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(12) United States Patent (10) Patent No.: US 8,765.426 B2 Tao et al. (45) Date of Patent: Jul. 1, 2014

(54) PANTOTHENIC ACID BIOSYNTHESIS IN 7,629,156 B2 12/2009 Viitanen et al. ZYMOMONAS 7,741,084 B2 6, 2010 Viitanen et al. 7,741,119 B2 6, 2010 Viitanen et al. (75) Inventors: Luan Tao, Wallingford, PA (US); 7,897.396 B2 3/2011 Caimi et al. - 7,932,063 B2 4/2011 Dunson et al. Jean-Francois Tomb, Wilmington, DE T.998.722 B2 8/2011 Viitanen et al. (US); Paul V. Vitanen, West Chester, PA 2005.0089973 A1 4, 2005 Yocum et al. (US) 2005/0221466 A1 10, 2005 Liao et al. 2009/0031453 A1 1/2009 Jessen et al. (73) Assignee: E I du Pont de Nemours and Company, Wilmington, DE (US) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this W. w862. A. 1928. patent is extended or adjusted under 35 U.S.C. 154(b) by 130 days. WO WO2O1007 5241 A1 T 2010 (21) Appl. No.: 13/433,343 OTHER PUBLICATIONS (22) Filed: Mar. 29, 2012 Feldmann et al., Pentose Metabolism in Zymomonas Mobilis Wild Type and Recombinant Strains (1992) Appl Microbiol Biotechnol (65) Prior Publication Data 38: 354-361. Kanehisa et al. KEGG: Kyoto Encyclopedia of Genes and Genomes US 2013/0078694A1 Mar. 28, 2013 (2000) Nucleic Acids Research vol. 28 No. 1, 27-30. Related U.S. Application Data RNKanehisa et al.N. (2002)3. NucleicN. A.Acids RRes 34,30, 42-46.D554.357 (60) Provisional application No. 61/472.664, filed on Apr. Nipkow etal (1984) Appl Microbiol Biotechnol 19, 237-240. 7, 2011. Ramjee etal (1997) Biochem J 323, 661-669. Seo et al (2005) Nat Biotechnol 23, 63-68 Seo etal 2005 Nat (51) Int.nt. ClC. Biotechnol.pdf. CI2P I/00 (2006.01) Zhang et al (1995) Science 267, 240-243. CI2P 13/02 (2006.01) CI2P 17/04 (2006.01) CI2PI3/06 (2006.01) Primary Examiner — Kagnew H Gebreyesus CI2N 15/52 (2006.01) CI2N 9/90 (2006.01) (52) U.S. Cl. (57) ABSTRACT USPC ...... 435/161; 435/476; 435/252.3: 536/23.2 (58) Field of Classification Search Zymomonas 1S unable to synthesize pantothenic acid and None requires this essential vitamin in growth medium. Zymomo See application file for complete search history. nas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase (56) References Cited were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pan U.S. PATENT DOCUMENTS tothenic acid Supplementation in seed culture and fermenta 5,514,583 A 5/1996 Picataggio et al. tion media. 5,712,133 A 1/1998 Picataggio et al. 6,171,845 B1 1/2001 Elischweski et al. 7,223,575 B2 5/2007 Zhang et al. 17 Claims, 10 Drawing Sheets U.S. Patent US 8,765.426 B2

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s US 8,765,426 B2 1. 2 PANTOTHENIC ACID BIOSYNTHESIS IN ces cerevisiae YRH063c ORF encodes a protein having ZYMOMONAS ketopantoate reductase activity by complementation of a panE-ilvC mutant in E. coli. US 2005.0089973 discloses pro This application claims the benefit of U.S. Provisional ducing panto-compounds in microorganisms where existing Application 61/472.664, filed Apr. 7, 2011, and is incorpo biosynthetic pathways are manipulated, such as by overex rated by reference in its entirety. pressing ketopantoate reductase and aspartate alpha-decar boxylase. STATEMENT OF GOVERNMENT RIGHTS US 2005221466 discloses the use of cells with alanine 2,3-aminomutase activity, which converts alpha-alanine to This invention was made with United States government 10 beta-alanine, for production of pantothenate. support under Contract No. DE-FC36-07GO17056 awarded There remains a need for creating Zymomonas strains that by the Department of Energy. The government has certain are able to grow and produce ethanol in the absence of exter rights in this invention. nally supplied PA. These Zymomonas Strains may be used to improve and reduce the cost of ethanol production using this FIELD OF THE INVENTION 15 biocatalyst. The invention relates to the fields of microbiology and SUMMARY OF THE INVENTION genetic engineering. More specifically, Zymomonas was engineered for expression of enzymes to provide a pathway The invention provides recombinant Zymomonas cells that for pantothenate biosynthesis. express heterologous enzymes to provide a PA biosynthetic pathway. BACKGROUND OF THE INVENTION Accordingly, the invention provides a bacterial strain of the genus Zymomonas comprising a heterologous nucleic acid Production of ethanol by microorganisms provides an molecule encoding a polypeptide having 2-dehydropantoate alternative energy source to fossil fuels and is therefore an reductase activity and a heterologous nucleic acid molecule important area of current research. The bacteria Zymomonas 25 encoding a polypeptide having aspartate 1-decarboxylase naturally produces ethanol, and has been genetically engi activity. neered for improved ethanol production. Improvements In another embodiment the invention provides a process include elimination of competing pathways, utilization of for producing a Zymomonas Strain that synthesizes pan Xylose, and better performance in medium containing biom tothenic acid comprising: ass hydrolysate (for example: U.S. Pat. No. 7,741,119, U.S. 30 a) providing a bacterial Strain of the genus Zymomonas, Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, WO95/28476, b) introducing a heterologous nucleic acid molecule Feldmann etal. (1992) Appl. Microbiol. Biotechnol. 38: 354 encoding a polypeptide having 2-dehydropantoate reductase 361, Zhanget al. (1995) Science 267:240-243, and US 2009 activity; and 0203099 A1). The hydrolysate produced from lignocellulosic c) introducing a heterologous nucleic acid molecule encod and cellulosic biomass can provide an abundantly available, 35 ing a polypeptide having aspartate 1-decarboxylase activity; low cost source of carbon substrates for biocatalyst fermen wherein steps b) and c) may be in either order or simulta tation to produce desired products. Biomass hydrolysate typi neous and wherein 2-dehydropantoate reductase and aspar cally includes xylose, as well as inhibitors of fermentation. tate 1-decarboxylase activities are expressed in the strain. For economical fermentative production, it is desired that a biocatalyst does not require addition of any costly nutrients to In yet another embodiment the invention provides a 40 method for producing ethanol comprising: growth and production media. In particular, it is desired that a) providing the recombinant bacterial strain of the genus no vitamin Supplements be required for seed or production Zymomonas comprising a heterologous nucleic acid mol biocatalyst cultures. Zymomonas requires Supplementation of ecule encoding a polypeptide having 2-dehydropantoate pantothenic acid (PA; also pantothenate, vitamin Bs, 3-(2,4- reductase activity and a heterologous nucleic acid molecule dihydroxy-3.3-dimethylbutanoyl)aminopropanoic acid) in encoding a polypeptide having aspartate 1-decarboxylase growth medium, being unable to synthesize this nutrient (Seo 45 activity; and et al. (2005) Nat. Biotechnol. 23:63-68; Nipkow etal. (1984) b) contacting the strain of (a) with fermentation medium Appl. Microbiol. Biotechnol. 19:237-240 and references therein). PA is an important cellular component as it is under conditions whereby the strain produces ethanol. required for the synthesis of coenzyme-A (CoA), a compound BRIEF DESCRIPTION OF THE FIGURES, with many important cellular functions. For many animals it 50 is an essential nutrient, while many plants express enzymes BIOLOGICAL DEPOSITS AND SEQUENCE for the synthesis of PA. DESCRIPTIONS E. coli is able to synthesize PA and the biosynthetic path way is known. E. coli genes encoding enzymes of the path Applicants have made the following biological deposits way have been identified. Increased production of pantothen under the terms of the Budapest Treaty on the International ate has been achieved by overexpressing genes in the 55 Recognition of the Deposit of Microorganisms for the Pur biosynthetic pathway of microorganisms that naturally pro poses of Patent Procedure: duce pantothenate. Disclosed in WO 2003006664 is increas ing expression of coding regions in a Bacillus that naturally Information on Deposited Strains produces D-pantothenic acid, such as ybbT, ywkA, yimC, yts.J. mcdh, cysK, iol.J., pdhD, yuiE, dhas, adk, yush, yah.J. 60 ychK, and/orych-I for increased pantothenic acid produc tion. In addition, panE, ylbO, panB, panD, panC, ilvB, ilvN. Depositor International alsS, ilvC, ilvD, serA, serC., ywp.J. and/or gly A may be Identification Depository Date of increased in expression. U.S. Pat. No. 6,171,845 discloses Reference Designation Deposit amplification of nucleotide sequences encoding keptopan 65 Zymomonas ZW658 ATCC No PTA-7858 Sep. 12, 2006 toate reductase, in particular panE, in pantothenic acid pro ducing microorganisms. It was shown that the Saccharomy US 8,765,426 B2 3 4 FIG. 1 is a diagram of a pantothenic acid biosynthetic SEQ ID NO:6 is the nucleotide sequence of the E. coli pathway with bold arrows marking activities that may be panD open reading frame encoding aspartate 1-decarboxy present in Zymomonas and arrows with “X” marking absent lase. activities. The numbers with the arrows are EC numbers of SEQ ID NO:7 is the amino acid sequence of the E. coli enzymes that perform the shown reaction. Gene names asso 5 panD encoded aspartate 1-decarboxylase. ciated with the EC numbers are given in Some cases. SEQ ID NO:8 is the nucleotide sequence of a stretch of FIG. 2 shows conserved amino acid positions, using one DNA that corresponds to the small, stabilizing stem-loop letter abbreviations, of aspartate 1-decarboxylase polypep sequence that immediately follows the Xylose isomerase tides in a general structure diagram (A), in a representative (xylA) stop codon in the E. coli XylA/B operon. sequence (B), and in the E. coli aspartate 1-decarboxylase 10 amino acid sequence (C). TABLE 1 FIG. 3 shows conserved amino acid positions, using one letter abbreviations, of 2-dehydropantoate reductase 2-dehydropantoate reductases used in first alignment polypeptides in a general structure diagram (A), in a repre 15 SEQ ID sentative sequence (B), in the E. coli 2-dehydropantoate Accession number Organism NO reductase amino acid sequence based on a ten sequence align gi|53803269 refYP 114934.1| Methylococcus capsulatus 9 ment (C), and in the E. coli 2-dehydropantoate reductase gi78223840|refYP 385587.1 Geobacter metaireducerns 10 GS-15 amino acid sequence based on a 648 sequence alignment (D). gil 19113647 refNP 596855.1| Schizosaccharomyces pombe 11 FIG. 4 shows a graph of growth curves of wild type gi|6321854|refNP 011930.1| Saccharomyces cerevisiae 12 Zymomonas strain ZW1 grown in minimal medium Supple S288c mented with different concentrations of pantothenic acid gi|7353.8792|refYP 299159.1| Ralstonia eutropha JMP134 13 gi2O7722086 refYP 002252524. Ralstonia solanacearum 14 (PA), after a PA-depletion step. MoIK2 FIG. 5 shows a graph of growth curves of wild type gil 1943.67655 refYP 002030265. Stenotrophomonas 15 Zymomonas strain ZW1(A) and strain ZW1/PanED#1 (also 25 maitophilia R551-3 referred to as ZEDi 1)(B) grown in minimal medium with or gi29376939|refNP 816093.11 Enterococcus faecalis V583 16 without 2.5 mg/L pantothenic acid supplementation, after a gil 16078575 refNP 389394.1| Bacillus subtilis subsp. subtilis PA-depletion step. str. 168) FIG. 6 shows a graph of growth curves of Zymomonas strain ZW801-4 in (A) minimal medium with and without 2.5 30 SEQ ID NO:18 is a representation of a conserved amino mg/L pantothenic acid Supplementation, after a PA-depletion acid sequence for aspartate 1-decarboxylase showing the step, and in (B) minimal medium containing 15 mg/L p-ami highly conserved amino acid positions, without notation of nobenzoic acid and different concentrations of pantothenic less conserved amino acid positions and with insertion posi acid, after a PA-depletion step. tions omitted. FIG. 7 shows a graph of growth curves of Zymomonas 35 SEQ ID NO:19 is a representation of a conserved amino strains ZW801-4 (A) and ZW 801-4/Pan EDH1(B) grown in acid sequence for 2-dehydropantoate reductase showing the minimal medium Supplemented with 15 mg/L p-aminoben highly conserved amino acid positions, without notation of Zoic acid (PABA), 2.5 mg/L pantothenic acid, both com less conserved amino acid positions and with insertion posi pounds, or neither, after a PA-depletion step. tions omitted. The invention can be more fully understood from the fol 40 lowing detailed description and the accompanying sequence DETAILED DESCRIPTION descriptions which form a part of this application. The following sequences conform with 37 C.F.R. 1.821 The following definitions may be used for the interpreta 1.825 (“Requirements for Patent Applications Containing tion of the claims and specification: Nucleotide Sequences and/or Amino Acid Sequence Disclo 45 As used herein, the terms “comprises.” “comprising.” sures—the Sequence Rules') and are consistent with World “includes.” “including.” “has.” “having.” “contains” or “con Intellectual Property Organization (WIPO) Standard ST.25 taining.” or any other variation thereof, are intended to cover (2009) and the sequence listing requirements of the EPO and a non-exclusive inclusion. For example, a composition, a PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex mixture, process, method, article, or apparatus that comprises C of the Administrative Instructions). The symbols and for 50 a list of elements is not necessarily limited to only those mat used for nucleotide and amino acid sequence data comply elements but may include other elements not expressly listed with the rules set forth in 37 C.F.R.S 1.822. or inherent to Such composition, mixture, process, method, SEQ ID NO:1 is the nucleotide sequence of a synthetic article, or apparatus. Further, unless expressly stated to the chimeric E. coli panE and panD operon. contrary, “or” refers to an inclusive or and not to an exclusive SEQ ID NO:2 is the nucleotide sequence of the GI pro 55 or. For example, a condition A or B is satisfied by any one of moter from the Actinoplanes missouriensis Xylose isomerase the following: A is true (or present) and B is false (or not gene. present), A is false (or not present) and B is true (or present), SEQ ID NO:3 is the nucleotide sequence of the E. coli and both A and B are true (or present). panE open reading frame encoding 2-dehydropantoate reduc Also, the indefinite articles “a” and “an preceding an tase. 60 element or component of the invention are intended to be SEQ ID NO:4 is the amino acid sequence of the E. coli nonrestrictive regarding the number of instances (i.e. occur panE encoded 2-dehydropantoate reductase (strain K-12 Sub rences) of the element or component. Therefore “a” or “an str. MG 1655; gil 16128410|refNP 414959.11). should be read to include one or at least one, and the singular SEQ ID NO:5 is the nucleotide sequence of a stretch of word form of the element or component also includes the DNA that is upstream from the start codon for the Z. mobilis 65 plural unless the number is obviously meant to be singular. glyceraldehyde 3-phosphate dehydrogenase gene that The term “invention” or “present invention” as used herein includes the Shine-Delgarno sequence is a non-limiting term and is not intended to refer to any single US 8,765,426 B2 5 6 embodiment of the particular invention but encompasses all The terms “plasmid' and “vector” as used herein, refer to possible embodiments as described in the specification and an extra chromosomal element often carrying genes which the claims. are not part of the central metabolism of the cell, and usually As used herein, the term “about modifying the quantity of in the form of circular double-stranded DNA molecules. Such an ingredient or reactant of the invention employed refers to 5 elements may be autonomously replicating sequences, variation in the numerical quantity that can occur, for genome integrating sequences, phage or nucleotide example, through typical measuring and liquid handling pro sequences, linear or circular, of a single- or double-stranded cedures used for making concentrates or use solutions in the DNA or RNA, derived from any source, in which a number of real world; through inadvertent error in these procedures: nucleotide sequences have been joined or recombined into a 10 unique construction which is capable of introducing a pro through differences in the manufacture, source, or purity of moter fragment and DNA sequence for a selected gene prod the ingredients employed to make the compositions or carry uct along with appropriate 3' untranslated sequence into a out the methods; and the like. The term “about also encom cell. passes amounts that differ due to different equilibrium con The term “operably linked’ refers to the association of ditions for a composition resulting from a particular initial 15 nucleic acid sequences on a single nucleic acid fragment So mixture. Whether or not modified by the term “about’, the that the function of one is affected by the other. For example, claims include equivalents to the quantities. In one embodi a promoter is operably linked with a coding sequence when it ment, the term “about” means within 10% of the reported is capable of affecting the expression of that coding sequence numerical value, preferably within 5% of the reported (i.e., that the coding sequence is under the transcriptional numerical value. control of the promoter). Coding sequences can be operably "Gene' refers to a nucleic acid fragment that expresses a linked to regulatory sequences in sense or antisense orienta specific protein or functional RNA molecule, which may tion. optionally include regulatory sequences preceding (5' non The term “selectable marker” means an identifying factor, coding sequences) and following (3' non-coding sequences) usually an antibiotic or chemical resistance gene, that is able the coding sequence. “Native gene' or “wildtype gene' refers 25 to be selected for based upon the marker gene's effect, i.e., to a gene as found in nature with its own regulatory resistance to an antibiotic, wherein the effect is used to track sequences. “Chimeric gene' refers to any gene that is not a the inheritance of a nucleic acid of interest and/or to identify native gene, comprising regulatory and coding sequences that a cell or organism that has inherited the nucleic acid of inter are not found together in nature. Accordingly, a chimeric gene eSt. may comprise regulatory sequences and coding sequences 30 As used herein the term “codon degeneracy” refers to the that are derived from different sources, or regulatory nature in the genetic code permitting variation of the nucle sequences and coding sequences derived from the same otide sequence without affecting the amino acid sequence of Source, but arranged in a manner different than that found in an encoded polypeptide. The skilled artisan is well aware of nature. "Endogenous gene' refers to a native gene in its the “codon-bias' exhibited by a specific host cell in usage of natural location in the genome of an organism. A “foreign' 35 nucleotide codons to specify a given amino acid. Therefore, gene refers to a gene not normally found in the host organism, when synthesizing a gene for improved expression in a host but that is introduced into the host organism by gene transfer. cell, it is desirable to design the gene Such that its frequency Foreign genes can comprise native genes inserted into a non of codon usage approaches the frequency of preferred codon native organism, or chimeric genes. usage of the host cell. “Promoter' or “Initiation control regions’ refers to a DNA 40 The term "codon-optimized as it refers to genes or coding sequence capable of controlling the expression of a coding regions of nucleic acid molecules for transformation of Vari sequence or functional RNA. In general, a coding sequence is ous hosts, refers to the alteration of codons in the gene or located 3' to a promoter sequence. Promoters may be derived coding regions of the nucleic acid molecules to reflect the in their entirety from a native gene, or be composed of differ typical codon usage of the host organism without altering the ent elements derived from different promoters found in 45 polypeptide encoded by the DNA. nature, or even comprise synthetic DNA segments. It is The term “carbon substrate' or "fermentable carbon Sub understood by those skilled in the art that different promoters strate” refers to a carbon source capable of being metabolized may direct the expression of a gene in different tissues or cell by microorganisms. A type of carbon Substrate is "ferment types, or at different stages of development, or in response to able Sugars' which refers to oligosaccharides and monosac different