical SAM enzyme in the cell results in increased production of a radical SAM enzyme product.

11. The method of claim 10, wherein the radical SAM enzyme comprises alanine 2,3- aminomutase and the radical SAM enzyme product comprises beta-alanine.

12. The method of claim 11, wherein the cell comprises at least one exogenous nucleic acid molecule that encodes the alanine 2,3-aminomutase, wherein the alanine 2,3-aminomutase is capable of producing beta-alanine from alpha-alanine.

13. The method of claim 10, wherein the radical SAM enzyme comprises alanine 2,3- aminomutase and the radical SAM enzyme product comprises 3-hydroxypropionic acid (3- HP).

14. The method of claim 13, wherein the cell comprises at least one exogenous nucleic acid that encodes an alanine 2,3-aminomutase that produces beta-alanine from alpha-alanine, and wherein the 3-HP is produced from the beta-alanine.

15. The method of claim 13, wherein the cell produces 3-HP.

16. The method of claim 13, wherein the cell further comprises:

CoA transferase or CoA synthetase activity; beta-alanyl-CoA ammonia lyase activity;

3 -HP-CoA dehydratase activity; and 3-hydroxypropionyl-CoA hydrolase activity.

17. The method of claim 13, wherein the cell farther comprises: beta alanine-2-oxoglutarate aminotransferase activity; and 3 -HP dehydrogenase activity.

18. The method of claim 10, wherein the radical SAM enzyme comprises alanine 2,3- aminomutase and the radical SAM enzyme product comprises pantothenic acid.

19. The method of claim 18, wherein the cell further comprises alpha-ketopantoate hydroxymethyltransferase activity alpha-ketopantoate reductase activity; and pantothenate synthase activity.

20. The method of claim 10, wherein the radical SAM enzyme comprises arginine 2,3- aminomutase and the radical SAM enzyme product comprises beta-arginine.

21. The method of claim 10, wherein the radical SAM enzyme comprises biotin synthase and the radical SAM enzyme product comprises biotin.

22. The method of claim 10, wherein the radical SAM enzyme comprises lipoate synthase and the radical SAM enzyme product comprises lipoic acid.

23. The method of claim 10, wherein the radical SAM enzyme comprises lysine 2,3- aminomutase and the radical SAM enzyme product comprises beta-lysine.

24. The method of claim 1, wherein the cell is a prokaryotic cell.

25. The method of claim 1, wherein the cell is a eukaryotic cell.

26. A transformed cell comprising increased radical SAM enzyme activity, wherein expression of a nucleic acid molecule encoding flavodoxin, ferredoxin, flavodoxin reductase, or a combination thereof is increased in the cell.

27. The transformed cell of claim 26, wherein an exogenous promoter controls expression of the nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof.

28. The transformed cell of claim 26, wherein the nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof comprises an exogenous nucleic acid molecule.

29. The transformed cell of claim 26, wherein the expression of a nucleic acid molecule that encodes flavodoxin, ferredoxin, flavodoxin reductase, or combination thereof, is increased by at least 50%.

30. The transformed cell of claim 26, wherein the activity of the radical SAM enzyme is increased by at least 20%.

31. The transformed cell of claim 26, wherein the cell further comprises an exogenous nucleic acid molecule encoding the radical SAM enzyme.

32. The transformed cell of claim 26, wherein the radical SAM enzyme comprises: biotin synthase, lipoate synthase, or an aminomutase that interconverts alpha and beta amino acids.

33. The transformed cell of claim 32, wherein the aminomutase that interconverts alpha and beta amino acids comprises alanine 2,3-aminomutase, arginine 2,3 -aminomutase, or lysine 2,3-aminomutase.

34. The transformed cell of claim 30, wherein increasing the activity of the radical SAM enzyme in the cell by at least 20% results in increased production of a radical SAM enzyme product by the cell.

35. The transformed cell of claim 34, wherein the radical SAM enzyme comprises lipoate synthase, and wherein the radical SAM enzyme product comprises lipoic acid.

36. The transformed cell of claim 34, wherein the radical SAM enzyme comprises biotin synthase, and wherein the radical SAM enzyme product comprises biotin.

37. The transformed cell of claim 34, wherein the radical SAM enzyme comprises lysine 2,3-aminomutase, and wherein the radical SAM enzyme product comprises beta-lysine.

38. The transformed cell of claim 30, wherein the radical SAM enzyme comprises alanine 2,3-aminomutase, and wherein the radical SAM enzyme product comprises beta-alanine, 3- HP, pantothenic acid, or combination thereof.