environmental conditions. Promoters which cause a 50 charides that can be used as a carbon Source by a microorgan gene to be expressed in most cell types at most times are ism in a fermentation process. commonly referred to as “constitutive promoters'. The term “lignocellulosic’ refers to a composition com The term “expression', as used herein, refers to the tran prising both lignin and cellulose. Lignocellulosic material Scription and stable accumulation of coding (mRNA) or func may also comprise hemicellulose. tional RNA derived from a gene. Expression may also refer to 55 The term “cellulosic’ refers to a composition comprising translation of mRNA into a polypeptide. “Overexpression cellulose and additional components, which may include refers to the production of a gene product in transgenic organ hemicellulose and lignin. isms that exceeds levels of production in normal or non The term “saccharification” refers to the production of transformed organisms. fermentable Sugars from polysaccharides. The term “transformation' as used herein, refers to the 60 The term “pretreated biomass” means biomass that has transfer of a nucleic acid fragment into a host organism, been Subjected to thermal, physical and/or chemical pretreat resulting in genetically stable inheritance. The transferred ment to increase the availability of polysaccharides in the nucleic acid may be in the form of a plasmid maintained in the biomass to saccharification enzymes. host cell, or some transferred nucleic acid may be integrated “Biomass” refers to any cellulosic or lignocellulosic mate into the genome of the host cell. Host organisms containing 65 rial and includes materials comprising cellulose, and option the transformed nucleic acid fragments are referred to as ally further comprising hemicellulose, lignin, starch, oli “transgenic' or “recombinant’ or “transformed organisms. gosaccharides and/or monosaccharides. Biomass may also US 8,765,426 B2 7 8 comprise additional components, such as protein and/or lipid. gency of the hybridization, mismatches between bases are Biomass may be derived from a single source, or biomass can possible. The appropriate stringency for hybridizing nucleic comprise a mixture derived from more than one source; for acids depends on the length of the nucleic acids and the example, biomass could comprise a mixture of corn cobs and degree of complementation, variables well known in the art. corn Stover, or a mixture of grass and leaves. Biomass The greater the degree of similarity or homology between two includes, but is not limited to, bioenergy crops, agricultural nucleotide sequences, the greater the value of Tm for hybrids residues, municipal Solid waste, industrial Solid waste, sludge of nucleic acids having those sequences. The relative stability from paper manufacture, yard waste, wood and forestry (corresponding to higher Tm) of nucleic acid hybridizations waste. Examples of biomass include, but are not limited to, decreases in the following order: RNA:RNA, DNA:RNA, corn cobs, crop residues such as corn husks, corn Stover, 10 DNA:DNA. For hybrids of greater than 100 nucleotides in grasses, wheat, wheat Straw, barley Straw, hay, rice straw, length, equations for calculating Tm have been derived (see Switchgrass, waste paper, Sugar cane bagasse, Sorghum, com Sambrook et al., supra, 9.50-9.51). For hybridizations with ponents obtained from milling of grains, trees, branches, shorter nucleic acids, i.e., oligonucleotides, the position of roots, leaves, wood chips, sawdust, shrubs and bushes, Veg mismatches becomes more important, and the length of the etables, fruits, flowers and animal manure. 15 oligonucleotide determines its specificity (see Sambrook et “Biomass hydrolysate' refers to the product resulting from al., Supra, 11.7-11.8). In one embodiment the length for a saccharification of biomass. The biomass may also be pre hybridizable nucleic acid is at least about 10 nucleotides. treated or pre-processed prior to saccharification. Preferably a minimum length for a hybridizable nucleic acid The term "heterologous' means not naturally found in the is at least about 15 nucleotides; more preferably at least about location of interest. For example, a heterologous gene refers 20 nucleotides; and most preferably the length is at least about to a gene that is not naturally found in the host organism, but 30 nucleotides. Furthermore, the skilled artisan will recog that is introduced into the host organism by gene transfer. For nize that the temperature and wash Solution salt concentration example, a heterologous nucleic acid molecule that is present may be adjusted as necessary according to factors such as in a chimeric gene is a nucleic acid molecule that is not length of the probe. naturally found associated with the other segments of the 25 The term “complementary' is used to describe the relation chimeric gene, such as the nucleic acid molecules having the ship between nucleotide bases that are capable of hybridizing coding region and promoter segments not naturally being to one another. For example, with respect to DNA, adenosine associated with each other. is complementary to thymine and cytosine is complementary As used herein, an "isolated nucleic acid molecule' is a to guanine. polymer of RNA or DNA that is single- or double-stranded, 30 The term “percent identity”, as known in the art, is a rela optionally containing synthetic, non-natural or altered nucle tionship between two or more polypeptide sequences or two otide bases. An isolated nucleic acid molecule in the form of or more polynucleotide sequences, as determined by compar a polymer of DNA may be comprised of one or more seg ing the sequences. In the art, “identity also means the degree ments of cDNA, genomic DNA or synthetic DNA. of sequence relatedness between polypeptide or polynucle A nucleic acid fragment is “hybridizable' to another 35 otide sequences, as the case may be, as determined by the nucleic acid fragment, such as a cDNA, genomic DNA, or match between Strings of Such sequences. “Identity” and RNA molecule, when a single-stranded form of the nucleic “similarity” can be readily calculated by known methods, acid fragment can anneal to the other nucleic acid fragment including but not limited to those described in: 1.) Computa under the appropriate conditions of temperature and Solution tional Molecular Biology (Lesk, A. M., Ed.) Oxford Univer ionic strength. Hybridization and washing conditions are well 40 sity: NY (1988); 2.) Biocomputing: Informatics and Genome known and exemplified in Sambrook, J., Fritsch, E. F. and Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Com Maniatis, T. Molecular Cloning: A Laboratory Manual. 2" puter Analysis of Sequence Data, Part I (Griffin, A. M., and ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence N.Y. (1989), particularly Chapter 11 and Table 11.1 therein Analysis in Molecular Biology (von Heinje, G., Ed.) Aca (entirely incorporated herein by reference). The conditions of 45 demic (1987); and 5.) Sequence Analysis Primer (Gribskov, temperature and ionic strength determine the 'stringency of M. and Devereux, J., Eds.) Stockton: NY (1991). the hybridization. Stringency conditions can be adjusted to Preferred methods to determine identity are designed to screen for moderately similar fragments (such as homologous give the best match between the sequences tested. Methods to sequences from distantly related organisms), to highly similar determine identity and similarity are codified in publicly fragments (such as genes that duplicate functional enzymes 50 available computer programs. Sequence alignments and per from closely related organisms). Post-hybridization washes cent identity calculations may be performed using the MegA determine Stringency conditions. One set of preferred condi lign program of the LASERGENE bioinformatics computing tions uses a series of washes starting with 6xSSC, 0.5% SDS suite (DNASTAR Inc., Madison, Wis.). at room temperature for 15 min, then repeated with 2xSSC, Multiple alignment of the sequences is performed using the 0.5% SDS at 45° C. for 30 min, and then repeated twice with 55 “Clustal method of alignment” which encompasses several 0.2xSSC, 0.5% SDS at 50° C. for 30 min. A more preferred varieties of the algorithm including the “Clustal V method of set of Stringent conditions uses higher temperatures in which alignment” corresponding to the alignment method labeled the washes are identical to those above except for the tem Clustal V (described by Higgins and Sharp, CABIOS. 5:151 perature of the final two 30 min washes in 0.2xSSC, 0.5% 153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., SDS was increased to 60° C. Another preferred set of highly 60 8:189-191 (1992)) and found in the MegAlign v8.0 program stringent conditions uses two final washes in 0.1 xSSC, 0.1% of the LASERGENE bioinformatics computing suite SDS at 65° C. An additional set of stringent conditions (DNASTAR Inc.). Formultiple alignments, the default values include hybridization at 0.1xSSC, 0.1% SDS, 65° C. and correspond to GAPPENALTY=10 and GAP LENGTH PEN washes with 2xSSC, 0.1% SDS followed by 0.1xSSC, 0.1% ALTY=10. Default parameters for pairwise alignments and SDS, for example. 65 calculation of percent identity of protein sequences using the Hybridization requires that the two nucleic acids contain Clustal method are KTUPLE=1, GAP PENALTY=3, WIN complementary sequences, although depending on the strin DOW=5 and DIAGONALS SAVED=5. For nucleic acids US 8,765,426 B2 9 10 these parameters are KTUPLE=2, GAPPENALTY=5, WIN by Sambrook, J. and Russell, D., Molecular Cloning: A Labo DOW-4 and DIAGONALS SAVED–4. After alignment of ratory Manual. Third Edition, Cold Spring Harbor Labora the sequences using the Clustal V program, it is possible to tory Press, Cold Spring Harbor, N.Y. (2001); and by Silhavy, obtain a “percent identity” by viewing the “sequence dis T. J., Bennan, M. L. and Enquist, L. W., Experiments with tances” table in the same program. Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Additionally the “ClustalW method of alignment' is avail Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et. al., able and corresponds to the alignment method labeled Clustal Short Protocols in Molecular Biology, 5' Ed. Current Proto W (described by Higgins and Sharp, CABIOS. 5:151-153 cols, John Wiley and Sons, Inc., N.Y., 2002. (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191 Appendix 1, which is incorporated herein by reference, is a (1992); Thompson, J. D. et al. Nucleic Acid Research, 22 10 listing of Accession numbers and annotated identities of 648 (22): 4673-4680, 1994) and found in the MegAlign v8.0 2-dehydropantoate reductases of 250-350 amino acids that program of the LASERGENE bioinformatics computing have an E-value of 0.00001 or smaller to the E. coli 2-dehy suite (DNASTAR Inc.). Default parameters for multiple dropantoate reductase of SEQ ID NO:4, with 95% identity alignment (stated as protein/nucleic acid (GAP PEN and 95% overlap redundancy cutoffs. ALTY=10/15, GAP LENGTH PENALTY=0.2/6.66, Delay 15 Appendix 2, which is incorporated herein by reference, is a Divergen Seqs (%)—30/30, DNA Transition Weight=0.5, Pro listing of Accession numbers and annotated identities of 693 tein Weight Matrix-Gonnet Series, DNA Weight aspartate 1-decarboxylases of 120-150 amino acids that have Matrix=IUB). After alignment of the sequences using the an E-value of 0.00001 or smaller to the E. coli aspartate ClustalW program, it is possible to obtain a “percent identity” 1-decarboxylase of SEQ ID NO:7, with 95% identity and by viewing the “sequence distances” table in the same pro 95% overlap redundancy cutoffs. gram. The present invention relates to engineered strains of It is well understood by one skilled in the art that many Zymomonas that have the ability to grow without pantothenic levels of sequence identity are useful in identifying polypep acid (PA; also called pantothenate and vitamin Bs) supple tides, from other species, wherein Such polypeptides have the mentation in growth and production medium. A challenge for same or similar function or activity. Useful examples of per 25 providing an economical process for fermentation by cent identities include, but are not limited to: 50%, 55%, 60%, Zymomonas to produce ethanol is to reduce the requirement 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer for vitamin Supplementation in the medium, specifically of percentage from 50% to 100% may be useful in identifying PA, thereby reducing the cost of growth and/or production polypeptides of interest, such as 50%, 51%, 52%, 53%, 54%, medium. Zymomonas strains disclosed herein are genetically 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, 63%, 64%, 30 engineered to synthesize pantothenic acid. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, Pantothenic Acid Biosynthesis 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, Zymomonas is known to lack the natural ability to synthe 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, size pantothenic acid and therefore requires the presence of 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid frag this vitamin in medium used for growth of this bacteria. PA is ments not only have the above identities but typically encode 35 require for CoA (Coenzyme A) production, and is therefore a polypeptide having at least 50 amino acids, preferably at critical for carbohydrate, protein and fatty acid metabolism. least 100 amino acids, and more preferably at least 125 amino Applicants analyzed the Zymomonas genome for the acids. potential to encode enzymes of a pantothenic acid biosyn The term "sequence analysis Software” refers to any com thetic pathway. The complete sequence of the Zymomonas puter algorithm or software program that is useful for the 40 genome is known (Seo et al. (2005) Nat. Biotechnol. 23:63 analysis of nucleotide or amino acid sequences. “Sequence 68; NCBI Reference: NC 006526.2) and open reading analysis software may be commercially available or inde frames (ORFs) have been annotated as encoding proteins pendently developed. Typical sequence analysis software will with defined function where possible, based on sequence include, but is not limited to: 1.) the GCG suite of programs analysis. The presence of ORFs potentially encoding (Wisconsin Package Version 9.0, Genetics Computer Group 45 enzymes that could function in a pantothenic acid biosyn (GCG), Madison, Wis.); 2.) BLASTP. BLASTN, BLASTX thetic pathway was analyzed using KEGG analysis (Kyoto (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) Encyclopedia of Genes and Genomes; Kanehisa et al. (2002) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Nucleic Acids Res. 30:42-46; Kanehisa and Goto (2000) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); Nucleic Acids Res. 28:27-30; Kanehisa et al. (2006) Nucleic and 5.) the FASTA program incorporating the Smith-Water 50 Acids Res. 34:D354-357). KEGG provides knowledge-based man algorithm (W. R. Pearson, Comput. Methods Genome methods for uncovering higher-order systemic behaviors of Res., Proc. Int. Symp. (1994), Meeting Date 1992, 111-20. the cell and the organism from genomic and molecular infor Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within mation, as stated by KEGG. the context of this application it will be understood that where The KEGG analysis showed that the Zymomonas genome sequence analysis Software is used for analysis, that the 55 has the potential for encoding proteins with activities for results of the analysis will be based on the “default values” of Some steps in a pantothenic acid biosynthetic pathway (see the program referenced, unless otherwise specified. As used FIG.1, bold arrows). The genome includes ORFs predicted to herein “default values' will mean any set of values or param encode proteins with activities that may produce 2-dehydro eters that originally load with the software when first initial pantoate and L-aspartate. The EC group to which each ized. 60 enzyme activity in the pathway belongs, and in some cases the The term "E-value', as known in the art of bioinformatics, name of the gene encoding the enzyme, is shown in the is “Expect-value' which provides the probability that a match pathway diagram of FIG. 1. will occur by chance. It provides the statistical significance of No ORF was found with the potential to encode a protein the match to a sequence. The lower the E-value, the more with activity that would convert 2-dehydropantoate to pan significant the hit. 65 toate, and no ORF was found with the potential to encode a Standard recombinant DNA and molecular cloning tech protein with activity that would convert L-aspartate to beta niques used herein are well known in the art and are described alanine. These two steps are marked with an X in the FIG. 1 US 8,765,426 B2 11 12 diagram. Pantoate and beta-alanine are ligated together to 075241 (US 2011/0014670), which are herein incorporated produce pantothenic acid in a known pantothenic acid bio by reference. Any of the disclosed strains, including for synthetic pathway, Such as from E. coli. Pantoate-beta-ala example ATCC31821/pZB5, ZW658 (ATCC #PTA-7858), nine ligase is encoded by the panC gene in many organisms, ZW800, ZW801-4, ZW801-4: Ahim.A, AcRi3, ZW705, or including in E. coli. The Zymomonas genome does have the other ethanol-producing strains of Zymomonas, may be used potential for encoding a pantoate-beta-alanine ligase. How as host cells for expression of heterologous nucleic acid mol ever, the protein encoded by the ORF annotated as panC has ecules encoding a polypeptide having 2-dehydropantoate only 46% amino acid sequence identity with the E. coli panC reductase activity and encoding a polypeptide having aspar gene encoded pantoate-beta-alanine ligase. With presumably tate 1-decarboxylase activity, which enables pantothenic acid no pantoate and beta-alanine Substrates available in the cell, 10 biosynthesis. the native function of the protein encoded by the ORF anno Enzyme Activities tated as panC is unknown. Any nucleic acid molecule encoding a polypeptide having Conversion of 2-dehydropantoate to pantoate in E. coli is 2-dehydropantoate reductase activity may be used in the catalyzed by 2-dehydropantoate reductase, which is encoded present strains. Enzymes with 2-dehydropantoate reductase by the panE ORF in many organisms including in E. coli 15 activity are also called 2-dehydropantoate 2-reductase, (SEQID NO:3). Conversion of L-aspartate to beta-alanine is 2-oxopantoate reductase, ketopantoate reductase, ketopan catalyzed by aspartate 1-decarboxylase, which is encoded by toic acid reductase, KPA reductase, and KPR. The reaction the panD ORF in many organisms including in E. coli (SEQ catalyzed by this enzyme activity is: ID NO:6). Whether expression of these two activities in Zymomonas cells would confer the ability to synthesize PA (R)-pantoate+NADP'<-->2-dehydropantoate+ was unknown, since that outcome necessitates the assump NADPH-H tion that the presumed existing native enzymes, that sequence The 2-dehydropantoate reductase enzyme is classified as analysis speculates can participate in PA biosynthesis, actu EC 1.1.1.169. A nucleic acid molecule encoding any enzyme ally do encode functional enzymes that do catalyze reactions belonging to this EC group having 2-dehydropantoate reduc of a portion of a PA biosynthetic pathway in Zymomonas. 25 tase activity may be used in the present strains. Upon experimental analysis as disclosed herein, Appli Any nucleic acid molecule encoding a polypeptide having cants have discovered that Zymomonas cells engineered for aspartate 1-decarboxylase activity may be used in the present expression of the E. coli panE and panD coding regions are strains. Enzymes with aspartate 1-decarboxylase activity are able to grow in medium that does not contain PA. This result also called aspartate alpha-decarboxylase, L-aspartate alpha Suggests that said Zymomonas cells have a complete func 30 decarboxylase, aspartic alpha-decarboxylase, L-aspartate tional PA biosynthetic pathway and synthesize PA. Further, 1-carboxy-lyase, ADC, Asp)C, and Dgad2. The reaction the engineered wild type Zymomonas cells grow as well in catalyzed by this enzyme activity is: medium that does not contain PA as wild type Zymomonas cells grow in the same medium Supplemented with a non L-aspartates-->beta-alanine--CO2 limiting amount of PA. 35 The aspartate 1-decarboxylase enzyme is classified as EC In the present recombinant Zymomonas bacterial strains, a 4.1.1.11. A nucleic acid molecule encoding any enzyme nucleic acid molecule encoding a polypeptide having 2-de belonging to this EC group having aspartate 1-decarboxylase hydropantoate reductase activity and a nucleic acid molecule activity may be used in the present strains. The protein trans encoding a polypeptide having aspartate 1-decarboxylase lated from the E. coli panD gene is an inactive protein called activity are introduced. These polypeptides are encoded by 40 the pi-protein (Ramjee et al (1997) Biochem. J. 323:661 heterologous nucleic acid molecules that are introduced into 669). This protein is autocatalytically self-processed into two the Zymomonas cell. Subunits (alpha and beta) that form the active enzyme. Host Zymomonas Cells Polypeptides with 2-dehydropantoate reductase activity or Heterologous nucleic acid molecules encoding polypep aspartate 1-decarboxylase activity may be identified using tides with 2-dehydropantoate reductase activity and aspartate 45 bioinformatics and/or experimental methods. Amino acid 1-decarboxylase activity may be introduced into any strain of sequences of these polypeptides can be readily found by EC Zymomonas, such as Zymomonas mobilis, to create a pan number, gene name, and/or enzyme name using databases tothenic acid biosynthesis pathway. Wild type Zymomonas that are well known to one of skill in the art including NCBI strains naturally produce ethanol and may be used as a host (National Center for Biotechnology Information: Bethesda, for introduction of said nucleic acid molecules. In other 50 Md.), BRENDA (The Comprehensive Enzyme Information embodiments the Zymomonas host strains are recombinant System; Technical University of Braunschweig Dept. of Bio strains engineered to be improved biocatalysts for ethanol informatics), and Swiss-Prot (Swiss Institute of Bioinformat production and comprise a number of genetic modifications ics; Lausanne, Switzerland). In addition, amino acid that enhance the production of ethanol. Host strains may be sequences of these polypeptides can be readily found based strains engineered in one or more of the following ways, in 55 on a known sequence using bioinformatics, including any combination. Z. mobilis Strains have been engineered to sequence analysis Software such as BLAST sequence analy utilize Xylose, a Sugar found in biomass hydrolysate, for sis using for example the E. coli sequences (2-dehydropan ethanol production (U.S. Pat. No. 5,514,583, U.S. Pat. No. toate reductase: SEQ ID NO:4; aspartate 1-decarboxylase: 5,712,133, WO95/28476, Feldmann et al. (1992) Appl. SEQID NO:7). Microbiol. Biotechnol. 38: 354-361, Zhanget al. (1995) Sci 60 The following analysis of polypeptide sequences identified ence 267:240-243). Ethanol has been produced by geneti a structure that is common to 2-dehydropantoate reductases cally modified Zymomonas in lignocellulosic biomass belonging to EC 1.1.1.169. First the amino acid sequences of hydrolysate fermentation media (U.S. Pat. No. 7,932,063). ten 2-dehydropantoate reductases (SEQID NOs: 9-17 and 4) Genetically modified strains of Z. mobilis with improved with experimentally verified function and/or characterized Xylose utilization and/or production of ethanol are disclosed 65 structure as identified in the BRENDA database (BRaunsch in U.S. Pat. No. 7,223,575, U.S. Pat. No. 7,741,119, U.S. Pat. weig ENzyme Database; Cologne University BioInformatics No. 7,897,396, U.S. Pat. No. 7,998,722, and WO2010/ Center; Scheer et al. (2011) Nucleic Acids Res. 39:670-676) US 8,765,426 B2 13 14 and the Protein Data Bank database (RCSB PDB: Berman et align a candidate sequence with SEQID NO:4, allowing for al. (2000) Nucleic Acids Res. 28:235-242) were aligned using extensions, insertions and deletions such as in positions indi Clustal W with the following parameters: Slow/Accurate cated in the structure diagram in FIG. 3A, such that the Pairwise Parameters: Gap Opening 10, Gap Extend=0.1 presence of the conserved amino acids of FIG. 3D can be Protein weight matrix Gonnet 250; Multiple Parameters: Gap determined. Any polypeptide having at least 10, at least 11, at Opening 10, Gap Extension=0.2, Protein Weight least 12, or all 13 of these 13 conserved amino acids when Matrix Gonnet series. From this multiple sequence align compared to SEQ ID NO.4 and having 2-dehydropantoate ment, the amino acid positions with 90% to 100% conserva reductase activity may be used in the present strains. tion as a single amino acid among the ten sequences were Nucleic acid molecules that may be used in the present identified and used to provide the conserved structure dia 10 strains include those encoding any protein having 2-dehydro gram shown in FIG. 3A. In this figure the conserved amino pantoate reductase activity, including for example: 1) those acids are indicated as G (glycine), L (leucine), K (lysine), N belonging to EC 1.1.1.169; 2) those with experimentally veri (asparagine), E (glutamic acid), S (serine), and D (aspartic fied function and/or characterized structure (SEQ ID NOs: acid). All of the amino acids shown are 100% conserved 9-17 and 4); 3) those having conserved structure of FIG. 3A, except the two asparagines (N) which are each 90% con 15 represented as a sequence in FIG. 3B (SEQ ID NO:19); 4) served. The dashed lines represent positions in the multiple those having ten or more of the conserved amino acids high sequence alignment where insertions and deletions, including lighted in SEQ ID NO:4 in FIG. 3C; 5) those with at least N-terminal and C-terminal extensions, occur in one or more about 95% sequence identity to any of the 648 proteins listed of the ten analyzed 2-dehydropantoate reductase amino acid in Appendix 1; and 6) those having ten or more of the con sequences belonging to EC 1.1.1.169. The conserved struc served amino acids highlighted in SEQID NO:4 in FIG. 3D. ture of FIG.3A is represented as a sequence in FIG. 3B (SEQ The following analysis of polypeptide sequences identified ID NO:19). In this sequence the dashed positions are omitted, a structure that is common to aspartate 1-decarboxylases and notation of other conserved amino acids, which are not as belonging to EC 4.1.1.11. A BLAST search was performed highly conserved, is omitted. using the E. coli aspartate 1-decarboxylase (SEQ ID NO:7) The amino acids at the conserved positions are highlighted 25 against publicly available sequences, and protein sequence in the E. coli 2-dehydropantoate reductase amino acid matches with an E-value of 0.00001 or smaller were sequence (SEQID NO:4) in FIG. 3C and are Gat position 7. extracted. Matched protein sequences in the range of 120-150 Gat position 9, Gat position 12, L at position 19, Kat position amino acids were retained. Sequence redundancy was 72, Nat position 98, G at position 99, Kat position 176, Nat reduced to 95% identity and 95% overlap. This filtering position 180, Nat position 184, Eat position 210, Sat position 30 resulted in 493 sequences, which are listed in Appendix 2 by 244, Dat position 248, and Eat position 256. One of skill in their accession numbers. A multiple sequence alignment was the art will be readily able to align a candidate sequence with performed using Clustal W with the following parameters: SEQID NO:4, allowing for extensions, insertions and dele Slow/Accurate Pairwise Parameters: Gap Opening 10, Gap tions such as in positions indicated in the structure diagram of Extend=0.1, Protein weight matrix Gonnet 250; Multiple FIG. 3A, such that the presence of the conserved amino acids 35 Parameters: Gap Opening 10, Gap Extension=0.2, Protein highlighted in FIG. 3C can be determined. Any polypeptide Weight Matrix Gonnet series. A sequence logo was gener having at least 10, at least 11, at least 12, at least 13, or all 14 ated by LOGO extraction using Weblogo, a publicly available of these 14 conserved amino acids when compared to SEQID web based application (Crooks etal (2004) Genome Research NO.4 and having 2-dehydropantoate reductase activity may 14:1188–1190); Schneider and Stephens (1990) Nucleic be used in the present strains. 40 Acids Res. 18:6097-6100). According to the provided infor A BLAST search was performed using the E. coli 2-dehy mation, each logo consists of stacks of symbols, one stack for dropantoate reductase (SEQID NO:4) against publicly avail each position in the sequence. The overall height of the stack able sequences, and protein sequence matches with an indicates the sequence conservation at that position, while the E-value of 0.00001 or smaller were extracted. Matched pro height of symbols within the stack indicates the relative fre tein sequences in the range of 250-350 amino acids were 45 quency of each amino or nucleic acid at that position. The retained. Sequence redundancy was reduced to 95% identity percent frequency of each amino acid at each position was and 95% overlap. This filtering resulted in 648 sequences, calculated for the set of 493 sequences. which are listed in Appendix 1 by their accession numbers. A From this analysis, the most highly conserved amino acid multiple sequence alignment was performed using Clustal W positions were identified as those having a single amino acid with the same parameters used above. All of the amino acid 50 occurring in at least 99% of the 493 sequences analyzed, and positions identified above in the ten sequence alignment were were used to provide the conserved structure diagram shown also highly conserved among the 648 sequences except the L in FIG. 2A. In this figure the conserved amino acids are at position 19. In addition Dat position 248 is replaced with indicated as K (lysine), H (histidine), Y (tyrosine), G (gly S in about 4% of the sequences and the Nsat positions 180 and cine), S (serine), R (arginine), T (threonine), N (asparagine), 184 have some variation. Thus characterization of the broader 55 and I (isoleucine). The dashed lines represent positions in the group of 2-dehydropantoate reductases provided a structure multiple sequence alignment where insertions and deletions, with conserved amino acids highlighted in the E. coli 2-de including N-terminal and C-terminal extensions, occur in one hydropantoate reductase amino acid sequence (SEQ ID or more of the aligned 493 aspartate 1-decarboxylase amino NO:4) shown in FIG.3D. The Nsat positions 180 and 184 are acid sequences belonging to EC 4.1.1.11. The conserved underlined but not bolded to represent some amino acid varia 60 structure of FIG. 2A is represented as a sequence in FIG. 2B tion at those positions. At position 248, the presence of S (SEQ ID NO:18). In this sequence the dashed positions are instead of D is included in the conserved structure. The con omitted, and notation of other conserved amino acids, which served amino acids are G at position 7, G at position 9, G at are not as highly conserved, is omitted. position 12, K at position 72, Nat position 98, G at position The amino acids at the most highly conserved positions are 99, Kat position 176, Nat position 180, Nat position 184, E 65 highlighted in the E. coli aspartate 1-decarboxylase amino at position 210, Sat position 244, D or Sat position 248, and acid sequence (SEQ ID NO:7) in FIG. 2C and are K at E at position 256. One of skill in the art will be readily able to position 9, Hat position 11, Yat position 22, Gat position 24, US 8,765,426 B2 15 16 S at position 25, G at position 52, R at position 54. T at operably linked to a promoter and a termination sequence. In position 57, Yat position 58, N at position 72, G at position an operon, typically a ribosome binding site is located 73, and I at position 86. One of skill in the art will be readily upstream of the start codons for all open reading frames in the able to align a candidate sequence with SEQID NO:7, allow operon. Promoters that may be used in chimeric genes and ing for extensions, insertions and deletions such as in posi operons are promoters that are expressed in Zymomonas cells tions indicated in the structure diagram of FIG. 2A, such that Such as the promoters of Z. mobilis glyceraldehyde-3-phos the presence of the conserved amino acids highlighted in FIG. phate dehydrogenase gene (GAP promoter), Z. mobilis eno 2C can be determined. Any polypeptide having at least 8, at lase gene (ENO promoter), and the Actinoplanes missourien least 9, at least 10, at least 11, or all 12 of these 12 conserved sis Xylose isomerase gene (GI promoter). Termination signals amino acids when compared to SEQ ID NO:7 and having 10 are also those that are expressed in the target cell. aspartate 1-decarboxylase activity may be used in the present Chimeric genes oran operon for 2-dehydropantoate reduc strains. tase and aspartate 1-decarboxylase expression are typically Of the 12 most highly conserved amino acid positions constructed in or transferred to a vector for further manipu shown in FIG. 2B, five of these occurred in 100% of the 493 lations. Vectors are well known in the art. Particularly useful sequences aligned. These 100% conserved positions are Kat 15 for expression in Zymomonas are vectors that can replicate in position 9,Y at position 22, Gat position 24, Tat position 57. both E. coli and Zymomonas, such as pZB188 which is and Y at position 58. In one embodiment a polypeptide that described in U.S. Pat. No. 5,514,583. Vectors may include may be used has all five of these 100% highly conserved plasmids for autonomous replication in a cell, and plasmids amino acid positions when compared to the E. coli aspartate for carrying constructs to be integrated into bacterial 1-decarboxylase amino acid sequence (SEQID NO:7). genomes. Plasmids for DNA integration may include trans Nucleic acid molecules that may be used in the present posons, regions of nucleic acid sequence homologous to the strains include those encoding any protein having aspartate target bacterial genome, or other sequences Supporting inte 1-decarboxylase activity, including for example: 1) those gration. An additional type of vector may be a transposome belonging to EC 4.1.1.11: 2); 2) those with 95% sequence produced using, for example, a system that is commercially identity to the E. coli aspartate 1-decarboxylase (SEQ ID 25 available from EPICENTRE(R). It is well known how to NO:7); 3) those having conserved structure of FIG. 2A, rep choose an appropriate vector for the desired target host and resented as a sequence in FIG. 2B (SEQID NO:18): 4) those the desired function. having eight or more of the conserved amino acids high Vectors carrying the desired coding regions are introduced lighted in SEQID NO:7 in FIG. 2C; 5) those having the five into Zymomonas cells using known methods such as elec conserved amino acid positions Kat position 9, Yat position 30 troporation, freeze-thaw transformation, calcium-mediated 22, G at position 24, Tat position 57, and Y at position 58, as transformation, or conjugation. The coding regions may be compared to the E. coli aspartate 1-decarboxylase of SEQID maintained on a plasmid in the cell, or integrated into the NO:7; and 6) those with at least about 95% sequence identity genome. Integration methods may be used that are well to any of the 493 proteins listed in Appendix 2. known in the art Such as homologous recombination, trans DNA sequences encoding polypeptides with 2-dehydro 35 poson insertion, or transposome insertion. In homologous pantoate reductase activity or aspartate 1-decarboxylase recombination, DNA sequences flanking a target integration activity may also be identified using bioinformatics and/or site are placed bounding a spectinomycin-resistance gene, or experimental methods. Coding sequences can be found in other selectable marker, and the chimeric genes or operon for databases including NCBI (ibid.) using gene name and/or expression, leading to insertion of the selectable marker and enzyme name as is well know to one of skill in the art. Genes 40 the expression sequences into the target genomic site. In encoding 2-dehydropantoate reductase have multiple names, addition, the selectable marker may be bounded by site-spe including for example panE or ApbA in E. coli, and PAN5 in cific recombination sites, so that after expression of the cor Saccharomyces cerevisiae. In addition, nucleic acid responding site-specific recombinase, the resistance gene is sequences encoding these polypeptides can be readily found excised from the genome. based on a known sequence using bioinformatics, including 45 Transformed Zymomonas strains expressing 2-dehydro sequence analysis Software such as BLAST sequence analy pantoate reductase and aspartate 1-decarboxylase may be sis using for example the E. coli sequences (2-dehydropan readily identified by their ability to grow in medium lacking toate reductase: SEQ ID NO:3: aspartate 1-decarboxylase: PA. A wild type strain of Zymomonas mobilis engineered as SEQ ID NO:6). Experimental methods include those based described in examples herein was able to grow in minimal on nucleic acid hybridization. 50 medium lacking PA as well as the wild type strain grew with Nucleic acid molecules encoding 2-dehydropantoate Supplementation of 2.5 mg/L PA (i.e. a saturating concentra reductase and aspartate 1-decarboxylase are found in numer tion of this vitamin). In a strain of Zymomonas mobilis pre ous organisms including, for example, in some bacteria (ex viously engineered for expression of Xylose utilization cluding Zymomonas), yeast, and plants. A coding region enzyme activities and adapted to growth on xylose (U.S. Pat. sequence from one of these sources, which is heterologous to 55 No. 7,629,156), and engineered for improved ethanol produc Zymomonas, may be used directly or it may be optimized for tion through disruption of the endogenous glucose-fructose expression in Zymomonas. For example, it may be codon oxidoreductase (U.S. Pat. No. 7,741,119) gene, expressing optimized for optimal protein expression in Zymomonas, and/ 2-dehydropantoate reductase and aspartate 1-decarboxylase or introns may be removed if present in a eukaryotic coding also conferred the ability to grow in minimal medium lacking region, both of which are well known to one skilled in the art. 60 PA. However supplementation with p-aminobenzoic acid Expression of Enzyme Activities (PABA) was required for growth of this strain due to disrup For expression, a nucleic acid molecule encoding a tion of the pabB gene encoding p-aminobenzoate synthase polypeptide having 2-dehydropantoate reductase activity and Subunit I, which occurred in previous engineering steps. a nucleic acid molecule encoding aspartate 1-decarboxylase Ethanol Production by Pantothenic Acid Producing Strain are each constructed in a chimeric gene with operably linked 65 The present engineered Zymomonas Strain expressing promoter and typically a termination sequence. Alternatively 2-dehydropantoate reductase and aspartate 1-decarboxylase the coding regions are constructed as part of an operon that is may be used as a biocatalyst in fermentation to produce US 8,765,426 B2 17 18 ethanol. The Zymomonas Strain is brought in contact with derland, Mass., or Deshpande, Mukund V., Appl. Biochem. medium containing a carbon Substrate. Typically one or more Biotechnol., 36, 227, (1992), herein incorporated by refer Sugars provide the carbon Substrate. In one embodiment the CCC. medium may be a minimal medium with no addition of a Commercial production of ethanol may also be accom complex ingredient that contains PA Such as yeast extract, or plished with a continuous culture. Continuous cultures are PA itself, such that the medium lacks PA. Alternatively, the open systems where a culture medium is added continuously medium may contain an amount of PA that is Suboptimal for to a bioreactor and an equal amount of conditioned medium is growth and/or production of Zymomonas strains not engi removed simultaneously for processing. Continuous cultures neered for pantothenic acid production. In one embodiment a generally maintain the cells at a constant high liquid phase seed culture is grown in minimal medium lacking PA or in 10 density where cells are primarily in log phase growth. medium having a sub-optimal amount of PA. The seed culture Continuous or semi-continuous culture allows for the is then used to inoculate a larger fermentation culture. The modulation of one factor or any number of factors that affect fermentation medium may lack PA or have a sub-optimal cell growth or end product concentration. For example, one amount of PA. Alternatively, the fermentation medium may 15 method will maintain a limiting nutrient such as the carbon contain an adequate amount of PA for growth and/or produc source or nitrogen level at a fixed rate and allow all other tion of Zymomonas strains not engineered for pantothenic parameters to moderate. In other systems a number of factors acid production. affecting growth can be altered continuously while the cell Seed culture medium and/or fermentation medium may