39. The transformed cell of claim 38, wherein the cell comprises at least one exogenous nucleic acid molecule that encodes the alanine 2,3-aminomutase, wherein the alanine 2,3- aminomutase is capable of producing beta-alanine from alpha-alanine.

40. The transformed cell of claim 39, wherein the cell produces 3-HP.

41. The transformed cell of claim 40, wherein the cell further comprises:

CoA transferase or CoA synthetase activity; beta-alanyl-CoA ammonia lyase activity; 3 -HP-CoA dehydratase activity; and 3-hydroxypropionyl-CoA hydrolase activity.

42. The transformed cell of claim 40, wherein the cell further comprises: beta alanine-2-oxoglutarate aminotransferase activity; and 3-HP dehydrogenase activity.

43. The transformed cell of claim 39, wherein the cell produces pantothenic acid.

44. The transformed cell of claim 43, wherein the cell further comprises alpha-ketopantoate hydroxymethyltransferase activity alpha-ketopantoate reductase activity; and pantothenate synthase activity.

45. The transformed cell of claim 34, wherein the radical SAM enzyme comprises arginine 2,3-aminomutase, and wherein the radical SAM enzyme product comprises beta-arginine.

46. The transformed cell of claim 26, wherein the cell is a prokaryotic cell.

47. The transformed cell of claim 46, wherein the prokaryotic cell is a Lactobacillus, Lactococcυs, Bacillus, or Escherichia cell.

48. The transformed cell of claim 47, wherein the prokaryotic cell is Escherichia coli.

49. The transformed cell of claim 25, wherein the cell is a eukaryotic cell.

50. The transformed cell of claim 49, wherein the eukaryotic cell is a yeast, plant, or animal cell.

51. The transformed cell of claim 49, wherein the cell is a plant cell.

52. A plant comprising the transformed cell of claim 51.

53. The transformed cell of claim 26, wherein the cell comprises the characteristics of a cell deposited as American Type Culture Collection No PTA-6837.

54. A method for making 3-HP, comprising: purifying beta-alanine from the cell of claim 39; contacting the beta-alanine with a peptide comprising CoA transferase activity to form beta-alanyl-CoA; contacting the beta-alanine CoA with a peptide comprising beta-alanyl-CoA ammonia lyase activity to form acrylyl-CoA; contacting the acrylyl-CoA with a peptide comprising 3HP-CoA dehydratase activity to form 3 -HP-CoA; and contacting 3-HP-CoA with a peptide comprising CoA transferase activity, 3- hydroxypropionyl-CoA hydrolase activity to make 3-HP.

55. A method for making 3-HP, comprising: purifying beta-alanine from the cell of claim 39; contacting the beta-alanine with a peptide comprising beta-alanine - 2-oxoglutarate aminotransferase activity to form malonic semialdehyde; and contacting the malonic semialdehyde with a peptide comprising 3-HP dehydrogenase activity to make 3-HP.

56. A method for making 3-HP, comprising: transfecting the cell of claim 40, with a nucleic acid encoding a peptide comprising CoA transferase activity, with a nucleic acid encoding a peptide comprising beta-alanyl- CoA ammonia lyase activity, and with a nucleic acid encoding a peptide comprising CoA transferase activity, 3-hydroxypropionyl-CoA hydrolase activity; and culturing the transfected cell to allow the transfected cell to make 3-HP.

57. A method for making 3-HP, comprising: transfecting the cell of claim 40, with a nucleic acid encoding a peptide comprising beta-alanine-2-oxoglutarate aminotransferase activity and with a nucleic acid encoding a peptide comprising 3-HP dehydrogenase activity; and culturing the transfected cell to allow the transfected cell to make 3-HP.

58. A method for making pantothenate, comprising: purifying beta-alanine from the cell of claim 39; and contacting the beta-alanine with alpha-ketopantoate hydroxymethyltransferase, alpha-ketopantoate reductase, and pantothenate synthase to make pantothenate.

59. A method for making pantothenate, comprising: transfecting the cell of claim 39 with a nucleic acid encoding a peptide comprising alpha-ketopantoate hydroxymethyltransferase activity, a nucleic acid encoding a peptide comprising alpha-ketopantoate reductase activity, and a nucleic acid encoding a peptide comprising pantothenate synthase activity; and culturing the transfected cell to allow the transfected cell to make pantothenate.