concentration, measured by medium turbidity, is kept con contain biomass hydrolysate which provides mixed Sugars as stant. Continuous systems strive to maintain steady state a carbon Source, typically including glucose, Xylose, and growth conditions and thus the cell loss due to medium being arabinose. It is desirable that the present engineered Zymomo drawn off must be balanced against the cell growth rate in the nas Strain also expresses enzyme activities for utilization of culture. Methods of modulating nutrients and growth factors Xylose, or of Xylose and arabinose. When the mixed Sugars for continuous culture processes as well as techniques for concentration is high Such that growth is inhibited, the 25 maximizing the rate of product formation are well known in medium may include Sorbitol, mannitol, or a mixture thereof the art of industrial microbiology and a variety of methods are as disclosed in U.S. Pat. No. 7,629,156. Galactitol or ribitol detailed by Brock, supra. may replace or be combined with sorbitol or mannitol. The Ethanol may be produce in simultaneous saccharification present Zymomonas Strain grows in the medium where fer and fermentation (SSF) where pretreated biomass is saccha mentation occurs and ethanol is produced. The fermentation 30 rified producing hydrolysate containing fermentable Sugars is run without supplemented air, oxygen, or other gases concurrently with ethanol production by the present (which may include conditions such as anaerobic, microaero Zymomonas Strain. bic, or microaerophilic fermentation), for at least about 24 In one embodiment the present Zymomonas strain is grown hours, and may be run for 30 or more hours. The timing to 35 in shake flasks in minimal medium lacking PA at about 30°C. reach maximal ethanol production is variable, depending on to about 37° C. with shaking at about 150 rpm in orbital the fermentation conditions. Typically, if inhibitors are shakers and then transferred to a 10 L seed fermentor con present in the medium, as may be present in hydrolysate taining a similar medium. The seed culture is grown in the medium, a longer fermentation period is required. The fer seed fermentor anaerobically until ODoo is between 3 and 6. mentations may be run attemperatures that are between about 40 when it is transferred to the production fermentor where the 30° C. and about 37° C., at a pH of about 4.5 to about 7.5. fermentation parameters are optimized for ethanol produc The present Zymomonas Strains may be grown in medium tion. Typical inoculum volumes transferred from the seed without PA supplementation in laboratory scale fermenters, tank to the production tank range from about 2% to about 20% and in scaled up fermentation where commercial quantities of V/v. Typical fermentation medium contains biomass hydroly ethanol are produced. Where commercial production of etha 45 sate. A final concentration of about 5 mM sorbitol or mannitol nol is desired, a variety of culture methodologies may be is present in the medium. The fermentation is controlled at pH applied. For example, large-scale production from the present 5.0-6.0 using caustic solution (Such as ammonium hydroxide, Zymomonas strains may be produced by both batch and con potassium hydroxide, or Sodium hydroxide) and either Sulfu tinuous culture methodologies. A classical batch culturing ric or phosphoric acid. The temperature of the fermentor is method is a closed system where the composition of the 50 controlled at 30° C.-35°C. In order to minimize foaming, medium is set at the beginning of the culture and not subjected antifoam agents (any class—silicone based, organic based to artificial alterations during the culturing process. Thus, at etc) are added to the vessel as needed. An antimicrobial, to the beginning of the culturing process the medium is inocu which the present Zymomonas strain has tolerance, may be lated with the desired organism and growth or metabolic used optionally to minimize contamination. activity is permitted to occur adding nothing to the system. 55 Any set of conditions described above, and additionally Typically, however, a “batch' culture is batch with respect to variations in these conditions that are well known in the art, the addition of carbon Source and attempts are often made at are suitable conditions for production of ethanol by a pan controlling factors such as pH and oxygen concentration. tothenic acid producing Zymomonas strain. A variation on the standard batch system is the Fed-Batch system. Fed-Batch culture processes are also suitable for 60 EXAMPLES growth of the present Zymomonas Strains and comprise a typical batch system with the exception that the substrate is The present invention is further defined in the following added in increments as the culture progresses. Batch and Examples. It should be understood that these Examples, Fed-Batch culturing methods are common and well known in while indicating preferred embodiments of the invention, are the art and examples may be found in Biotechnology: A 65 given by way of illustration only. From the above discussion Textbook of Industrial Microbiology, Crueger, Crueger, and and these Examples, one skilled in the art can ascertain the Brock, Second Edition (1989) Sinauer Associates, Inc., Sun essential characteristics of this invention, and without depart US 8,765,426 B2 19 20 ing from the spirit and scope thereof, can make various that is upstream from the start codon for the Z. mobilis glyc changes and modifications of the invention to adapt it to eraldehyde 3-phosphate dehydrogenase gene that includes various uses and conditions. the Shine-Delgarno sequence; nts 1140-1520 (SEQID NO:6) General Methods correspond to the E. colipanD open reading frame (GenBank The meaning of abbreviations is as follows: “kb' means accession no. AAC73242) that codes for aspartate 1-decar kilobase(s), “bp” means base pairs, “nts’ means nucleotides, boxylase; and nts 1543-1577 (SEQID NO:8) correspond to “hr” means hour(s), “min' means minute(s), “sec’ means the stabilizingxylA stem-loop structure described above. The second(s), “d' means day(s), “L” means liter(s), “ml” means GI-PanED operon DNA fragment was synthesized by Gene milliliter(s), "LL means microliter(s), 'ug' means micro script (Piscataway, N.J.). gram(s), “ng” means nanogram(s), ''g' means gram(s). 10 “mM” means millimolar, “uM” means micromolar, “nm' Example 2 means nanometer(s), “Limol” means micromole(s), “pmol” means picomole(s), “OD' or “OD600” means optical density Construction of the Shuttle Vector Used for at 600 nm, “rpm” is revolutions per minute, '-' means GI-PanED Operon Expression in Z. mobilis, and approximately. 15 Generation of PanBD Strains Shake Flask Experiments with Minimal Media Unless otherwise noted, all experiments described below To introduce the GI-PanED operon into Z. mobilis, the were conducted in shake flasks (15-ml loosely-capped, coni synthetic DNA molecule described above was digested with cal shaped test tubes) using PA-depleted cells and a synthetic NcoI and Not, and the resulting fragment was ligated into the growth medium, MM-G5, that does not contain pantothenic unique NcoI and NotI sites of the plasmid shuttle vector acid. MM-G5 is a modified version of a minimal medium that pZB188/aadA. As described in US 2009-0246876 A1, which is described in Goodman et al. (1982) Applied and Environ is herein incorporated by reference, pZB188/aadA is vector mental Microbiology 44:496-498). It contains 50 g/L glu pZB188 described in U.S. Pat. No. 5,514,583, which is herein cose, 2 g/L KHPO, 1 g/L MgSO (7H20) 2.5 g/L (NH), incorporated by reference, which is able to replicate in Z. SO, 0.5g/L NaCl, 50 mg/LCaCl (2H2O), 1 mg/L NaMoC) 25 mobilis and E. coli since it has origins of replication for both (2H,0), 5 mg/L FeSO (7HO), and 1 mg/L each of pyridox bacterial species, with an added spectinomycin resistance ine, nicotinic acid, biotin and thiamine; with the final pH DNA fragment. To generate non-methylated plasmid DNA brought to 5.9 with KOH, and the solution was filtered for transformation of Z. mobilis, pZB188/aadA-GlpanEpanD through a 0.2 um membrane. It is also important to adjust the was introduced into chemically competent E. coli SCS110 pH to -5.9 with KOH after dissolving the first five ingredients 30 cells (Stratagene, San Diego, Calif.), and transformants were in close to the final volume of deionized water to avoid pre selected on LB medium that contained spectinomycin (100 cipitation of the other components. To deplete intracellular ug/ml). Isolated non-methylated plasmid DNA was then elec and carryover PA, cells from agar plates or glycerol Stocks troporated into ZW1 (ATCC #31821) and ZW801-4. A were inoculated into MM-G5 medium to an OD of 0.1-0.3 detailed description of the construction of thexylose-utilizing and the cultures were incubated at 33°C. (~150 rpm) until the 35 recombinant strain, ZW801-4, starting from the wild type cells stopped growing (~14-20 hrs). However, depending on parent strain, ZW1, is provided in U.S. Pat. No. 7,741,084, the history of the cells, the volume and density of the initial which is herein incorporated by reference. Strain ZW801-4 inoculum and the extent of growth that occurred during the was derived from strain ZW800, which was derived from incubation period, complete depletion of PA may require a strain ZW658, all as described in U.S. Pat. No. 7,741,084. second growth period in fresh MM-G5 medium and/or a 40 ZW658 was constructed by integrating two operons, Pxy longer incubation period. Unless stated otherwise, spectino 1AB and Ptaltkt, containing four xylose-utilizing genes mycin (200 g/ml) was included in the growth media for all encoding Xylose isomerase (XylA), xylulokinase (XylB), tran experiments that were performed with the plasmid-bearing saldolase (tal), and transketolase (tkt), into the genome of Strains ZW1/PanBDH1 and 801 /PanBDH1. ZW1 (rename of strain ZM4; ATCC #31821) via sequential 45 transposition events to produce strain X13L3, which was Example 1 renamed ZW641, and followed by adaptation on selective media containing xylose. ZW658 was deposited under the Construction of the Synthetic GipanEpanD Operon Budapest Treaty as ATCC #PTA-7858. In ZW658, the gene encoding glucose-fructose oxidoreductase was insertionally To complete a putative pathway for pantothenic acid (PA) 50 inactivated using host-mediated, double-crossover, homolo biosynthesis in Z. mobilis we designed a synthetic 1620 bp gous recombination and spectinomycin resistance as a select DNA fragment (SEQ ID NO: 1) that codes for an artificial, able marker to create strain ZW800. The spectinomycin chimeric E. coli panE and panD operon (referred to below as resistance marker, which was bounded by loxP sites, was either the “GipanEpanD operon” or the “GI-PanED operon”). removed by site specific recombination using Cre recombi The 5' end of the operon contains the A. missouriensis (ATCC 55 nase to create strain ZW801-4. As disclosed in commonly 14538)GI promoter and there is a stretch of DNA at the 3' end owned and co-pending US Patent Application Publication that corresponds to the Small, stabilizing stem-loop sequence #US 20090246846, which is herein incorporated by refer that immediately follows the xylose isomerase (xylA) stop ence, ZW648 has much more xylose isomerase activity codon in the E. coli XylA/B operon. The synthetic DNA (about 4-fold higher) than ZW641 (represented by X13bC fragment also has NcoI and Spel sites at its 5' end and NotI 60 strain) due to a point mutation in the promoter (Pgap) that and EcoRI sites at its 3' end that can be used for cloning drives expression of the XylA coding region. purposes. With reference to the DNA sequence of SEQ ID Transformants were selected on agar plates that contained NO:1, nts 23-209 (SEQID NO:2) correspond to the GI pro mRM3-G5 media (50 g/L glucose, 10 g/L yeast extract (con moter; nts 210-1121 (SEQID NO:3) correspond to the E. coli tains PA), 2 g/L KHPO, 1 g/L MgSO) and 200 ug/ml of panE open reading frame (Gen Bank accession number 65 spectinomycin. The resulting ZW1 and ZW801-4 strains that AAC73528) that codes for 2-dehydropantoate reductase; nts harbor the pZB 188/aadA-GlpanEpanD shuttle vector were 1122-1139 (SEQ ID NO:5) correspond to a stretch of DNA named ZW1/PanED #1 and 801/PanED #1, respectively. It US 8,765,426 B2 21 22 should be noted that two primary transformants for each growth curve for ZW1 when a saturating concentration of PA strain were evaluated in the shake flask experiments was present. These results clearly demonstrate that with intro described below. Since both transformants behaved essen duction of panE and panD genes Zymomonas was able to tially the same in both cases, only the results that were synthesize PA, and PA was made in an amount sufficient to obtained with ZW1/PanED#1 and 801/PanED#1 are pre Support the maximum growth rate in minimal medium lack sented below. ing PA. Example 3 Example 5

Growth of ZW1 in MM-G5 Medium Requires PA 10 Effect of the Synthetic GI-PanED Operon in Supplementation ZW8O1-4 The ZW1 strain froman mRM3-G5 plate that contained 50 ZW 801-4 has two vitamin requirements for growth in g/L glucose, 10 g/L yeast extract, 2 g/L KHPO4, 1 g/L minimal medium. Growth experiments using ZW801-4 were MgSO and 1.5% agar was inoculated into 20 ml of MM-G5 15 carried out as described in General Methods and the resulting (described in General Methods) and the culture was incu growth curves are shown in FIG. 6. As seen in FIG. 6A, when bated for ~19 hours at 33°C. (150 rpm) to deplete carryover ZW801-4 was depleted of pantothenic acid in MM-G5 and pantothenic acid. The OD600 increased from 0.178 to 0.408 transferred to the same medium it failed to grow, similar to the during the incubation period. An aliquot of the PA-depleted results that were obtained with ZW1. However, very little cells was then diluted with fresh MM-G5 medium to an growth was also observed when pantothenic acid (2.5 mg/L) OD600 of 0.035, and 10-ml aliquots of the resulting culture was added to the growth medium during the second incuba were distributed to eight 15-ml conical test tubes that con tion period (FIG. 6A). There is a genetic basis for this obser tained various amounts of pantothenic acid (0, 0.025, 0.063, vation that is related to strain construction. Like other ZW641 0.125, 0.25, 0.63, 2.5, or 5 mg/L, final concentrations). After derivatives, ZW801-4 cannot synthesize p-aminobenzoic this step the eight cultures were incubated at 33° C. at 150 25 acid (PABA), which is a vitamin that is required for folic acid rpm, and growth was monitored by following changes in biosynthesis and hence is essential. As described in U.S. Pat. optical density (OD) at 600 nm as a function of time. As No. 7,741,084, the first step in the construction of ZW641 was shown in FIG. 4, both the exponential growth rate and maxi the integration of a synthetic Ptaltkt operon (encoding E. mum cell density increased in a dose related manner with coli transaldolase and transketolase under the control of the Z. increasing concentrations of pantothenic acid until Saturation 30 mobilis P. promoter) into the ZW1 chromosome. The was achieved and growth was no longer limited by this vita operon was introduced by a transposon that randomly inte min. It was clear from this experiment that a concentration of grates into DNA, and the transposon insertion site for the ~2.5 mg/L PA or higher is able to Support maximum growth strain that was selected for further metabolic engineering is in of ZW1 in MM-G5 medium under the conditions employed. the open reading frame of the Z. mobilis pabB gene that codes Growth was not observed unless the cells were supplemented 35 for p-aminobenzoate synthase, Subunit I, which is required with pantothenic acid. for biosynthesis of PABA. The Ptaltkt transposon insert is located between nts 102021 and 102022 of GenBank acces Example 4 sion number AE008692, as determined by whole genome DNA sequence analysis. Since the disrupted pabB gene does Effect of the Synthetic GI-PanED Operon in ZW1 40 not appear to be functional, ZW641 and all strains that were derived from it require two vitamins for growth in minimal Growth curves for ZW1 and ZW1/PanED #1 in the pres media, namely PA and PABA. ence and absence of Supplemented pantothenic acid were A titration experiment was conducted with ZW801-4 to assayed. The protocol for this experiment was as follows. Two determine the optimal concentration of PA for growth of in 10-ml MM-G5 cultures were started for each of Strains ZW1 45 MM-G5 medium that contains a saturating concentration of and ZW 1/PanED #1. One was supplemented with PA (2.5 PABA (15 mg/L.; FIG.6B). Strain ZW801-4 cells were inocu mg/L) while the other received an equivalent volume of ster lated into 20 ml of MM-G5 medium and the culture was ille water. The initial ODs for all four cultures were ~0.1. After incubated for ~19 hours at 33°C. (150 rpm) to deplete intra a 15-hr incubation period at 33°C. (150 rpm), aliquots of cellular and carryover pantothenic acid. During the incuba these cultures were used to start new 10-ml cultures that 50 tion period the OD increased from 0.143 to 0.364. The PA contained the same growth media as the original cultures; the depleted culture was then diluted with MM-G5 medium to an initial OD was -0.05 in all cases. The new cultures were OD of -0.035 and PABA was added to a final concentration of incubated at 33°C. (150 rpm) and growth was monitored by 15 mg/L. Aliquots (10 ml) of the cell suspension were dis OD600. The resulting exponential growth curves are shown tributed to eight 15-ml conical tubes that contained various in FIG. 5. 55 concentrations of PA (ranging from 0-5 mg/L), and the result Consistent with previous results, when ZW1 was depleted ing cultures were incubated at 33°C. (150 rpm) to monitor of pantothenic acid in MM-G5 medium and then transferred growth. As shown in FIG. 6B, the optimal concentration of PA to fresh medium that had the same composition, it failed to for growth of ZW801-4 in MM-G5 medium that contains a grow (FIG. 5A). In contrast, when ZW1 was transferred to saturating concentration of PABA was ~2.5 mg/L, similar to medium that contained 2.5 mg/L of pantothenic acid, the cells 60 the requirement for ZW1 (FIG. 4). grew exponentially with a doubling time of about 2 hours to GI-PanBD Operon in ZW801-4 a final OD of ~1.2 (FIG. 5A). Very different results were Each of Strains ZW801-4 and ZW801-4/PanBDH1 was obtained with the ZW1 derivative that contains the synthetic inoculated into 10 ml of MM-G5 and the cultures were incu GI-PanED operon. As shown in FIG. 5B, ZW1/PanED#1 bated at 33° C. for 15 hrs to deplete pantothenic acid and (ZEDi 1) grew with the same kinetics in MM-G5 medium in 65 partially deplete PABA; the initial OD was -0.1 in both cases. the presence or absence of supplemented PA. Indeed, both Following this step the cultures were diluted with the same growth curves for this strain were virtually identical to the growth medium to an OD of -0.04, and quadruplicate 10-ml US 8,765,426 B2 23 24 aliquots of each cell Suspension were distributed to eight >gil 146310553 refYP 001175627.1 2-dehydropantoate 15-ml conical tubes. The tubes were then supplemented with 2-reductase Enterobacter sp. 638 PA, PABA both vitamins, or neither, and the resulting cultures >gil 206579053 refYP 0022401.06.1 2-dehydropantoate were incubated at 33°C. to monitor growth at 600 nm. The 2-reductase Klebsiella pneumoniae 342 final concentrations of PA and PABA when present were 2.5 >gil 161615377|refYP 001589342.1 hypothetical protein mg/L and 15 mg/L, respectively, and the no vitamin control SPAB 03148 Salmonella enterica subsp. enterica sero cultures received an equivalent volume of sterile water. The var Paratyphi B str. SPB7 resulting growth curves are shown in FIG. 7. >gi311280686|refYP 003942.917.1 2-dehydropantoate Consistent with previous results, ZW801-4 only grew 2-reductase Enterobacter cloacae SCF1 when PA and PABA were both added to the growth medium 10 >gil4930741gb|AAA56681.1 ApbA protein Salmonella (FIG. 7A). The small amount of growth that occurred in the enterica Subsp. enterica serovar Tiphimurium/ culture that was only supplemented with PA is the result of >gi260596793 refYP 003209364.1 2-dehydropantoate carryover PABA, since there is always a small amount of 2-reductase Cronobacter turicensis Z3032 residual PABA after the PA-depletion step during the first 15 >gil 156935026|refYP 001438942.1 hypothetical protein incubation period in MM-G5 medium (i.e. the cells use up all ESA 02877 Cronobacter sakazakii ATCC BAA-894 the PA before they run out of PABA). In contrast to the above >gil 188534647|refYP 001908444.1 2-dehydropantoate results, the ZW801-4 strain that contained the synthetic GI 2-reductase (KPA reductase) Erwinia tasmaniensis Et1/ PanED operon only required PABA for growth (FIG. 7B)) 99 since it was able to synthesize pantothenic acid. >gil 157369326|refYP 001.477315.1 2-dehydropantoate Note that the ZW801-4 culture that was supplemented with 2-reductase Serratia proteamaculans 568 both vitamins grew slightly better in MM-G5 medium than >gi259909271 refYP 002649627.1 2-dehydropantoate the corresponding culture of ZW801-4/PanED#1 (FIG. 7A 2-reductase (KPA reductase) Erwinia pyrifoliae Ep1/96 versus 7B). The most likely explanation for this result is >gi317047160|refYP 0041 14808.1 2-dehydropantoate “plasmid burden', which is often observed with Z. mobilis 25 2-reductase Pantoea sp. At-9b) and other bacterial strains (Kim et al. (2000) Applied and >gi270263558|refZP 06191827.1| 2-dehydropantoate Environmental Microbiology 66:186-193 and references 2-reductase Serratia odorifera 4RX13 therein). This phenomenon, whereby energy that would oth >gi13043.95487|refZP 07377370.1| 2-dehydropantoate erwise be available for growth is diverted to plasmid replica 2-reductase Pantoea sp. aE tion and maintenance, would likely be far more pronounced 30 >gi3007 15579|refYP 00374,0382.11 2-dehydropantoate in minimal medium compared to rich medium. 2-reductase (KPA reductase) Erwinia billingiae Eb661 The key finding in this experiment is that ZW801-4/ >gi293392557|refZP 06636877.1| 2-dehydropantoate PanED#1 grew with the same kinetics and to the same cell 2-reductase Serratia odorifera DSM 4582 density in the presence and absence of added pantothenic acid >gi318604615emb|CBY26113.1 2-dehydropantoate 2-re when the growth medium contained a saturating concentra 35 ductase Yersinia enterocolitica Subsp. palearctica Y11 tion of PABA. Taken together the above results clearly indi >gi238754221 refZP 04615578.1 2-dehydropantoate cate that co-expression of the E. coli panB and panD coding 2-reductase Yersinia ruckeri ATCC 29473 regions in wild type and recombinant strains of Z. mobilis >gi238783622|refZP 04627643.1 2-dehydropantoate allowed growth under conditions where pantothenic acid was 2-reductase Yersinia bercovieri ATCC 43970 limiting. 40 >gi238750992|refZP 046.12488.1 2-dehydropantoate 2-reductase Yersinia rohdei ATCC 43380 APPENDIX 1 >gi238791418 refZP 04635056.1 2-dehydropantoate 2-reductase Yersinia intermedia ATCC 29909 Polypeptides of 250-350 Amino Acids with E Value >gi22124921|refNP 668344.1 2-dehydropantoate 2-re of 0.00001 or Smaller to SEQID NQ:4 with 95% 45 ductase Yersinia pestis KIM 10 Identity and 95% Overlap Redundancy Cut-offs >gi2916.16534|refYP 003519276.1| PanE Pantoea ananatis LMG 20103 >gil 16128410|refNP 414959.1 2-dehydropantoate reduc >gi227113453|refZP 03827 109.1| 2-dehydropantoate tase, NADPH-specific Escherichia coli str. K-12 substr 2-reductase Pectobacterium carotovorum Subsp. brasil MG1655 50 iensis PBR1692 >gil 126031569.pdb|2OFPIA Chain A, Crystal Structure Of >gi261822519 refYP 00326.0625.1 2-dehydropantoate Escherichia Coli Ketopantoate Reductase In A Ternary 2-reductase Pectobacterium wasabiae WPP163 Complex With Nadp+ And Pantoate >gi238759275 refZP 04620442.1 2-dehydropantoate >gil 15714.6959|refYP 001454278.1 2-dehydropantoate 2-reductase Yersinia aldovae ATCC 35236 2-reductase Citrobacter koseri ATCC BAA-895 55 >gi271499585 refYP 003332610.1 2-dehydropantoate >gil 168232408 |refZP 02657466.1 2-dehydropantoate 2-reductase Dickeya dadantii Echi586 2-reductase Salmonella enterica Subsp. enterica serovar >gi322834061 refYP 004214068.1 2-dehydropantoate Kentucky str. CDC 191 2-reductase Rahnella sp.Y.9602 >gi283764244|refYP 003364109.1 2-dehydropantoate >gi1317492927|refZP 07951351.1| 2-dehydropantoate 2-reductase Citrobacter rodentium ICC168 60 2-reductase Enterobacteriaceae bacterium 9 2 >gi283834256|refZP 06353997.1| 2-dehydropantoate 54FAA 2-reductase Citrobacter youngae ATCC 29220 >gi307129910|refYP 003881926.1 2-dehydropantoate >gi261341017|refZP 05968875.1 2-dehydropantoate reductase, NADPH-specific Dickeya dadantii 3937 2-reductase Enterobacter cancerogenus ATCC 35316 >gi251790660|refYP 003005381.1 2-dehydropantoate >gi296.101546|refYP 003611692.1 2-dehydropantoate 65 2-reductase Dickeya zeae Ech1591 2-reductase Enterobacter cloacae Subsp. cloacae ATCC >gi242240305 refYP 002988486.1 2-dehydropantoate 13047 2-reductase Dickeya dadantii Ech703 US 8,765,426 B2 25 26 >gi269138340|refYP 003295.040.1 2-dehydropantoate >gi258620951 IrefZP 05715985.1| 2-dehydropantoate 2-reductase Edwardsiella tarda EIB202 2-reductase Vibrio mimicus VM573 >gi85058639|refYP 45.4341.1 2-dehydropantoate 2-re >gil 59711325|refYP 204101.1 2-dehydropantoate 2-re ductase Sodalis glossinidius Str. morsitans ductase Vibrio fischeri ES114 >gi290474680|refYP 003467560.1 2-dehydropantoate >gi319999267 lgb|AAO10217.2 2-dehydropantoate 2-re reductase Xenorhabdus bovienii SS-2004 ductase Vibrio vulnificus/ >gi294635317|refZP 06713814.1 2-dehydropantoate >gi258627307|refZP 05722091.1| 2-dehydropantoate 2-reductase Edwardsiella tarda ATCC 23685 2-reductase Vibrio mimicus VM603 >gi300722004|refYP 003711284.1 2-dehydropantoate >gi84388932|refZP 0099.1140.12-dehydropantoate 2-re 10 ductase Vibrio splendidus 12B01 reductase, NADPH-specific, alternative pyrimidine bio >gi269103241 refZP 06155936.1 2-dehydropantoate synthesis Xenorhabdus nematophila ATCC 19061 2-reductase Photobacterium damselae Subsp. damselae >gi253990908 |refYP 003042264.1 2-dehydropantoate CIP 102761 2-reductase Photorhabdus asymbiotica Subsp. asymbi >gi269960517|refZP 06174889.1 conserved hypotheti otica ATCC 43949 15 cal protein Vibrio harveyi 1DA3 >gi268592049 refZP 06126270.1 2-dehydropantoate >gi297580947|refZP 06942872.1 2-dehydropantoate 2-reductase Providencia rettgeri DSM 1131) 2-reductase Vibrio cholerae RC385 >gi261346377|refZP 0597.4021.1 2-dehydropantoate >gi209694510|refYP 002262438.1 2-dehydropantoate 2-reductase Providencia rustigianii DSM 4541 2-reductase Alivibria salmonicida LFI 1238 >gi37527742|refNP 93.1087.1 2-dehydropantoate 2-re >gil 163802380|refZP 02196274.1 2-dehydropantoate ductase Photorhabdus luminescens subsp. laumondii 2-reductase Vibrio sp. AND4 TTO1 >gi260773428 refZP 05882344.1 2-dehydropantoate >gi226329087|ref ZP 03804605.1 hypothetical protein 2-reductase Vibrio metschnikovii CIP 69.14 PROPEN 02990 Proteus penneri ATCC 35198 >gil 153833983 |refZP 01986650.1 2-dehydropantoate >gil 183601047 refZP 02962540.1 hypothetical protein 25 2-reductase Vibrio harveyi HYO1 PROSTU 04668 Providencia Stuartii ATCC 25827 >gil 1491891.54|refZP 01867442.1 2-dehydropantoate >gi212709183|ref ZP 033173.11.1 hypothetical protein 2-reductase Vibrio Shilonii AK1 PROVALCAL 00216 Providencia alcalifaciens DSM >gi260775331 refZP 05884228.1 2-dehydropantoate 30120 2-reductase Vibrio coralliilyticus ATCC BAA-450 >gi2273581 17|refZP 03842458.1 2-dehydropantoate 30 >gi261253775 refZP 05946348.1 2-dehydropantoate 2-reductase Proteus mirabilis ATCC 2.9906 2-reductase Vibrio orientalis CIP 102891 >gi9467701 1|refYP 588722.11 2-dehydropantoate 2-re >gi1312884367|refZP 07744073.1 2-dehydropantoate ductasebaumannia cicadellinicola Str. He (Homalodisca 2-reductase Vibrio Caribbenthicus ATCC BAA-2122 Coagulata) >gil 156975556 refYP 001.446463.1 2-dehydropantoate >gi32490895|refNP 871149.1 hypothetical protein 35 2-reductase Vibrio harveyi ATCC BAA-1116 WGLp146 Wigglesworthia glossinidia endosymbiont of >gi218708705 refYP 002416326.1 2-dehydropantoate Glossina brevipalpis/ 2-reductase Vibrio splendidus LGP32 >gi292.493929|refYP 003533073.11 2-dehydropantoate >gi1308.125784 refZP 05777325.2 2-dehydropantoate 2-reductase Candidatus Riesia pediculicola USDA 2-reductase Vibrio parahaemolyticus K5030 >gi89073146|refZP 01159685.1 2-dehydropantoate 2-re 40 >gi1323497930|refZP 08102939.1| 2-dehydropantoate ductase Photobacterium sp. SKA34 2-reductase Vibrio Sinaloensis DSM 21326 >gi90579842|refZP 01235650.12-dehydropantoate 2-re >gi315181023 lgb|ADT87937.1 2-dehydropantoate 2-re ductase Vibrio angustum S1.4 ductase Vibrio furnissii NCTC 11218 >gi90410911|refZP 01218925.12-dehydropantoate 2-re >gi86145667|refZP 01.063997.12-dehydropantoate 2-re ductase Photobacterium profiindum 3TCK 45 ductase Vibrio sp. MED222 >gi54308007|refYP 129027.1 2-dehydropantoate 2-re >gi237807568|refYP 002892008.11 2-dehydropantoate ductase Photobacterium profiindum SS9 2-reductase Tolumonas auensis DSM 9187 >gi323491692|refZP 08096870.1 2-dehydropantoate >gi254507837|refZP 05119967.1| 2-dehydropantoate 2-reductase Vibrio brasillensis LMG 20546 2-reductase Vibrio parahaemolyticus 16 >gil 148979964|refZP 01815804.1 2-dehydropantoate 50 >gil 145300108 |refYP 001142949.1 2-dehydropantoate 2-reductase Vibrionales bacterium SWAT-3 2-reductase Aeromonas salmonicida Subsp. salmonicida >gil 153217426|refZP 01951177.1| 2-dehydropantoate A449 2-reductase Vibrio cholerae 1567 >gil 117618856|refYP 855669.1 2-dehydropantoate 2-re >gil 91223419 refZP 01258684.12-dehydropantoate 2-re ductase Aeromonas hydrophila Subsp. hydrophila ATCC ductase Vibrio alginolyticus 12G01 55 7966 >gi28899137|refNP 798742.1 2-dehydropantoate 2-re >gil 109897.687 IrefYP 660942.1 2-dehydropantoate 2-re ductase Vibrio parahaemolyticus RIMD 2210633 ductase Pseudoalteromonas atlantica T6c >gi229521149 refZP 04410569.1 2-dehydropantoate >gil 1676251 12|refYP 0016754.06.1 2-dehydropantoate 2-reductase Vibrio cholerae TM 11079-80 2-reductase Shewanella halifaxensis HAW-EB4 >gil 153825996|refZP 01978663.1| 2-dehydropantoate 60 >gil 14991 1104|refZP 01899731.1 2-dehydropantoate 2-reductase Vibrio cholerae MZO-2 2-reductase Moritella sp. PE36 >gi229513947|refZP 04403409.1 2-dehydropantoate >gi254480183 refZP 05093431.1 2-dehydropantoate 2-reductase Vibrio cholerae TMA 21 2-reductase marine gamma proteobacterium HTCC2148 >gi261211422|refZP 05925710.1 2-dehydropantoate >gil 116052432|refYP 792743.1 2-dehydropantoate 2-re 2-reductase Vibrio sp. RC341 65 ductase Pseudomonas aeruginosa UCBPP-PA14 >gil 197335509|refYP 002155481.1| 2-dehydropantoate >gi221 134751|refZP 03561054.1 2-dehydropantoate 2-reductase Vibrio fischeri MJ11 2-reductase Glaciecola sp. HTCC2999 US 8,765,426 B2 27 28 >gil 157962933 refYP 001502967. 11 2-dehydropantoate >gil 1493.78127|refZP 01895846.1 2-dehydropantoate 2-reductase Shewanella pealeana ATCC 700345 2-reductase Marinobacter algicola DG893 >gil 157376577|refYP 001475177.1| 2-dehydropantoate >gi253576112|refZP 04853444.1 2-dehydropantoate 2-reductase Shewanella sediminis HAW-EB3 2-reductase Paenibacillus sp. oral taxon 786 str. D14 >gi294142129IrefYP 003558107.1 2-dehydropantoate >gi70732101|refYP 261857.1 2-dehydropantoate 2-re 2-reductase Shewanella violacea DSS12 ductase Pseudomonas fluorescens Pf-5 >gil 90409046|refZP 01217173.1 ketopantoate reductase i304310467|refYP 003810065.1 2-dehydropantoate Psychromonas sp. CNPT3 2-reductase Ketopantoate reductase)/gamma proteobac >gil 120553890|refYP 958241.1 2-dehydropantoate 2-re terium HaN1 ductase Marinobacter aquaeolei VT8) 10 >gil 153002110|refYP 001367791.1 2-dehydropantoate >gi21263.4008 |refYP 002310533.1 2-dehydropantoate 2-reductase Shewanella baltica OS185 2-reductase Shewanella piezotolerans WP3 >gil 104783439|refYP 609937.1 2-dehydropantoate 2-re >gi3021896.12|refZP 07266285.1 2-dehydropantoate ductase Pseudomonas entomophila L48 2-reductase Pseudomonas Syringae pv. Syringae 642 >gil 163750766|refZP 02158001.1| 2-dehydropantoate 15 >gi288931618 |refYP 003435678.1 2-dehydropantoate 2-reductase Shewanella benthica KT99 2-reductase Ferroglobus placidus DSM 10642 >gil 71278126|refYP 267839.1 2-dehydropantoate 2-re >gil 127513823|refYP 001095020.1 2-dehydropantoate ductase Colwellia psychrerythraea 34H. 2-reductase Shewanella loihica PV-4 >gil 152984521|refYP 0013503.04.1 2-dehydropantoate >gi24375310|refNP 719353.1 2-dehydropantoate 2-re 2-reductase Pseudomonas aeruginosa PA7 ductase Shewanella Oneidensis MR-1 >gil 146291921|refYP 001182345.1 2-dehydropantoate >gi2545.15212|refZP 05127273.1| 2-dehydropantoate 2-reductase Shewanella putrefaciens CN-32 2-reductase gamma proteobacterium NOR5-3 >gi307304742|refZP 07584492.1 2-dehydropantoate >gi237799306|refZP 04587767.1| 2-dehydropantoate 2-reductase Shewanella baltica BA 175 2-reductase Pseudomonas Syringae pv. Oryzae Str. 1, 6 >gil 196157569|refYP 002127058.1 2-dehydropantoate 25 >gi294101101|refYP 003552959.1 2-dehydropantoate 2-reductase Alteromonas macleodii Deep ecotype 2-reductase Aminobacterium colombiense DSM 12261 >gil 146283482|refYP 001173635.1 2-dehydropantoate >gil 1194.68495 refZP 01611586.1 putative 2-dehydro 2-reductase Pseudomonas Stutzeri A1501 pantoate reductase Alteromonadales bacterium TW-7 >gi226943468 refYP 002798.541.1 2-dehydropantoate >gi1312879258|refZP 07739058.1| 2-dehydropantoate 2-reductase Azotobacter vinelandii DJ 30 2-reductase Aminomonas paucivorans DSM 12260 >gi229592315|refYP 00287.4434.1 2-dehydropantoate >gi28871529 refNP 794148.1 2-dehydropantoate 2-re 2-reductase Pseudomonas fluorescens SBW25 ductase Pseudomonas syringae pv. tomato str. DC3000 >gil 126667530|refZP 01736500.1| 2-dehydropantoate >gi298488549|refZP 07006579.1| 2-dehydropantoate 2-reductase Marinobacter sp. ELB17 2-reductase Pseudomonas Savastanoi pv. Savastanoi >gil 14854.9578|refYP 001269680.1| 2-dehydropantoate 35 NCPPB 3335) 2-reductase Pseudomonas putida F1 >gil 114561913|refYP 74.9426.1 2-dehydropantoate 2-re >gi312962772|ref ZP 07777260.1 ketopantoate reductase ductase Shewanella frigidimarina NCIMB 400 ApbA Pseudomonas fluorescens WH6 >gil 119477336|refZP 01617527.1| 2-dehydropantoate >gil 77.460643|refYP 350150.1 2-dehydropantoate 2-re 2-reductase marine gamma proteobacterium HTCC2143 ductase Pseudomonas fluorescens Pf)-1 40 >gi282165330|refYP 003357715.1| 2-dehydropantoate >gi91792130|refYP 561781.1 2-dehydropantoate 2-re 2-reductase Methanocella paludicola SANAE ductase Shewanella denitrificans OS217 >gil 94502098 |refZP 01308601.1 2-dehydropantoate 2-re >gil 167035489|refYP 001670720.1 2-dehydropantoate ductase Oceanobacter sp. RED65 2-reductase Pseudomonas putida GB-1 >gi269792076 refYP 003316980.1 2-dehydropantoate >gi89093977|refZP 01166922. 12-dehydropantoate 2-re 45 2-reductase Thermanaerovibrio acidaminovorans DSM ductase Oceano spirillum sp. MED92 6589 >gi324101840lgb|EGB99375.1 2-dehydropantoate 2-re >gi1322383702|refZP 08057453.1| 2-dehydropantoate ductase Pseudomonas sp. TJI-51 2-reductase-like protein Paenibacillus larvae Subsp. lar >gi83644750|refYP 433185.1 2-dehydropantoate 2-re vae B-3650 ductase Hahella cheiuensis KCTC 2396 50 >gil 119775691 refYP 928431.1 2-dehydropantoate 2-re >gil 146305972|refYP 001186437.1 2-dehydropantoate ductase Shewanella amazonensis SB2B 2-reductase Pseudomonas mendocina ymp >gi308048598 |refYP 003912164.1 2-dehydropantoate >gil 170727979|refYP 001762005.11 2-dehydropantoate 2-reductase Ferrimonas balearica DSM 9799 2-reductase Shewanella woodvi ATCC 51908 >gi85712652|refZP 01043698.12-dehydropantoate 2-re >gil 170720144|refYP 001747832.1 2-dehydropantoate 55 ductase Idiomarina baltica OS145 2-reductase Pseudomonas putida W619 >gi22954.1211|refZP 04430271.1 2-dehydropantoate >gil 113971493 refYP 735286.1 2-dehydropantoate 2-re 2-reductase Bacillus coagulans 36D1 ductase Shewanella sp. MR-4 >gi77361202|refYP 340777.1 2-dehydropantoate reduc >gil 119945922|refYP 943602.11 2-dehydropantoate 2-re tase Pseudoalteromonas haloplanktis TAC 125 ductase Psychromonas ingrahamii 37 60 >gil 11499285 refNP 070523.1 2-dehydropantoate 2-re >gi288573554 refZP 06391911.1| 2-dehydropantoate ductase Archaeoglobus fulgidus DSM 4304 2-reductase Dethiosulfovibrio peptidovorans DSM >gi254427620|refZP 05041327.1 2-dehydropantoate 11002 2-reductase Alcanivorax sp. DG881 >gil 117919232|refYP 868424.1 2-dehydropantoate 2-re >gi317151157 reflXP 001824478.2 2-dehydropantoate ductase Shewanella sp. ANA-3 65 2-reductase Aspergillus oryzae RIB40 >gi311694203 lgb|ADP97076.1 2-dehydropantoate 2-re >gi254281804|refZP 04956772.1 2-dehydropantoate ductase marine bacterium HP15 2-reductase gamma proteobacterium NOR51-B US 8,765,426 B2 29 30 >gi315125903 refYP 004067906.1 2-dehydropantoate >gi242399000|refYP 002994424.1 2-dehydropantoate reductase Pseudoalteromonas sp. SM9913 2-reductase Thermococcus Sibiricus MM 739 >gi300311 176|refYP 003775268.1| 2-dehydropantoate >gil 1903479041gb|EDK40261.2 hypothetical proten 2-reductase Herbaspirillum seropedicae SmR1 PGUG 04359 Pichia guilliermondii ATCC 6260) >gi251797891|refYP 003012622.1 2-dehydropantoate 5 >gi301055411|refYP 003793622.1 putative 2-dehydro 2-reductase Paenibacillus sp. JDR-2 pantoate 2-reductase Bacillus anthracis CI >gi307544890|refYP 003897369.1| 2-dehydropantoate >gi300712827|refYP 03738639.11 2-dehydropantoate 2-reductase Halomonas elongata DSM 2581 2-reductase Halalkalicoccus jeotgali B3 >gi301064244|refZP 07204687.1 2-dehydropantoate >gil 156052.196 reflXP 001592059.1 hypothetical protein 2-reductase delta proteobacterium NaphS2 10 SS1G 07507 Sclerotinia sclerotiorum 1980 >gi825238961emb|CA|78619.1 ketopantoate reductase >gi85704948|refZP 01036049.1 ketopantoate reductase uncultured delta proteobacterium/ ApbA Roseovarius sp. 217 >gi315646013 refZP 07899134.1 2-dehydropantoate >gi295399703 |refZP 06809684.1| 2-dehydropantoate 2-reductase Paenibacillus vortex V453 2-reductase Geobacillus thermoglucosidasius C56 >gi229061526|refZP 04.198870.1 2-dehydropantoate 15 YS93 2-reductase Bacillus cereus AH603 >gi254573604|refXP 0024939.11.1 hypothetical protein >gi229162856|refZP 04290813.1 2-dehydropantoate Pichia pastoris GS115 2-reductase Bacillus cereus R309803 >gi299132097 refZP 07025292.1| 2-dehydropantoate >gi228960 138|refZP 04121802.1 2-dehydropantoate 2-reductase Afipia sp. 1 NLS2 2-reductase Bacillus thuringiensis serovar pakistani Str. >gi307354061 refYP 003895112.1 2-dehydropantoate T13001) 2-reductase Methanoplanus petrolearius DSM 11571 >gi324327820gb|ADY23080.1 2-dehydropantoate 2-re >gi261405647|refYP 003241888.1 2-dehydropantoate ductase Bacillus thuringiensis serovar finitimus YBT-020 2-reductase Paenibacillus sp. Y412MC10 >gi282857022|refZP 06266273.1 2-dehydropantoate >gi28958.1665 refYP 0034.80131.1 2-dehydropantoate 2-reductase Pyramidobacter piscolens W5455 25 2-reductase Natrialba magadii ATCC 43099 >gi56460968|refYP 156249.1 2-dehydropantoate 2-re >gi212696972|ref ZP 03305100.1 hypothetical protein ductase Idiomarina loihiensis L2TR ANHYDRO 01535 Anaerococcus hydrogenalis DSM >gi302342758 |refYP 003807287.1| 2-dehydropantoate 7454) 2-reductase Desulfarculus baarsii DSM 2075 >gi70730838|refYP 260579.1 2-dehydropantoate 2-re >gi260655057 refZP 05860545.1| 2-dehydropantoate 30 ductase Pseudomonas fluorescens Pf-5 2-reductase Jonquetella anthropi E3 33 E1 >gi225575149 refZP 03783.759.1 hypothetical protein >gi297180479gb|ADI16693.1 ketopantoate reductase un RUMHYD 03238 Blautia hydrogenotrophica DSM cultured gamma proteobacterium HF0010 05D02 10507 >gi2291 17417|refZP 04246793.1 2-dehydropantoate >gi296504414|refYP 0036661 14.1 2-dehydropantoate 2-reductase Bacillus cereus Rock1-3 35 2-reductase Bacillus thuringiensis BMB171 >gi294.501030|refYP 003564730.1 2-dehydropantoate >gil 187780219|ref ZP 02996692.1 hypothetical protein 2-reductase Bacillus megaterium QMB.1551 CLOSPO 03815 Clostridium sporogenes ATCC 15579 >gi310643005 refYP 003947763.1 2-dehydropantoate >gil 56475560) refYP 157149.1 ketopantoate reductase 2-reductase Paenibacillus polymyxa SC2 ApbA Aromatoleum aromaticum EbN1 >gil 121704788 |refXP 001270657.1 2-dehydropantoate 40 >gi57641903 |refYP 184361.1 2-dehydropantoate 2-re 2-reductase, putative Aspergillus clavatus NRRL 1 ductase Thermococcus kodakarensis KOD1 >gil 1940 15015 refZP 03053632.1 2-dehydropantoate >gi296242388 |refYP 003649875.1 2-dehydropantoate 2-reductase Bacillus pumilus ATCC 7061 2-reductase Thermosphaera aggregains DSM 11486 >gil 121534373|refZP 01666197.1 2-dehydropantoate >gi229031552|refZP 04187552.1| 2-dehydropantoate 2-reductase Thermosinus carboxydivorans Nor1 45 2-reductase Bacillus cereus AH1271 >gi300854.950|refYP 003779934.1 ketopantoate reduc >gil 152996054 refYP 001340889.1 2-dehydropantoate tase PanE/ApbA Clostridium ljungdahlii DSM 13528 2-reductase Marinomonas sp. MWYL1 >gi228909746|refZP 04073569.1 2-dehydropantoate >gil 88704128|refZP 01101843.1 Ketopantoate reductase 2-reductase Bacillus thuringiensis IBL 200 Apb A/PanE Congregibacter litoralis KT71 >gil 153938200 refYP 001390518.11 2-dehydropantoate 50 >gil 149181743|refZP 01860235.1 2-dehydropantoate 2-reductase Clostridium botulinum F Str. Langeland 2-reductase Bacillus sp. SG-1 >gil 51243923 |refYP 063807.1 2-dehydropantoate 2-re >gi322371463|refZP 08046012.1 2-dehydropantoate ductase Desulfotalea psychrophila LSV54 2-reductase Haladaptatus paucihalophilus DX253 >gi88858567|refZP 01.133209.1 putative 2-dehydropan >gil 110833490|refYP 692349.1 2-dehydropantoate 2-re toate reductase Pseudoalteromonas tunicata D2 55 ductase Alcanivorax borkumensis SK2 >gi241895418 refZP 04782714.1 2-dehydropantoate >gi241663589 refYP 00298.1949.1 2-dehydropantoate 2-reductase Weissella paramesenteroides ATCC 33313 2-reductase Ralstonia pickettii 12D >gi229019124 refZP 04175958.1 2-dehydropantoate >gil 18977768|refNP 579 125.1 2-dehydropantoate 2-re 2-reductase Bacillus cereus AH1273 ductase Pyrococcus furiosus DSM 3638 >gi313124944|refYP 004035208.1 ketopantoate reduc 60 >gil 110798.963|refYP 695239.1 2-dehydropantoate 2-re tase Halogeometricum borinquense DSM 11551 ductase Clostridium perfingens ATCC 13124 >gi 308069898 |refYP 003871503.11 2-dehydropantoate >gil 169782143 reflXP 001825534.1 2-dehydropantoate 2-reductase Paenibacillus polymyxa E681 2-reductase family protein Aspergillus Oryzae RIB40 >gil 134299186|refYP 001112682.1 2-dehydropantoate >gil 157692185 refYP 001486647.1 2-dehydropantoate 2-reductase Desulfotomaculum reducens MI-1 65 2-reductase Bacillus pumilus SAFR-032 >gil 116750179|refYP 84.6866.1 2-dehydropantoate 2-re >gil 15606805|refNP 214186.1 hypothetical protein ductase Syntrophobacter fitmaroxidans MPOB aq 1727 Aquifex aeolicus VF5 US 8,765,426 B2 31 32 >gil 163782247|ref ZP 02177245.1 hypothetical protein >gi2122246.16|refYP 002307852.1 2-dehydropantoate HG1285 05655 Hydrogenivirga sp. 126-5-R1-1) 2-reductase Thermococcus Onnurineus NA1 >gi317129310|refYP 004095592.1 2-dehydropantoate 2 >gi302335576|refYP 003.800783.11 2-dehydropantoate reductase Bacillus cellulosilyticus DSM 2522 2-reductase Olsenella uli DSM 7084 >gi22.9174587|refZP 04302117.1 2-dehydropantoate >gil 154150658 |refYP 001404276.1 2-dehydropantoate 2-reductase Bacillus cereus MM3 2-reductase Candidates Methanoregula boomei 6A8 >gi229086485 refZP 04218657.1 2-dehydropantoate >gi284166943|refYP 003405222.1 2-dehydropantoate 2-reductase Bacillus cereus Rock3-44 2-reductase Haloterrigena turkmenica DSM5511 >gil 167044940lgb|ABZ09606.1 putative Ketopantoate >gil 172057979|refYP 001814439.1 2-dehydropantoate reductase PanE/ApbA uncultured marine microorganism 10 2-reductase Exiguobacterium Sibiricum 255-15 HF4000 APKG8D23 >gi313637013 lgb|EFS02587.1 2-dehydropantoate 2-re >gil 15896.190|refNP 349539.1 ketopantoate reductase ductase Listeria seeligeri FSL S4-171 PanE/ApbA Clostridium acetobutyllicum ATCC 824 >gi228998702|refZP 04158289.1 2-dehydropantoate >gi324103300gb|EGC00636.1 2-dehydropantoate 2-re 2-reductase Bacillus mycoides Rock3-17 ductase Pseudomonas sp. TJI-51 15 >gil 152976282|refYP 001375799.11 2-dehydropantoate >gil 14520967|refNP 126442.1 2-dehydropantoate 2-re 2-reductase Bacillus cereus subsp. cytotoxis NVH 391 ductase Pyrococcus abyssi GE51 98) >gi222097366|refYP 002531423.1| 2-dehydropantoate >gi921 14269|refYP 574197.1 2-dehydropantoate 2-re 2-reductase Bacillus cereus Ol ductase Chromohalobacter salexigens DSM 3043) >gi31964.9677|refZP 08003833.1| 2-dehydropantoate >gi240102379 refYP 002958688.1 2-dehydropantoate 2-reductase Bacillus sp. 2. A 57 CT2 2-reductase Thermococcus gammatolerans EJ3 >gi258514829|refYP 003191051.1 2-dehydropantoate >gil 2068894.43 refYP 002249396.1 2-dehydropantoate 2-reductase Desulfotomaculum acetoxidans DSM 771 2-reductase Thermodesulfovibrio yellowstonii DSM >gi87121684|refZP 01.077770.1 ketopantoate reductase 11347 Marinomonas sp. MED121 25 >gi297583937|refYP 0036997 17.1| 2-dehydropantoate >gil 145589722|refYP 011563.19.1 2-dehydropantoate 2-reductase Bacillus selenitireducens MLS10 2-reductase Polynucleobacter necessarius Subsp. asymbi >gi270290296 refZP 06196521.1 2-dehydropantoate oticus QLW-P1DMWA-1 2-reductase Pediococcus acidilactici 7 4 >gi254.173247|refZP 0487.9920.1 2-dehydropantoate >gil 147919508 |refYP 686752.1 putative 2-dehydropan 2-reductase Thermococcus sp. AM4 30 toate 2-reductase uncultured methanogenic archaeon >gil 145592200|refYP 001154202.1 2-dehydropantoate RC-1 2-reductase Pyrobaculum arsenaticum DSM 13514 >gi313632475 lgb|EFR994.93.1 2-dehydropantoate 2-re >gi91788789|refYP 549741.1 2-dehydropantoate 2-re ductase Listeria seeligeri FSLN 1-067 ductase Polaromonas sp. JS666 >gi266623089 refZP 06116024.1 2-dehydropantoate >gi257387161 refYP 003176934.1 2-dehydropantoate 35 2-reductase Clostridium hathewayi DSM 13479 2-reductase Halomicrobium mukohataei DSM 12286 >gil 186470939|refYP 001862257.1 2-dehydropantoate il 18314044|refNP 5607 11.1 2-dehydropantoate 2-re 2-reductase Burkholderia phymatum STM815 ductase (ketopantoate reductase) Pyrobaculum aerophi >gi91975083|refYP 567742.1 2-dehydropantoate 2-re lum str. IM2 ductase Rhodopseudomonas palustris Bisb5 >gi319646112|ref ZP 08000342.1|Y1bQ protein Bacillus 40 >gil 168186958 |refZP 02621593.1 2-dehydropantoate sp. BT1B CT2) 2-reductase Clostridium botulinum C str. Eklund >gil 168232151 refZP 02657209.1 2-dehydropantoate >gil 171316249|refZP 02905471.1 2-dehydropantoate 2-reductase Salmonella enterica Subsp. enterica serovar 2-reductase Burkholderia ambifaria MEX-5 Kentucky str. CDC 191 >gil 11844.4120|refYP 877052.1 2-dehydropantoate 2-re >gi323488622|refZP 08093866.1 2-dehydropantoate 45 ductase Clostridium novyi NT 2-reductase Planococcus donghaensis MPA1 U2 >gi304403996 |refZP 07385658.1 2-dehydropantoate >gi226313441|refYP 002773335.1 2-dehydropantoate 2-reductase Paenibacillus curdlanolyticus YK9 2-reductase Brevibacillus brevis NBRC 100599 >gil 14591 192|refNP 143268.1 2-dehydropantoate 2-re >gi887.99068|refZP 011 14649,12-dehydropantoate 2-re ductase Pyrococcus horikoshii OT3 ductase Reinekea sp. MED297 50 >gi39937620) refNP 949896.1 putative 2-dehydropan >gi242813341|refXP 00248.6147.1 2-dehydropantoate toate 2-reductase Rhodopseudomonaspalustris CGA009 2-reductase, putative Talaromyces stipitatus ATCC >gi257051985 reflyP 0031298.18.11 2-dehydropantoate 10500 2-reductase Halorhabdus utahensis DSM 12940 >gi225849819|refYP 002730053.1 2-dehydropantoate >gi297616632|refYP 003701791.1 2-dehydropantoate 2-reductase Persephonella marina EX-H1 55 2-reductase Syntrophothermus lipocalidus DSM 12680 >gi209664195 refYP 002288052.1 2-dehydropantoate >gil 124485202|refYP 0010298.18.1 hypothetical protein 2-reductase Oligotropha carboxidovorans OM5 Mlab 0375 Methanocorpusculum labreanum Z. >gil 188589152|refYP 001920485.1 2-dehydropantoate >gi1311029915 refZP 07708005.1| 2-dehydropantoate 2-reductase Clostridium botulinum E3 str. Alaska E43 2-reductase Bacillus sp. m3-13 >gil 119505092|refZP 01627168.1 2-dehydropantoate 60 >gi320355422|refYP 004196761.1 2-dehydropantoate 2-reductase marine gamma proteobacterium HTCC2080 2-reductase Desulfobulbus propionicus DSM 2032 >gil 15789904|refNP 279728.1 hypothetical protein >gi255654270|ref ZP 05399679.1 putative ketopantoate VNG0730C Halobacterium sp. NRC-1 reductase Clostridium difficile QCD-23m63 >gi300709487|refYP 0037353.01.1| 2-dehydropantoate >gi86747344|refYP 483840.1 2-dehydropantoate 2-re 2-reductase Halalkalicoccus jeotgali B3 65 ductase Rhodopseudomonas palustris HaA2 >gi269124038 |refYP 0.03306615.1 2-dehydropantoate >gi319796261|refYP 004157901.1 2-dehydropantoate 2-reductase Streptobacillus moniliformis DSM 12112 2-reductase Variovorax paradoxus EPS US 8,765,426 B2 33 34 >gi289435387|refYP 003465259.1 2-dehydropantoate >gi3197895.10|refYP 004151143.1 2-dehydropantoate 2-reductase Listeria seeligeri serovar 1/2b str. 2-reductase Thermovibrio ammonificans HB-1 SLCC3954 >gil 171320779|refZP 02909787.1| 2-dehydropantoate >gi284161230|refYP 003399853.1 2-dehydropantoate 2-reductase Burkholderia ambifaria MEX-5 2-reductase Archaeoglobus profiindus DSM 5631 >gi295705715 reflyP 003598790.1| 2-dehydropantoate >gil 163792409|refZP 02186386.1 2-dehydropantoate 2-reductase Bacillus megaterium DSM 319 2-reductase alpha proteobacterium BAL199 >gi256853805 refZP 05559170.1| 2-dehydropantoate >gi227535072|refZP 03965121.1| 2-dehydropantoate 2-reductase Enterococcus faecalis T8 2-reductase Lactobacillus paracasei Subsp. paracasei >gil 186472751|refYP 001860093.1 2-dehydropantoate ATCC 25302 10 2-reductase Burkholderia phymatum STM815 >gil 1720.65517|refYP 001816229.1 2-dehydropantoate >gil 164687814|ref ZP 0221 1842.1 hypothetical protein 2-reductase Burkholderia ambifaria MC40-6 CLOBAR 01458 Clostridium bartlettii DSM 16795 >gi56696485 refYP 166842.1 2-dehydropantoate 2-re >gi315147351 lgb|EFT91367.1 2-dehydropantoate 2-re ductase Ruegeria pomeroyi DSS-3 ductase Enterococcus faecalis TX4244 >gi21263.9667 refYP 002316187.1 2-dehydropantoate 15 >gi289450069|refYP 0.03474740.1 putative 2-dehydro 2-reductase Anoxybacillus flavithermus WK1 pantoate 2-reductase Clostridiales genomosp. BVAB3 str. >gil 7353.8792|refYP 299159.1 ketopantoate reductase UPII9-5 Apb A/PanE Ralstonia eutropha JMP134 >gil 126178741 refYP 001046706.1 2-dehydropantoate >gi288818570|refYP 003432918.1 2-dehydropantoate 2-reductase Methanoculleus marisnigri JR1 2-reductase Hydrogenobacter thermophilus TK-6 >gi313622979gb|EFR93276.1 2-dehydropantoate 2-re >gil 160941380|ref ZP 02088717.1 hypothetical protein ductase Listeria innocua FSL J1-023 CLOBOL 06273 Clostridium bolteae ATCC BAA-613 >gil 1971 17182|refYP 002137609.1 2-dehydropantoate >gil 167630796|refYP 001681295.1 2-dehydropantoate 2-reductase Geobacter bemidjiensis Bern 2-reductase Heliobacterium modesticaldun Icel 25 >gi257065623 reflyP 003 151879.11 2-dehydropantoate >gi89099594|refZP 01 172469.1 2-dehydropantoate 2-re 2-reductase Anaerococcus previotii DSM 20548 ductase Bacillus sp. NRRL B-14911 >gi221211405 refZP 03584384.1 2-dehydropantoate >gi227431961|ref ZP 03913981.1 possible 2-dehydro 2-reductase Burkholderia multivorans CGD1 pantoate 2-reductase Leuconostoc mesenteroides Subsp. >gil 121606900|refYP 984229.1 2-dehydropantoate 2-re Cremoris ATCC 19254 30 ductase Polaromonas naphthalenivorans CJ2 >gi2995.34661|ref ZP 07047993.1 putative 2-dehydro >gi229823430|ref ZP 04449499.1 hypothetical protein pantoate 2-reductase Lysinibacillus fusiformis ZC1 GCWU000282 00728 Catonella morbi ATCC 51271 >gi227552200 refZP 03982249.1 2-dehydropantoate >gi303256960|refZP 07342974.1 2-dehydropantoate 2-reductase Enterococcus faecium TX 1330 2-reductase Burkholderiales bacterium 1 1 47 >gi289595871|refYP 003482567.11 2-dehydropantoate 35 >gil 159042347 IrefYP 001541599.1 2-dehydropantoate 2-reductase Aciduliprofiindum boomei T469 2-reductase Caldivirga maquilingensis IC-167 >gil 138894643|refYP 001 125096.1 2-dehydropantoate >gi315155665 lgb|EFT99681.1 2-dehydropantoate 2-re 2-reductase Geobacillus thermodernitrificans NG80-2 ductase Enterococcus faecalis TX0043 >gi55379641|refYP 137491.1 2-dehydropantoate 2-re >gi1317496587|refZP 07954935.1| 2-dehydropantoate ductase Haloarcula marismortui ATCC 43049 40 2-reductase Gemella moribilum M424 >gi294.083831|refYP 003550588.1 2-dehydropantoate >gi323342656|refZP 08082888.1 2-dehydropantoate 2-reductase Candidatus Puniceispirillum marinum 2-reductase Erysipelothrix rhusiopathiae ATCC 19414 IMCC1322 >gi239817916|refYP 002946826.1 2-dehydropantoate >gil 119898762|refYP 933975.1 2-dehydropantoate 2-re 2-reductase Variovorax paradoxus S110 ductase Azoarcus sp. BH72 45 >gi254169372|refZP 04876201.1 2-dehydropantoate >gil 119872861|refYP 93.0868.1 2-dehydropantoate 2-re 2-reductase Aciduliprofiundum boomei T469 ductase Pyrobaculum islandicum DSM 4184 >gi307594721|refYP 003901038.11 2-dehydropantoate >gi257456159|refZP 05621356.1| 2-dehydropantoate 2-reductase Vulcanisaeta distributa DSM 14429 2-reductase Treponema vincentii ATCC 35580 >gi297530725 reflyP 003672000. 11 2-dehydropantoate >gi257867166|ref ZP 056468.19.1 ketopantoate reductase 50 2-reductase Geobacillus sp. C56-T3 Enterococcus casselliflavus EC30 >gi300173017|refYP 003772183.11 2-dehydropantoate >gil 126459006|refYP 001055284.1 2-dehydropantoate 2-reductase Leuconostoc gasicomitatum LMG 18811 2-reductase Pyrobaculum calidifontis JCM 11548 >gi525.49536lgb|AAU83385.1 hypothetical protein >gi239826511|refYP 002949135.1 2-dehydropantoate GZ27G5 16 uncultured archaeon GZfos27G5 2-reductase Geobacillus sp. WCH7O 55 >gil 119094200gb|ABL61022.1 ketopantoate reductase >gi91790339|refYP 551291.1 2-dehydropantoate 2-re uncultured marine bacterium HF10 25F10 ductase Polaromonas sp. JS666 >gil 162146807|refYP 001601268.1 putative 2-dehydro >gi256829670|refYP 003158398.1 2-dehydropantoate pantoate 2-reductase Gluconacetobacter diazotrophicus 2-reductase Desulfomicrobium baculatum DSM 4028 PAI 5) >gi315659442|refZP 07912305.1 2-dehydropantoate 60 >gi256963612|ref ZP 05567763.1 ketopantoate reductase 2-reductase Staphylococcus lugdunensis M23590 Apb A/PanE Enterococcus faecalis HIP1 1704 >gil 150403611 refYP 001330905.1 2-dehydropantoate >gi313617966lgb|EFR90131.1 2-dehydropantoate 2-re 2-reductase Methanococcus maripaludis C7 ductase Listeria innocua FSL S4-378 >gi259047992|refZP 05738393.1| 2-dehydropantoate >gi313679570|refYP 004057309.11 2-dehydropantoate 2-reductase Granulicatella adiacens ATCC 49175 65 2-reductase Oceanithermus profiindus DSM 14977 >gi24214890|refNP 712371.1 2-dehydropantoate 2-re >gi1313.885617|refZP 07819367.1| 2-dehydropantoate ductase Leptospira interrogans serovar Lai Str. 566.01 2-reductase (Eremococcus coleocola ACS-139-V-Col8 US 8,765,426 B2 35 36 >gil 163790016|refZP 02184451.1 2-dehydropantoate >gi297.527045|refYP 003669069.1 2-dehydropantoate 2-reductase Carnobacterium sp. AT7 2-reductase Staphylothermus hellenicus DSM 12710 >gi225870636|refYP 002746583.1 ketopantoate reduc >gi1303233096|refZP 07319769.1| 2-dehydropantoate tase Streptococcus equi Subsp. equi 4047 2-reductase Atopobium vaginae PB189-T1-4 >gi1320527125 refZP 08028312.1| 2-dehydropantoate 5 >gi253699477 refYP 003020666.1 2-dehydropantoate 2-reductase Solobacterium moorei F0204 2-reductase Geobacter sp. M21 >gi316936042|refYP 004111024.1 2-dehydropantoate 2 >gi293401734|refZP 06645875.1 2-dehydropantoate reductase Rhodopseudomonas palustris DX-1 2-reductase Erysipelotrichaceae bacterium 5 2 >gi209559212|refYP 002285684.1 2-dehydropantoate 54FAA 10 >gi225849463|refYP 002729628.1 2-dehydropantoate 2-reductase Streptococcus pyogenes NZ131 2-reductase Sulfurihydrogenibium azorense AZ-Ful >gi1315304102|refZP 07874502.1| 2-dehydropantoate >gi227893553 refZP 04011358.1| 2-dehydropantoate 2-reductase Listeria ivanovii FSL F6-596 2-reductase Lactobacillus ultunensis DSM 16047 >gi3233.73091 lgb|ADX45360.1 2-dehydropantoate 2-re >gi85544557.pdb|2EW2|A Chain A, Crystal Structure Of ductase Acidovorax avenae subsp. avenae ATCC 19860 15 The Putative 2-Dehydropantoate 2- Reductase From >gil 169782199 refiXP 001825562. 1 2-dehydropantoate Enterococcus Faecalis 2-reductase family protein Aspergillus oryzae RIB40 >gil 146318978 |refYP 001 198690.1 2-dehydropantoate >gil 188996324|refYP 001930575..1 2-dehydropantoate 2-reductase Streptococcus suis 05ZYH33 2-reductase Sulfurihydrogenibrum sp.YO3AOP1 >gil 124266554|refYP 001020558.1 2-dehydropantoate >gil 206561131|refYP 02231896.1 putative ketopantoate 2-reductase Methylibium petroleiphilum PM1 reductase Burkholderia cenocepacia J2315 >gi283768342|refZP 0634.1254.1 2-dehydropantoate >gi56807918|refZP 003.65743.1 COG 1893: Ketopan 2-reductase Bulleidia extructa W1219 toate reductase Streptococcus pyogenes M49 591 >gi315223029 refZP 07864908.1 2-dehydropantoate >gil 120610087|refYP 969765.1 2-dehydropantoate 2-re 2-reductase Streptococcus anginosus F0211 ductase Acidovorax avenae subsp. citrulli AAC00-1 25 >gi207857980|refYP 002244631.1 2-dehydropantoate >gi52548816|gb|AAU82665.1 conserved hypothetical pro 2-reductase Salmonella enterica Subsp. enterica serovar tein uncultured archaeon GZfos19A5 Enteritidis str. P125109 >gi70725616|refYP 252530.1 2-dehydropantoate 2-re >gil 134293442|refYP 001117178.1 2-dehydropantoate ductase Staphylococcus haemolyticus JCSC 1435 2-reductase Burkholderia vietnamiensis G4 >gi292654245 refYP 003534142.1 2-dehydropantoate 30 >gi52550479.gb|AAU84328.1 conserved hypothetical pro 2-reductase Haloferax volcanii DS2 tein uncultured archaeon GZfos9D1 >gi288931405 refYP 003435465.11 2-dehydropantoate >gi324965.1531gb|ADY55932.1 2-dehydropantoate 2-re 2-reductase Ferroglobus placidus DSM 10642 ductase Syntrophobotulus glycolicus DSM 8271 >gil 1504.00258 |refYP 001324025.1 2-dehydropantoate >gi296331086 refZP 06873560.1 2-dehydropantoate 2-reductase Methanococcus vannielii SB 35 2-reductase Bacillus subtilis subsp. spizizeni ATCC >gil 126466272|refYP 001041381.1 2-dehydropantoate 6633 2-reductase Staphylothermus marinus F1 >gi228474704|refZP 04059435.1 2-dehydropantoate >gi221068483|refZP 0354.4588.1 2-dehydropantoate 2-reductase Staphylococcus hominis SK119 2-reductase Comamonas testosteroni KF-1 >gil 119896483|refYP 931696.1 2-dehydropantoate 2-re >gi29982 1547|ref ZP 07053435.1 possible 2-dehydro 40 ductase Azoarcus sp. BH72 pantoate 2-reductase Listerigrayi DSM 20601 >gil 297181166lgb|ADI17362.1 ketopantoate reductase un >gi290508302|refZP 06547673.1| 2-dehydropantoate cultured delta proteobacterium HF0070 30B07 2-reductase Klebsiella sp. 1 1 55 >gil 16933.3685 refZP 028.60878.1 hypothetical protein >gi297.570429|refYP 003691773.11 2-dehydropantoate ANASTE 00069 Anaerofustis stercorihominis DSM 2-reductase Desulfurivibrio alkaliphilus AHT2 45 17244 >gil 169827000 refYP 001697 158.1 | putative 2-dehydro >gil 171184791 refYP 001793710.1 2-dehydropantoate pantoate 2-reductase Lysinibacillus sphaericus C3-41 2-reductase Thermoproteus neutrophilus V24Sta >gi238503586|refXP 002383026.1 2-dehydropantoate >gil 116334695 refYP 796222.1 2-dehydropantoate 2-re 2-reductase family protein, putative Aspergillus flavus ductase Lactobacillus brevis ATCC 367 NRRL3357 50 >gi45358235|refNP 987792.1 binding-proteindependent >gil 116873476|refYP 850257.1 2-dehydropantoate 2-re transport system inner membrane protein Methanococcus ductase Listeria wellshimeri serovar 6b str. SLCC5334 maripaludis S2 >gi242556790.pdb|3HWRIA Chain A, Crystal Structure Of >gil 15673305 refNP 267479.1 2-dehydropantoate 2-re Pane APBA family ketopantoate reductase (Yp ductase Lactococcus lactis Subsp. lactis II 1403 29915.9.1) From Ralstonia Eutropha Jmp134 55 >gil 167569002|refZP 02361876.1 2-dehydropantoate >gi302387383 refYP 003823205.11 2-dehydropantoate 2-reductase Burkholderia Oklahomensis C6786 2-reductase Clostridium saccharolyticum WM1 >gi222444726|ref ZP 03607241.1 hypothetical protein >gi307726219 refYP 003909432.1 2-dehydropantoate METSMIALI 00339 Methanobrevibacter Smithii DSM 2-reductase Burkholderia sp. CCGE1003 2375 >gi257871049|ref ZP 05650702.1 ketopantoate reductase 60 >gil 148642093 refYP 001272606.1 ketopantoate reduc Enterococcus gallinarum EG2 tase. ApbA Methanobrevibactersmithii ATCC 35061 >gil 145254262|refXP 001398576.1 2-dehydropantoate >gi56964.133 refYP 175864.1 2-dehydropantoate 2-re 2-reductase family protein Aspergillus niger CBS 513.88 ductase Bacillus clausii KSM-K16 >gi227515951 IrefZP 03.946000.1| 2-dehydropantoate >gil 76801 123 refYP 326131.1 2-dehydropantoate 2-re 2-reductase Atopobium vaginae DSM 15829 65 ductase Natronomonas pharaonis DSM 2160 >gi254247377|ref ZP 0494.0698.1 Ketopantoate reductase >gi291563922emb|CBL42738.1 ketopantoate reductase Burkholderia cenocepacia PC184 butyrate-producing bacterium SS3/4 US 8,765,426 B2 37 38 >gil 126649737|refZP 01721973.1| 2-dehydropantoate >gi310827874|refYP 003.960231.1 hypothetical protein 2-reductase Bacillus sp. B14905 ELI 2285 Eubacterium limosum KIST612 >gil 125623962|refYP 001032445.1 2-dehydropantoate >gil 121595444|refYP 98.7340.1 2-dehydropantoate 2-re 2-reductase Lactococcus lactis subsp. Cremoris MG 1363 ductase Acidovorax sp. JS42 >gi264677329|refYP 003277235.1 2-dehydropantoate >gi29375143|refNP 814296.1 2-dehydropantoate 2-re 2-reductase Comamonas testosteroni CNB-2 ductase Enterococcus faecalis V583 >gil 163793943 |ref ZP 02187917.1 putative ketopantoate >gi1313899882|refZP 07833,385.1| 2-dehydropantoate reductase alpha proteobacterium BAL 199 2-reductase Clostridium sp. HGF2 >gi229543594|ref ZP 04432654. 1 2-dehydropantoate >gil 187920203 refYP 001889234.1 2-dehydropantoate 10 2-reductase Burkholderia phytofirmans PsN 2-reductase Bacillus coagulans 36D1 >gi27381507|refNP 773036.1 2-dehydropantoate 2-re >gi 208435758.pdb|3EGOIA Chain A, Crystal Structure Of ductase Bradyrhizobium japonicum USDA 110 Probable 2-Dehydropantoate 2-Reductase Pane From >gi13.00768503 refZP 07078402.1| 2-dehydropantoate Bacillus Subtilis 2-reductase Lactobacillus plantarum Subsp. plantarum >gi86143831|refZP 01062207.1 hypothetical protein 15 ATCC 14917 MED217 01020 Leeuwenhoekiella blandensis >gil 197295211 reflyP 002153752.1 | putative 2-dehydro MED217 pantoate 2-reductase Burkholderia cenocepacia J2315 >gi 260946531|refXP 002617563.1 hypothetical protein >gi225410102|ref ZP 03761291.1 hypothetical protein CLUG 03007 Clavispora lusitaniae ATCC 42720 CLOSTASPAR 05323 Clostridium asparagiforme >gil 15615142|refNP 243445.1 2-dehydropantoate 2-re DSM 15981 ductase Bacillus halodurans C-125 >gi222 152909|refYP 02562086.1 2-dehydropantoate >gi239625263 refZP 04668294.1 2-dehydropantoate 2-reductase Streptococcus uberis 0140J 2-reductase Clostridiales bacterium 1 7 47 FAA >gi300854058 |refYP 003779042.11 2-dehydropantoate >gi315038258 |refYP 004.031826.1 2-dehydropantoate 2-reductase Clostridium liungdahlii DSM 13528 2-reductase Lactobacillus amylovorus GRL 1112 25 >gi237653536|refYP 002889650.1| 2-dehydropantoate >gi300310480|refYP 003774572.11 2-dehydropantoate 2-reductase Thauera sp. MZ1T 2-reductase Herbaspirillum seropedicae SmR1 >gi259047521|refZP 05737922.1| 2-dehydropantoate >gil 167893.095 refZP 02480497.1 2-dehydropantoate 2-reductase Granulicatella adiacens ATCC 49175 2-reductase Burkholderia pseudomallei 7894 >gi319957266|refYP 004168529.1 2-dehydropantoate >gi89894746|refYP 518233.1) hypothetical protein 30 2-reductase Nitratifactor salsuginis DSM 16511 DSY2000 Desulfitobacterium hafniense Y51 >gi320355269|refYP 004.196608.1 2-dehydropantoate >gi319762040|refYP 004125977.1| 2-dehydropantoate 2-reductase Desulfobulbus propionicus DSM 2032 2-reductase Alicycliphilus denitrificans BC >gi239636063|refZP 04677077.1| 2-dehydropantoate >gil 69250617|refZP 00605173.1 Ketopantoate reductase 2-reductase Staphylococcus warneri L37603 Apb A/PanE Enterococcus faecium DO 35 >gi320100944|refYP 004176536.1 ketopantoate reduc >gi288560576|refYP 003424062. 1 2-dehydropantoate tase Desulfurococcus mucosus DSM 2162 2-reductase PanE Methanobrevibacter ruminantium M1 >gil 169630635 refYP 001704284.1 ketopantoate reduc >gi70725181 refYP 252095.1 2-dehydropantoate 2-re tase ApbA/PanE Mycobacterium abscessus ATCC ductase Staphylococcus haemolyticus JCSC 1435 19977 >gi324098.987 Igb|EGB96980.1 2-dehydropantoate 2-re 40 >gi81427656|refYP 394.653.1 2-dehydropantoate 2-re ductase Pseudomonas sp. TJI-51 ductase Lactobacillus sakei Subsp. sakei 23K >gi86608338|refYP 477100.1 2-dehydropantoate 2-re >gi2418894.07|refZP 04776708.1 2-dehydropantoate ductase Synechococcus sp. JA-2-3Ba(2-13) 2-reductase Gemella haemolysans ATCC 10379 >gil679067991gb|AAY82862.1 putative ketopantoate >gi251811868|refZP 048263.41.1 2-dehydropantoate reductase uncultured bacterium MedeBAC46A06 45 2-reductase Staphylococcus epidermidis BCM >gi310778974|refYP 003967307.11 ketopantoate reduc HMP0060 tase IIvobacter polytropus DSM 2926 >gi218883653|refYP 002428035.1 2-dehydropantoate >gi242372095 refZP 04817669.1 2-dehydropantoate 2-reductase Desulfurococcus kamchatkensis 1221n 2-reductase Staphylococcus epidermidis M23864:W1 >gi229918541|refYP 002887187.1 2-dehydropantoate >gil 170749968 |refYP 001756228.1 2-dehydropantoate 50 2-reductase Exiguobacterium sp. AT1b) 2-reductase Methylobacterium radiotolerans JCM2831 >gi314939006|refZP 07846271.1 2-dehydropantoate >gil 167588290|refZP 02380678.1| 2-dehydropantoate 2-reductase Enterococcus faecium TXO133a()4 2-reductase Burkholderia ubonensis Bu >gil 69244336|refZP 00602804.1 Ketopantoate reductase >gi 78067381|refYP 370150.1 2-dehydropantoate 2-re Apb A/PanE Enterococcus faecium DO ductase Burkholderia sp. 383 55 >gi311068031|refYP 003972954.1 2-dehydropantoate2 >gil 124028371 refYP 001013691.1 ketopantoate reduc reductase Bacillus atrophaeus 1942 tase Hyperthermus butyllicus DSM 5456 >gi21984.7221|refYP 00246.1654.1 2-dehydropantoate >gi219848017|refYP 002462450.1 2-dehydropantoate 2-reductase Chloroflexus aggregains DSM 94.85 2-reductase Chloroflexus aggregains DSM 94.85 >gi295699875 reflyP 003607768.1| 2-dehydropantoate >gil 167580031|refZP 02372905.1| 2-dehydropantoate 60 2-reductase Burkholderia sp. CCGE1002 2-reductase Burkholderia thailandensis TXDOH >gi307730136|refYP 003907360.11 2-dehydropantoate >gi288553174 refYP 003425109.1 2-dehydropantoate 2-reductase Burkholderia sp. CCGE1003 2-reductase Bacillus pseudofirmus OF4 >gi261350733|refZP 05976150.1| 2-dehydropantoate >gil 186470810|refYP 001862128.1 2-dehydropantoate 2-reductase Methanobrevibacter Smithii DSM 2374 2-reductase Burkholderia phymatum STM815 65 >gi84489765|refYP 447997.1 putative 2-dehydropan >gi1313890559|refZP 07824187.1| 2-dehydropantoate toate 2-reductase Methanosphaera stadtmanae DSM 2-reductase Streptococcus pseudoporcinus SPIN 20026 3091) US 8,765,426 B2 39 40 >gi319893545 refYP 004150420.1 2-dehydropantoate >gil 121604457|refYP 98.1786.1 2-dehydropantoate 2-re 2-reductase Staphylococcus pseudintermedius HKU10 ductase Polaromonas naphthalenivorans CJ2 03 >gi308173476|refYP 003920181.1 2-dehydropantoate >gil 1973.02450|ref ZP 03167505.1 hypothetical protein 2-reductase Bacillus amyloliquefaciens DSM 7 RUMLAC 01.178 Ruminococcus lactaris ATCC 29176 >gi31993.9189 refZP 08013552.1| 2-dehydropantoate >gi258542366 refYP 003 187799.1 ketopantoate reduc 2-reductase Streptococcus anginosus 1 2 62CV tase Apb A/PanE Acetobacter pasteurianus IFO 3283-01 >gi295136259|refYP 003586935.1 ketopantoate reduc >gil 172061542|refYP 001809194.1 2-dehydropantoate tase PanE/ApbA Zunongwangia profinda SM-A87 2-reductase Burkholderia ambifaria MC40-6 >gil.94263023|refZP 01286842.1 2-dehydropantoate 2-re 10 ductase delta proteobacterium MLMS-1 >gil 15894883|refNP 348232.1 ketopantoate reductase >gi89896295 refYP 519782.11 hypothetical protein Clostridium acetobutyllicum ATCC 624 DSY3549 Desulfitobacterium hafniense Y51 >gi299132927|refZP 07026122.1 2-dehydropantoate >gi314934501 |refZP 0784 1860.1 2-dehydropantoate 2-reductase Afipia sp. 1 NLS2 2-reductase Staphylococcus Caprae C87 >gil 45657601|refYP 001687.1 2-dehydropantoate 2-re 15 >gil 167747028 |ref ZP 02419155.1 hypothetical protein ductase Leptospira interrogans serovar Copenhageni Str. ANACAC 01740 Anaerostipes caccae DSM 14662 Fiocruz L1-130 >gi258425014 |refZP 05687885.1 2-dehydropantoate >gi238024361|refYP 002.908593.1 2-dehydropantoate 2-reductase Staphylococcus aureus A9635 2-reductase Burkholderia glumae BGR1 >gi225180981|refZP 03734429.1 2-dehydropantoate >gil 154685930|refYP 001421091.1 2-dehydropantoate 2-reductase Dethiobacter alkaliphilus AHT 1 2-reductase Bacillus amyloliquefaciens FZEB42 >gi2961 11865 refYP 003622247.1 2-dehydropantoate >gil 126180322|refYP 00104.8287.1 2-dehydropantoate 2-reductase Leuconostoc kimchi IMSNU 11154 2-reductase Methanoculleus marisnigri JR1 >gi311319581 lgb|EFQ87840.1 hypothetical protein PTT >gil 161523150|refYP 001586079.1 2-dehydropantoate 16492 Pyranophora teres f. teres 0-1 2-reductase Burkholderia multivorans ATCC 17616 25 >gi225734175 pdb|3G17|A Chain A, Structure Of Putative >gi323528188 |refYP 0.04230340.1 2-dehydropantoate 2-Dehydropantoate 2-Reductase From Staphylococcus 2-reductase Burkholderia sp. CCGE1001 Aureus >gi2548.18916|refZP 05223917.1 2-dehydropantoate >gi1315283089 refZP 07871356.1| 2-dehydropantoate 2-reductase Mycobacterium intracellulare ATCC 13950 2-reductase Listeria marthii FSL S4-120 >gi313498.946 lgb|ADR60312.1 2-dehydropantoate 2-re 30 >gi2095438.68|refYP 002276097.1 2-dehydropantoate ductase Pseudomonas putida BIRD-1 2-reductase Gluconacetobacter diazotrophicus PAI 5 >gi22537498 |refNP 688349.1 2-dehydropantoate 2-re >gi1313113024 refZP 07798663.1| 2-denydropantoate ductase Streptococcus agalactiae 2603V/R 2-reductase Faecalibacterium cf. prausnitzii KLE1255 >gil 123403597 refiXP 001302267.1 Ketopantoate reduc >gi291523448 emb|CBK81741.1 ketopantoate reductase tase PanE/ApbA family protein Trichomonas vaginalis 35 Coprococcus catus GD/7 G3 >gi260584453|refZP 05852200.1 2-dehydropantoate >gil 189348029|refYP 00194 1225.1 2-dehydropantoate 2-reductase Granulicatella elegans ATCC 700633 2-reductase Burkholderia multivorans ATCC 17616 >gi261415325 refYP 003249008.1 2-dehydropantoate >gi222150304|refYP 00255.9457.1 2-dehydropantoate 2-reductase Fibrobacter succinogenos Subsp. succino 2-reductase homolog Macrococous caseolyticus 40 genes S85 JCSC5402 i45184780|refNP 982498.1| AAL044Cp Ashbya gos >gi90962337|refYP 536253.1 2-dehydropantoate 2-re sypii ATCC 10895 ductase Lactobacillus salivarius UCC118 >gi255034556 reflyP 003085177.1 | Ketopantoate reduc >gil 160900483 refYP 001566.065.1 2-dehydropantoate tase ApbA/PanE domain protein Dvadobacter fermentans 2-reductase Delfia acidovorans SPH-1 45 DSM 18053 >gi297588109|refZP 06946753.1| 2-dehydropantoate >gi264680193 refYP 0032801.03.1 2-dehydropantoate 2-reductase Finegoldia magna ATCC 53516 2-reductase Comamonas testosteroni CNB-2 >gi299533998 |refZP 07047350.1| 2-dehydropantoate >gil 148658426 refYP 001278.631.1 2-dehydropantoate 2-reductase Comamonas testosteroni S44 2-reductase Roseiflexus sp. RS-1 >gi291547410|emb|CBL20518.1 2-dehydropantoate 2-re 50 >gi242372246 refZP 04817820.1 2-dehydropantoate ductase Ruminococcus sp. SR 1/5 2-reductase Staphylococcus epidermidis M23864W1 >gil 118592425 |refZP 015498.17.1 2-dehydropantoate >gi225070608 |ref ZP 03779633.1 hypothetical protein 2-reductase Stappia aggregata IAM 12614 CLOHYLEM 06710 Clostridium hylemonae DSM >gi254826199 refZP 0523.1200.1 2-dehydropantoate 15053) 2-reductase Listeria monocytogenes FSLJ1-194 55 >gi209520519 refZP 03269277.1| 2-dehydropantoate >gil 116670444|refYP 831377.1 2-dehydropantoate 2-re 2-reductase Burkholderia sp. H160 ductase Arthrobacter sp. FB24 >gi217963807|refYP 002349485.1 2-dehydropantoate >gi89897521|refYP 521008.11 hypothetical protein 2-reductase Listeria monocytogenes HCC23 DSY4775 Desulfitobacterium hafniense Y51 >gi.224477588 |refYP 002635.194.1 putative 2-dehydro >gi293606857|refZP 06689205.1 2-dehydropantoate 60 pantoate 2-reductase Staphylococcus carnosus Subsp. 2-reductase Achromobacter piechaudii ATCC 43553 carnosus TM300 >gil 126730558 |refZP 01746368.1 2-dehydropantoate >gil 118462253 refYP 883754.1 2-dehydropantoate 2-re 2-reductase Sagittula stellata E-37 ductase Mycobacterium avium 104 >gi317133532|refYP 004.092846.1 2-dehydropantoate >gi313672571 refYP 004.050682.1 2-dehydropantoate 2-reductase Ethanoligenens harbinense YUAN-3 65 2-reductase Calditerrivibrio nitroreducens DSM 19672 >gil 167561774|refZP 02354690.1 2-dehydropantoate >gi857.14020 refZP 01045009.12-dehydropantoate 2-re 2-reductase Burkholderia Oklahomensis EO147 ductase Nitrobacter sp. Nb-31 1A US 8,765,426 B2 41 42 >gi258593851 emb|CBE70192.1 putative 2-dehydropan >gi295700080|refYP 003607973.11 2-dehydropantoate toate 2-reductase (Ketopantoate reductase) (KPA reduc 2-reductase Burkholderia sp. CCGE1002 tase) (KPR) NC10 bacterium Dutch sediment >gi209518576|refZP 03267396.1| 2-dehydropantoate >gil 160896993 refYP 001562575..1| 2-dehydropantoate 2-reductase Burkholderia sp. H160 2-reductase Delfia acidovorans SPH-1 >gi323528526|refYP 0.04230678.1 2-dehydropantoate >gi91779353|refYP 554561.1 ketopantoate reductase 2-reductase Burkholderia sp. CCGE1001 Apb A/PanE Burkholderia xenovorans LB400 >gi 312130704|refYP 003998044.1 ketopantoate reduc >gi 312795.995 refYP 004028917.1 Ketopantoate reduc tase apba/pano domain protein Leadbetterella byssophila tase Pan/ApbA family protein Burkholderia rhizoxinica DSM 17132 HKI 454 10 >gi33592466|refNP 8801.10.1 | putative ketopantoate >gi227544674|refZP 03.974723.1 2-dehydropantoate reductase Bordetella pertussis Tohama 1 2-reductase Lactobacillus reuteri CF48-3A >gil 15674.0709|refYP 001430838.1 2-dehydropantoate >gil 134098.436|refYP 001104097.1 2-dehydropantoate 2-reductase Roseiflexus castenholzii DSM 13941 2-reductase Saccharoporyspora erythraea NRRL 2338) >gil 18398.0885 refYP 001849176.1 ketopantoate reduc >gi222478734|refYP 002564971.1 2-dehydropantoate 15 tase, ApoA Mycobacterium marinum M 2-reductase Halorubrum lacusprofindi ATCC 49239 >gi1315225886|refZP 07867674.1| 2-dehydropantoate >gi301.061481|refZP 072022.45.1 2-dehydropantoate 2-reductase Parascardovia denticolens DSM 101.05 2-reductase delta proteobacterium NaphS2 >gil 170692978 |refZP 02884139.1 2-dehydropantoate >gi300024342|refYP 003756953.1 2-dehydropantoate 2-reductase Burkholderia graminis C4D1M 2-reductase Hyphomicrobium denitrificans ATCC 51888 >gil 184155218 |refYP 001843558.1 2-dehydropantoate >gi2544.43388 |refZP 05056864.1 2-dehydropantoate 2-reductase Lactobacillus fermentum IFO 3956 2-reductase Verrucomicrobiae bacterium DG1235 >gi229824124 refZP 04450193.1 hypothetical protein >gi254457455 refZP 05070883.1| 2-dehydropantoate GCWU000282 01428 Catonella morbi ATCC 51271 2-reductase Campylobacterales bacterium GD 1 >gi254557206 refYP 003063623.1 2-dehydropantoate >gil 189192294 refiXP 001932486.1 2-dehydropantoate 25 2-reductase Lactobacillus plantarum JDM1 2-reductase Pyrenophora tritici-repentis Pt-1C-BFP >gi888.12802|refZP 01128047.12-dehydropantoate 2-re >gil 114777349|ref ZP 01452346.1 putative 2-dehydro ductase Nitrococcus mobilis Nb-231 pantoate 2-reductase Mariprofiindus ferrooxydans PV-1 >gi297.625706|refYP 003687469.1 | Putative 2-dehydro >gil 108805776|refYP 645713.1 2-dehydropantoate 2-re pantoate 2-reductase (Ketopantoate reductase) (KPA ductase Rubrobacter xylanophilus DSM 9941 30 reductase) (KPR) Propionibacterium feudenreichii >gi324027764lgb|ADY14523.1 2-dehydropantoate 2-re subsp. Shermanii CIRM-BIA1 ductase Spirochaeta sp. Buddy >gil 163848886|refYP 001636930.1| 2-dehydropantoate >gi227509602|refZP 0393.9651.1| 2-dehydropantoate 2-reductase Chloroflexus aurantiacus J-10-fl 2-reductase Lactobacillus brevis Subsp. gravesensis >gi239817446 refYP 002946356.1 2-dehydropantoate ATCC 27305 35 2-reductase Vartovorax paradoxus S110 >gi256847249 refZP 05552695.1| 2-dehydropantoate >gi324314617Igb|ADY25732.1 2-dehydropantoate 2-re 2-reductase Lactobacillus Coleohominis 101-4-CHN ductase Deinococcus proteolyticus MRP >gi218289275 refZP 03493510.1| 2-dehydropantoate >gi314934649|refZP 07842008.1 2-dehydropantoate 2-reductase Alicyclobacillus acidocaldarius LAA1 2-reductase Staphylococcus Caprae C87 >gil 68164573 lgb|AAY87302.11 predicted ketopantoate 40 >gi 308270715emb|CBX27325.1 hypothetical protein reductase uncultured bacterium BAC17H8 N47 H21470 uncultured Desulfobacterium sp. >gil 150016117|refYP 001308371.1 2-dehydropantoate >gi228475460|refZP 04060178.1 2-dehydropantoate 2-reductase Clostridium beijerinckii NCIMB 8052 2-reductase Staphylococcus hominis SK119 >gi291550661|emb|CBL26923.1 ketopantoate reductase >gil 167618101|refZP 02386732.1 2-dehydropantoate Ruminococcus torques L2-14 45 2-reductase Burkholderia thailandensis Bt4 >gil 1638484.05 refYP 001636449.1 2-dehydropantoate >gil 197122126 refYP 002134077.1 2-dehydropantoate 2-reductase Chloroflexus aurantiacus J-10-fl 2-reductase Anaeromyxobacter sp. K >gi302337021|refYP 003802227.1 2-dehydropantoate >gi921 15818 refYP 575547.1 2-dehydropantoate 2-re 2-reductase Spirochaeta Smaragdinae DSM 11293 ductase Nitrobacter hamburgensis X14 >gi25399.5436|refYP 003047500.11 2-dehydropantoate 50 >gil 107025901 |refYP 623412.1 2-dehydropantoate 2-re 2-reductase Methylotenera mobilis JLW8) ductase Burkholderia cenocepacia AU 1054 >gi209966707 refYP 002299622.1 2-dehydropantoate >gil 167747378 |ref ZP 02419505.1 hypothetical protein 2-reductase Rhodospirillum centenum SW ANACAC 02097 Anaerostipes caccae DSM 14662 >gi309790241|ref ZP 0768.4811.1 hypothetical protein >gil 149188756 refZP 01867047.1 2-dehydropantoate OSCT 0762 Oscillochloris trichoides DG6 55 2-reductase Vibrio Shilonii AK1 >gil 159491174|refXP 001703548.1 ketopantoate reduc >gi2396284.05 refZP 04671436.1 ketopantoate reductase tase Chlamydomonas reinhardtii/ PanE/ApbA Clostridiales bacterium 1 7 47 FAA >gi288556204|refYP 003428.139.1 2-dehydropantoate >gi1315659888|refZP 07912747.1 2-dehydropantoate 2-reductase Bacillus pseudofirmus OF4 2-reductase Staphylococcus lugdunensis M23590 >gil 119960789|refYP 946726.1 2-dehydropantoate 2-re 60 >gi257454611|refZP 05619867.1 2-dehydropantoate ductase Arthrobacter aurescens TC1 2-reductase Enhydrobacter aerosaccus SK60 >gi221209631|refZP 03582612.1 2-dehydropantoate >gil 160940948 |ref ZP 02088288.1 hypothetical protein 2-reductase Burkholderia multivorans CGD1 CLOBOL 05840 Clostridium bolteae ATCC BAA-613 >gil 149927298|refZP 01915554.1| 2-dehydropantoate >gil 167588280|refZP 0238.0668.1 2-dehydropantoate 2-reductase Limnobacter sp. MED105 65 2-reductase Burkholderia ubonensis Bu >gi255017282|refZP 05289408.1| 2-dehydropantoate >gil 159125630gb|EDP50747.1 2-dehydropantoate 2-re 2-reductase Listeria monocytogenes FSL F2-515 ductase Aspergillus fumigatus A1163 US 8,765,426 B2 43 44 >gi871996.94 refYP 496951.1 2-dehydropantoate 2-re >gil 148271549|refYP 001221110.1 2-dehydropantoate ductase Novosphingobium aromaticivorans DSM 12444 2-reductase Clavibacter michiganensis Subsp. michigan >gi307727 196|refYP 003910409.11 2-dehydropantoate ensis NCPPB 382 2-reductase Burkholderia sp. CCGE1003 >gi86158581|refYP 465366.1 2-dehydropantoate 2-re >gil 14.9375503 refZP 01893273.1| 2-dehydropantoate ductase Anaeromyxobacter dehalogenans 2CP-C 2-reductase Marinobacter algicola DG893 >gi1307823565 refZP 07653794.1| 2-dehydropantoate >gi284992269|refYP 003410823.1 2-dehydropantoate 2-reductase Methylobacter tundripaludum SV96 2-reductase Geodermatophilus obscurus DSM 43160 >gil 169629992|refYP 001703641.1 2-dehydropantoate >gi319795820|refYP 004157460.1 2-dehydropantoate 2-reductase Mycobacterium abscessus ATCC 19977 2-reductase Vario vorax paradoxus EPS 10 >gil 114561980|refYP 749493.1 2-dehydropantoate 2-re >gi311696931 lgb|ADP99804.1 ketopantoate reductase ductase Shewanella frigidimarina NCIMB 400 Apb A/PanE marine bacterium HP15) >gil 1195.08832|refZP 01627984.1 2-dehydropantoate >gi220916894|refYP 002492198.1 2-dehydropantoate 2-reductase Nodularia spumigena CCY9414 2-reductase Anaeromyxobacter dehalogenans 2CP-1 15 >gi86740566|refYP 480966.1 2-dehydropantoate 2-re >gi29832362|refNP 826996.1 oxidoreductase Strepto ductase Frankia sp. Col3 myces avermitilis MA-4680 >gil 169594880|refXP 001790864.1 hypothetical protein >gi291246372|refYP 003505758.1 | putative 2-dehydro SNOG 00170 Phaeosphaeria nodorum SN15 pantoate 2-reductase PanE Staphylococcus simulans bv. >gi273797 15|refNP 771244.1 2-dehydropantoate 2-re Staphylolyticus/ ductase Bradyrhizobium japonicum USDA 110 >gi73661462|refYP 300243.1 2-dehydropantoate 2-re >gi284164004|refYP 003402283.1 2-dehydropantoate ductase Staphylococcus saprophyticus Subsp. saprophyti 2-reductase Haloterrigena turkmenica DSM5511 cus ATCC 15305 >gi241661691|refYP 00298.0051.1 2-dehydropantoate >gi266619728|refZP 06112663.1 2-dehydropantoate 2-reductase Ralstonia pickertti 12D 2-reductase Clostridium hathewayi DSM 13479 25 >gi319891385 refYP 004148260.1 2-dehydropantoate APPENDIX 2 2-reductase Staphylococcus pseudintermedius HKU 10-03 Polypeptides of 120-150 Amino Acids with E Value >gi258512824|refYP 0.03186258.1 2-dehydropantoate of 0.00001 or Smaller to SEQID NO:7 with 95% 2-reductase Alicyclobacillus acidocaldarius Subsp. aci 30 Identity and 95% Overlap Redundancy Cut-offs docaldarius DSM 446 >gil 15799815 refNP 285827.11 aspartate alpha-decar >gi2964.40847|refZP 06882926.1 2-dehydropantoate boxylase Escherichia coli 0157:H7 EDL933 2-reductase Clostridium lentocellum DSM5427 >gi39654551.pdb|1 PTOIA Chain A, Unprocessed Pyruvoyl >gi254254805 refZP 04948122.1 Ketopantoate reduc 35 Dependent Aspartate Decarboxylase With An Alanine tase Burkholderia dolosa AUO158 Insertion. At Position 26 >gi222053269|refYP 002535631.1| 2-dehydropantoate >gi283783924|refYP 003363789.1 aspartate L-decar 2-reductase Geobacter sp. FRC-32 boxylase Citrobacter rodentium ICC168 >gi890974.58|refZP 01 170347.12-dehydropantoate 2-re >gi290512515 refZP 06551881.1 aspartate 1-decarboxy ductase Bacillus sp. NRRL B-14911 40 lase Klebsiella sp. 1 1 55 >gi298506713 lgb|ADI85436.1 2-dehydropantoate 2-re >gi261338866|ref ZP 05966724.1 hypothetical protein ductase Geobacter sulfurreducens KN400 ENTCAN 05063 Enterobacter cancerogenus ATCC >gil 187919138|refYP 001888169.1 2-dehydropantoate 35316) 2-reductase Burkholderia phytofirmans Ps)N >gil 156935344|refYP 001439260.1 aspartate alpha-de >gi291246359|refYP 003505745.1 | putative 2-dehydro 45 carboxylase Cronobacter Sakazakii ATCC BAA-894 pantoate 2-reductase PanE Staphylococcus simulans bv. >gi283477352|emb|CAY73268.1 panDErwinia pyrifoliae Staphylolyticus/ DSM 12163) >gi260438889|refZP 05792705.1| 2-dehydropantoate >gi322834360|refYP 004214387.1 aspartate 1-decar 2-reductase Butyrivibrio crossotus DSM 2876 boxylase Rahnella sp.Y.9602 >gil 148654505 refYP 0012747 10.1 2-dehydropantoate 50 >gi291616317|refYP 003519059.1| PanD Pantoea 2-reductase Roseiflexus sp. RS-1 ananatis LMG 20103 >gil 162146827|refYP 0016.01288.1 2-dehydropantoate >gi317493244|ref ZP 07951666.1 aspartate 1-decarboxy 2-reductase Gluconacetobacter diazotrophicus PAI 5 lase Enterobacteriaceae bacterium 9 2 54FAA >gil 186473315|refYP 00186.0657.1 2-dehydropantoate >gi317046954|refYP 0041 14602.1 aspartate 1-decar 2-reductase Burkholderia phymatum STM815 55 boxylase Pantoea sp. At-9b) >gi209885679|refYP 002289536.1 2-dehydropantoate >gi251788657|refYP 003003378.1 aspartate alpha-de 2-reductase Oligotropha carboxidovorans OM5 carboxylase Dickeya zeae Ech1591 >gi90421544|refYP 529914.1 2-dehydropantoate 2-re >gi261820473|refYP 003258579.1 aspartate 1-decar ductase Rhodopseudomonas palustris Bisb18 boxylase Pectobacterium wasabiae WPP163 >gil 172063745 refYP 001811396.1 2-dehydropantoate 60 >gi293394524|ref ZP 06638820.1 aspartate 1-decarboxy 2-reductase Burkholderia ambifaria MC40-6 lase Serratia odorifera DSM 4582 >gi254555474|refYP 003061891.1 2-dehydropantoate >gi292489262|refYP 003532149.1 aspartate 1-decar 2-reductase Lactobacillus plantarum JDM1 boxylase Erwinia amylovora CFBP1430 >gi253701459|refYP 003022648.1 2-dehydropantoate >gi3007 15369|refYP 003740172.11 Aspartate 1-decar 2-reductase Geobacter sp. M21 65 boxylase Erwinia billingiae Eb661 >gil 108760447 refYP 631324.1 2-dehydropantoate 2-re >gil 197284104|refYP 002149976.1 aspartate alpha-de ductase Myxococcus xanthus DK 1622 carboxylase Proteus mirabilis HI4320 US 8,765,426 B2 45 46 >gi238918726|refYP 002932240.1 aspartate alpha-de >gi34497092 refNP 901307.1 aspartate alpha-decar carboxylase Edwardsiella ictaluri 93-146 boxylase Chromobacterium violaceum ATCC 12472 >gi85058464|refYP 454166.1 aspartate alpha-decar >gil 167585777|ref ZP 02378165.1 aspartate alpha-decar boxylase Sodalis glossinidius Str. morsitans' boxylase Burkholderia ubonensis Bu >gi294635062|ref ZP 06713578.1 aspartate 1-decarboxy >gi239815072|refYP 002943982.1 aspartate 1-decar lase Edwardsiella tarda ATCC 23685 boxylase Variovorax paradoxus S110 >gi320539340|ref ZP 08039009.1 aspartate 1-decarboxy >gi91774949|refYP 544705.11 aspartate alpha-decar lase Serratia symbiotica str. Tucson boxylase Methylobacillus flagellatus KT >gi37524865|refNP 928209.1 aspartate alpha-decar >gi2544.47307|ref ZP 05060774.1 aspartate 1-decarboxy boxylase Photorhabdus luminescens Subsp. laumondii 10 lase gamma proteobacterium HTCC5015 TTO1 >gi290476655 refYP 003469560.1 aspartate 1-decar >gil 186476949|refYP 0018584.19.1 aspartate alpha-de boxylase Xenorhabdus bovieni SS-2004 carboxylase Burkholderia phymatum STM815 >gil 183600092|ref ZP 0296.1585.1 hypothetical protein >gil 74318073|refYP 315813.1 aspartate alpha-decar PROSTU 03624 Providencia Stuartii ATCC 25827 15 boxylase Thiobacillus denitrificans ATCC 25259 >gi291326707 refZP 06125512.2 hypothetical protein >gi254429606 |ref ZP 05043313.1 aspartate 1-decarboxy PROVRETT 07565 Providencia rettgeri DSM 1131) lase Alcanivorax sp. DG881 >gi212711314|ref ZP 03319442.1 hypothetical protein >gi 78067243|refYP 370012.1 aspartate alpha-decar PROVALCAL 02386 Providencia alcalifaciens DSM boxylase Burkholderia sp. 383 30120 >gil 169796983|refYP 001714776.1 aspartate alpha-de >gil 149910516|ref ZP 01899.156.1 aspartate 1-decarboxy carboxylase Acinetobacter baumannii AYE) lase precursor Moritella sp. PE36 >gi307546720 reflyP 003899199.11 aspartate 1-decar >gil 148653905 refYP 001280998.1 aspartate alpha-de boxylase Halomonas elongata DSM 2581 carboxylase Psychrobacter sp. PRwf-1 >gi.224825655 ref ZP 03698759.1 aspartate 1-decarboxy >gi308050924|refYP 003.914490.1 L-aspartate 1-decar 25 lase Lutiella nitroferrum 2002 boxylase Ferrimonas balearica DSM 9799 >gi262369399|ref ZP 06062727.1 aspartate 1-decarboxy >gil 90406866|refZP 01215058.1 aspartate 1-decarboxy lase Acinetobacter johnsonii SH046 lase precursor Psychromonas sp. CNPT3 >gi262376653|ref ZP 06069881.1 aspartate 1-decarboxy >gi 71064749|refYP 263476.1 aspartate alpha-decar lase Acinetobacter lwoffii SH 145 boxylase Psychrobacter arcticus 273-4 30 >gi241766440|ref ZP 04764313.1 aspartate 1-decarboxy >gi237809047|refYP 002893487.1 aspartate 1-decar lase Acidovorax delafieldii 2AN boxylase Tolumonas auensis DSM 91.87 >gil 161524030|refYP 001579042.11 aspartate alpha-de >gi291613420|refYP 003523577.1 aspartate 1-decar carboxylase Burkholderia multivorans ATCC 17616 boxylase Sideroxydans lithotrophicus ES-1 >gi288942456 refYP 003444696.1 aspartate 1-decar >gi257455800|ref ZP 05621026.1 aspartate 1-decarboxy 35 boxylase Allochromatium vinosum DSM 180 lase Enhydrobacter aerosaccus SK60 >gi299137499|ref ZP 07030680.1 aspartate 1-decarboxy >gil 119944351 refYP 942031.1 aspartate 1-decarboxy lase Acidobacterium sp. MP5ACTX8 lase Psychromonas ingrahamii 37 >gi238028348 |refYP 002912579.1 hypothetical protein >gi297539336|refYP 003675105.1| aspartate 1-decar bglu 1 g28090 Burkholderia glumae BGR1 boxylase Methylotenera sp. 301 40 >gil 71901432|refZP 00683522.1 Aspartate decarboxy >g1iI257094.027 refYP 003167668.1 aspartate alpha-de lase LXylella fastidiosa Ann-1 carboxylase Candidatus Accumulibacter phosphatis >gil 71276366|refZP 00652643.1| Aspartate decarboxy clade IIA str. UW-1 lase LXylella fastidiosa Dixon >gi253997254|refYP 0030493.18.1 aspartate 1-decar >gil 94500961|refZP 01307486.1 aspartate 1-decarboxy boxylase Methylotenera mobilis JLW8 45 lase precursor Oceanobacter sp. RED65 >gil 121603845|refYP 981174.1 aspartate alpha-decar >gil 149927.699|ref ZP 01915951.1 aspartate alpha-decar boxylase Polaromonas naphthalenivorans CJ2 boxylase Limnobacter sp. MED 105 >gi220934.056 refYP 002512955.1| Aspartate 1-decar >gil 90020709|refYP 526536.1 aspartate 1-decarboxylase boxylase Thioalkalivibrio sp. HL-EbGR7 Saccharophagus degradans 2-40 >gi91786803 refYP 547755.11 aspartate alpha-decar 50 >gi254491956|ref ZP 05105.134.1 aspartate 1-decarboxy boxylase Polaromonas sp. JS666 lase Methylophaga thiooxidans DMS010 >gi253999879|refYP 003051942.1| aspartate 1-decar >gi92115182|refYP 575110.1 aspartate alpha-decar boxylase Methylovorus sp. SIP3-4 boxylase Chromohalobacter salexigens DSM 3043) >gil 167835820|ref ZP 02462703.1 aspartate alpha-decar >gi78485863|refYP 391788.1 aspartate alpha-decar boxylase Burkholderia thailandensis MSMB43 55 boxylase Thiomicrospira crunogena XCL-2 >gil 71908800 refYP 286387.1 aspartate alpha-decar >gi307826254|ref ZP 07656.463.1 aspartate 1-decarboxy boxylase Dechloromonas aromatica RCB lase Methylobacter tundripaludum SV96 >gi323525130|refYP 004227283.1 aspartate 1-decar >gi89093284|refZP 01166234.1 aspartate 1-decarboxy boxylase Burkholderia sp. CCGE1001 lase Oceano spirillum sp. MED92 >gi53803579|refYP 114732.1 aspartate alpha-decar 60 >gil 1462.83618 |refYP 001173771.1 aspartate alpha-de boxylase Methylococcus capsulatus Str. Bath carboxylase Pseudomonas Stutzeri A1501 >gi302879352|refYP 0.03847916.1 aspartate 1-decar >gi264679999|refYP 003279908.1 aspartate 1-decar boxylase Gallionella capsiferriformans ES-2 boxylase Comamonas testosteroni CNB-2 >gi8990 1803 refYP 524274.1 aspartate alpha-decar >gil 116052878 |refYP 793.195.1 aspartate alpha-decar boxylase Rhodoferax ferrireducens T118 65 boxylase Pseudomonas aeruginosa UCBPP-PA 14 >gi239820653|refYP 002947838.1 aspartate 1-decar >gil 160897278 |refYP 001562860.1 aspartate alpha-de boxylase Variovorax paradoxus S110 carboxylase Delfia acidovorans SPH-1 US 8,765,426 B2 47 48 >gil 121997.470|refYP 001002257.1 aspartate 1-decar >gil 15805744|refNP 294.441.1 aspartate alpha-decar boxylase Halorhodospira halophila SL1 boxylase Deinococcus radiodurans R1 >gi38372456 sp|Q848I5.1| PAND PSEFL RecName: >gil 197124548 |refYP 002136499.1 aspartate 1-decar Full=Aspartate 1-decarboxylase; boxylase Anaeromyxobacter sp. K >gi21231219 refNP 637136.1 aspartate alpha-decar >gi300310032|refYP 003774124.1 aspartate 1-decar boxylase Xanthomonas campestris pv. Campestris Str. boxylase precursor protein Herbaspirillum seropedicae ATCC 33.913 SmR1 >gi319949737|ref ZP 08023765.1 aspartate alpha-decar >gi77164404|refYP 342929.1 aspartate alpha-decar boxylase Dietzia cinnamea P4 boxylase Nitrosococcus oceani ATCC 19707 10 >gil 158706108sp|Q1 IYG7.2|PAND DEIGD RecName: >gi226940325|refYP 002795399.1| PanD Laribacter Full=Aspartate 1-decarboxylase; hongkongensis HLHK9 >gil 183985072|refYP 001853363.1 aspartate 1-decar >gi888.00686|refZP 011 16245.1 aspartate 1-decarboxy boxylase precursor PanD Mycobacterium marinum M lase precursor Reinekea sp. MED297 >gi291287339|refYP 003504.155.1 aspartate 1-decar >gil 194365298 |refYP 002027908.1 aspartate alpha-de 15 boxylase Denitrovibrio acetiphilus DSM 12809 carboxylase Stenotrophomonas maltophilia R551-3 >gi302870300|refYP 003838937.11 aspartate 1-decar >gi254469038 |ref ZP 05082444.1 aspartate 1-decarboxy boxylase Micromonospora aurantiaca ATCC 27029 lase beta proteobacterium KB13 >gi296040174|ref ZP 06832727.1 aspartate 1-decarboxy >gil 188576407|refYP 001913336.1 aspartate alpha-de lase Rhodococcus equi ATCC 33707 carboxylase Xanthomonas oryzae pv. oryzae PXO99A il 153870646|ref ZP 02000002.11 Aspartate decarboxy >gil 171059338|refYP 001791687.11 aspartate 1-decar lase Beggiatoa sp. PS boxylase Leptothrix cholodni SP-6 >gil 148271711|refYP 001221272.1 aspartate alpha-de >gil 121595787|refYP 987683.1 aspartate alpha-decar carboxylase Clavibacter michiganensis Subsp. michigan boxylase Acidovorax sp. JS42 ensis NCPPB 382 >gi289209202|refYP 003461268.1 aspartate 1-decar 25 >gi300690455 refYP 003751450.1 aspartate 1-decar boxylase Thioalkalivibrio sp. K90mix boxylase Ralstonia Solanacearum PSIO7 >gi213965138|ref ZP 03393336.1 aspartate 1-decarboxy >gi292491 194|refYP 003526633.1 aspartate 1-decar lase Corynebacterium amycolatum SK46 boxylase Nitrosococcus halophilus Nc4 >gi91787463|refYP 548415.1 aspartate alpha-decar >gi300703064|refYP 003744666.1 aspartate 1-decar boxylase Polaromonas sp. JS666 30 boxylase Ralstonia Solanacearum CFBP2957 >gi254784766|refYP 003072194.1 aspartate 1-decar >gil 18793.0038 |refYP 001900525.1 aspartate alpha-de boxylase Teredinibacter turnerae T7901 carboxylase Ralstonia pickettii 12J >gil 187478858 |refYP 786882.1 aspartate 1-decarboxy >gil 41406555|refNP 959391.1 aspartate alpha-decar lase Bordetella avium 197N boxylase Mycobacterium avium Subsp. paratuberculosis >gil 114776473|ref ZP 01451518.1 aspartate 1-decarboxy 35 K-10 lase precursor Mariprofindus ferrooxydans PV-1 >gil 169627646 refYP 0017012.95.1 aspartate 1-decar >gi226946298 |refYP 0028.01371.1 aspartate alpha-de boxylase precursor Mycobacterium abscessus ATCC carboxylase Azotobacter vinelandii DJ 19977 >gil 118594493 refZP 015.51840.1 aspartate 1-decarboxy >gil 1923.60161|refYP 001983429.1 aspartate 1-decar lase precursor Methylophilales bacterium HTCC2181 40 boxylase Cellvibrio japonicus Ueda107 >gi87118637|refZP 01.07453.6.1 putative aspartate 1 >gi320094682|ref ZP 08026439.1 aspartate 1-decarboxy decarboxylase Marinomonas sp. MED121 lase Actinomyces sp. oral taxon 178 str. F0338 >gi285017994|refYP 003375705.11 aspartate 1-decar >gi283778561|refYP 003369316.1 aspartate 1-decar boxylase precursor (aspartate alpha-decarboxylase) pro boxylase Pirellula stallevi DSM 6068 tein Xanthomonas albilineans GPE PC73 45 >gi256789458 |ref ZP 05527889.1 aspartate alpha-decar >gi8385.9460|refZP 00952981.1 aspartate 1-decarboxy boxylase Streptomyces lividans TK24 lase Oceanicaulis alexandrii HTCC2633 >gi291297.625 refYP 003508903.1 aspartate 1-decar >gi319786659|refYP 004146134.1 aspartate 1-decar boxylase Stackebrandtia nassauensis DSM 44728 boxylase Pseudoxanthomonas Suwonensis 11-1 >gi5598.0575|refYP 143872.1 aspartate alpha-decar >gil 152995292 refYP 001340127.1 aspartate 1-decar 50 boxylase Thermus thermophilus HB8 boxylase Marinomonas sp. MWYL1 >gil 120406311|refYP 956140.1 aspartate alpha-decar >gil 194290690|refYP 002006597.1 aspartate alpha-de boxylase Mycobacterium vanbaalenii PYR-1 carboxylase Cupriavidus taiwanensis LMG 19424 >gil 119475802|ref ZP 01616154.1 aspartate 1-decarboxy >gil 162449208 |refYP 001611575..1 hypothetical protein lase precursor marine gamma proteobacterium sce0938 Sorangium cellulosum Soce 56 55 HTCC2143 >gil 153003251 refYP 001377576.11 aspartate 1-decar >gil 114319727|refYP 74.1410.1 aspartate alpha-decar boxylase Anaeromyxobacter sp. Fw 109-5 boxylase Alkalilimnicola ehrlichii MLHE-1 >gi293604513 refZP 06686918.1 aspartate 1-decarboxy >gi90416572|refZP 01224.503.1 aspartate 1-decarboxy lase Achromobacter piechaudi ATCC 43553 lase precursor marine gamma proteobacterium >gi289704810|ref ZP 0650 1229.1 aspartate 1-decarboxy 60 HTCC2207) lase Micrococcus luteus SK58 >gil 184200024|refYP 001854231.1 aspartate 1-decar >gi237749062|ref ZP 04579542.1 aspartate 1-decarboxy boxylase precursor Kocuria rhizophila DC2201) lase Oxalobacter formigenes OXCC13 >gi72162261|refYP 289918.1 aspartate alpha-decar >gi255020069|refZP 05292141.1| Aspartate 1-decar boxylase Thermobifida fisca YX boxylase Acidithiobacillus caldus ATCC51756 65 >gil 154507911|ref ZP 02043553.1 hypothetical protein >gil 119962044|refYP 94.9406.1 aspartate 1-decarboxy ACTODO 00396 Actinomyces odontolyticus ATCC lase Arthrobacter aurescens TC1 17982 US 8,765,426 B2 49 50 >gi319442411|ref ZP 0799.1567.1 aspartate alpha-decar >gi220914411|refYP 002489720.1 aspartate 1-decar boxylase Corynebacterium variabile DSM 44702 boxylase Arthrobacter chlorophenolicus A6 >gi225028053|ref ZP 03717245.1 hypothetical protein >gil 108758834|refYP 634947.1 aspartate 1-decarboxy EUBHAL 02322 Eubacterium hallii DSM 3353 lase Myxococcus xanthus DK 1622 >gi27 1970326|refYP 003344522.1 aspartate 1-decar >gi269218264-IrefZP 061621 18.1 aspartate 1-decarboxy boxylase Streptosporangium roseum DSM 43021 lase Actinomyces sp. oral taxon 848 str. F0332 >gi2961383.04|refYP 003645547.1 aspartate 1-decar >gi311743353 refZP 07717160.1 aspartate 1-decarboxy boxylase Tsukamurella paurometabola DSM 20162 lase Aeromicrobium marinum DSM 15272 >gi226304123 refYP 002764081.1 aspartate 1-decar >gil 145596791|refYP 001161088.1 aspartate alpha-de boxylase Rhodococcus erythropolis PR4 10 carboxylase Salinispora tropica CNB-440 >gil 145588837|refYP 001155.434.1 aspartate alpha-de carboxylase Polynucleobacter necessarius Subsp. asym >gil 198284767 refYP 002221088.1 aspartate 1-decar bioticus QLW-P1DMWA-1 boxylase Acidithiobacillus ferrooxidans ATCC 53993 >gil 108801724|refYP 641921.1 aspartate alpha-decar >gi308178526|refYP 0039.17932.1 aspartate 1-decar boxylase Mycobacterium sp. MCS 15 boxylase Arthrobacter arilaitensis Re117 >gi323489711|ref ZP 08094938.1 aspartate 1-decarboxy >gi25506.5696 |ref ZP 05317551.1 aspartate 1-decarboxy lase Planococcus donghaensis MPA1 U2 lase Neisseria sicca ATCC 29256 >gi320450484 |refYP 004202580.1 aspartate 1-decar >gi888.10485 refZP 01125742.1 aspartate 1-decarboxy boxylase Thermus scotoductus SA-01 lase precursor Nitrococcus mobilis Nb-231 >gi240172844|ref ZP 0475.1503.1 aspartate alpha-decar >gil 158706097|sp|A1SDW8.2|PAND NOCSJ RecName: boxylase Mycobacterium kansasii ATCC 12478 Full=Aspartate 1-decarboxylase; >gil 148264137 IrefYP 001230843.1 aspartate alpha-de >gil 172041372|refYP 00180 1086.1 aspartate alpha-de carboxylase Geobacter uraniireducens Rf4 carboxylase Corynebacterium urealyticum DSM 7109 >gi302562209|ref ZP 07314551.1 aspartate 1-decarboxy >gi291297729|refYP 003509007.11 aspartate 1-decar lase Streptomyces griseoflavus Tu4000 25 boxylase Stackebrandtia nassauensis DSM 44728 >gil 1971 19382|refYP 002139809.1 aspartate alpha-de >gi294.675086 reflyP 003575702.11 aspartate 1-decar carboxylase Geobacter bemidjiensis Bem) boxylase Prevotella ruminicola 23 >gil 15610737|refNP 218 118.1 aspartate alpha-decar >gi2915197941emb|CBK75015.1 L-aspartate 1-decar boxylase Mycobacterium tuberculosis H37RV boxylase Butyrivibriofibrisolvens 16/4 >gil 159040208 |refYP 001539461.1 aspartate alpha-de 30 >gi 78042854|refYP 361172.1 aspartate alpha-decar carboxylase Salinispora arenicola CNS-205 boxylase Carboxydothermus hydrogenoformans Z-2901 >gi237746908IrefZP 04577388.1| aspartate-1-decar >gil 54022371|refYP 116613.1 aspartate alpha-decar boxylase Oxalobacter formigenes HOXBLS boxylase Nocardia farcinica IFM 10152 >gil 52080749|refYP 079540.1 aspartate alpha-decar >gil 153815600|ref ZP 01968268.1 hypothetical protein boxylase Bacillus licheniformis ATCC 14580 35 RUMTOR 01836 Ruminococcus torques ATCC 27756) >gi2961 13074|refYP 003627012.1 aspartate 1-decar >gil 119952798 |refYP 950285.1 aspartate 1-decarboxy boxylase Moraxella catarrhalis RH4 lase Arthrobacter aurescens TC1 >gil 111021388 |refYP 704360.1 aspartate alpha-decar >gil 118581933 refYP 903183.1 aspartate alpha-decar boxylase Rhodococcus jostii RHA1 boxylase Pelobacter propionicus DSM 2379 >gi298368957|ref ZP 06980275.1 aspartate 1-decarboxy 40 >gi 308174032|refYP 003920737.11 aspartate 1-decar lase Neisseria sp. oral taxon 014 str. F0314 boxylase Bacillus amyloliquefaciens DSM 7 >gi3154.46243 refYP 004079122.1 L-aspartate 1-decar >gi2274984.05 refZP 03928551.1 aspartate alpha-decar boxylase Mycobacterium sp. Spyr1 boxylase Acidaminococcus sp. D21 >gi297190372|ref ZP 06907770.1 aspartate 1-decarboxy >gi261880070|ref ZP 06006497.1 aspartate 1-decarboxy lase Streptomyces pristinaespiralis ATCC 25486 45 lase Prevotella bergensis DSM 17361 >gi315924 106 |ref ZP 07920332.1 aspartate 1-decarboxy >gi284989228 |refYP 003407782.1 aspartate 1-decar lase Pseudoramibacter alactolyticus ATCC 23263 boxylase Geodermatophilus obscurus DSM 43160 >gil 15677149|refNP 274302.1 aspartate alpha-decar >gi89099887|refZP 01 172759.1 aspartate 1-decarboxy boxylase Neisseria meningitidis MC58 lase precursor Bacillus sp. NRRL B-14911 >gi239980059|ref ZP 04702583.1 aspartate alpha-decar 50 >gi296271376 refYP 003654008.1 aspartate 1-decar boxylase Streptomyces albus J1074 boxylase Thermobispora bispora DSM 43833 >gil 15827028|refNP 301291.1 aspartate alpha-decar >gil 168698.967|ref ZP 02731244.1 hypothetical protein boxylase Mycobacterium leprae TN GobsU 05571 Gemmata obscuriglobus UQM 2246 >gil 116672429|refYP 833362.1 aspartate alpha-decar >gi220929206|refYP 002506115.1 aspartate 1-decar boxylase Arthrobacter sp. FB24 55 boxylase Clostridium cellulolyticum H10 >gi229918730|refYP 00288.7376.1 aspartate 1-decar >gi84495.005 |refZP 00994.124.1 aspartate 1-decarboxy boxylase Exiguobacterium sp. AT1b) lase precursor Janibacter sp. HTCC2649 >gi238020351 ref ZP 04600777.1 hypothetical protein >gi291547262|emb|CBL20370.1 L-aspartate 1-decarboxy GCWU000324 00231 Kingella oralis ATCC 51147) lase Ruminococcus sp. SR 1/5 >gil 163840966|refYP 001625371.1 aspartate alpha-de 60 >gi298246275 refZP 06970081.1 aspartate 1-decarboxy carboxylase Renibacterium salmoninarum ATCC 33209 lase Ktedonobacter racemifer DSM 44963 >gil68535357 refYP 250062.1 aspartate alpha-decar >gil 150017466|refYP 001309720.1 aspartate alpha-de boxylase Corynebacterium jeikeium K411 carboxylase Clostridium beijerinckii NCIMB 8052 >gil 117927421 refYP 871972.1 L-aspartate 1-decarboxy >gi300.934633 refZP 07149889.1 aspartate alpha-decar lase Acidothermus cellulolyticus 11B 65 boxylase orynebacterium resistens DSM 45100 >gil 124268753|refYP 001022757.1 aspartate alpha-de >gi269124634|refYP 003298004.1 aspartate 1-decar carboxylase Methylibium petroleiphilum PM1 boxylase Thermomonospora curvata DSM 43183 US 8,765,426 B2 51 52 >gi269128681|refYP 0.03302051.1 aspartate 1-decar >gi3234711511gb|ADX74836.1 L-aspartate 1-decarboxy boxylase Thermomonospora curvata DSM 43183 lase Arthrobacter phenanthrenivorans Sphe3 >gi307244703 |ref ZP 07526806.1 aspartate 1-decarboxy >gi25026691|refNP 736745.1 aspartate alpha-decar lase Peptostreptococcus stomatis DSM 17678 boxylase Corynebacterium efficiens YS-314 >gi282862735|ref ZP 06271796.1 aspartate 1-decarboxy >gil 16079298 |refNP 390122.1 aspartate alpha-decar lase Streptomyces sp. ACTE boxylase Bacillus subtilis subsp. subtilis str. 168 >gi227494386|ref ZP 03924702.1 aspartate 1-decarboxy >gi319790447 refYP 004152080.1 aspartate 1-decar lase Actinomyces coleocanis DSM 15436 boxylase Thermovibrio ammonificans HB-1 >gi268316317|refYP 003290036.1 aspartate 1-decar >gi251779952|ref ZP 04822872.1 aspartate 1-decarboxy boxylase Rhodothermus marinus DSM 4252 10 lase Clostridium botulinum E1 str. BoNTE Beluga >gi226312091|refYP 002771985.1 aspartate 1-decar >gil 167462328 |ref ZP 02327417.1 aspartate alpha-decar boxylase precursor Brevibacillus brevis NBRC 100599 boxylase Paenibacillus larvae subsp. larvae BRL >gi 308069472|refYP 003871077.1 | Aspartate 1-decar 230010) boxylase precursor (Aspartate alpha-decarboxylase) >gi225025397 refZP 03714589.1 hypothetical protein Paenibacillus polymyxia E681 15 EIKCOROL 02295 Eikenella corrodens ATCC 23834 >gil 121535059|ref ZP 01666876.1 aspartate 1-decarboxy >gi291279242|refYP 003496077.1 aspartate 1 -decar lase Thermosinus carboxydivorans Nor1 boxylase Deferribacter desulfuricans SSM1 >gi29376392 refNP 815546.1 aspartate alpha-decar >gi31 1896800ldb|BAJ29208.1 putative L-aspartate 1-de boxylase Enterococcus faecalis V583 carboxylase Kitasatospora setae KM-6054 >gil 172057787|refYP 001814247.1 aspartate 1-decar il 169827548|refYP 001697706.1 aspartate 1-decar boxylase Exiguobacterium Sibiricum 255-15 boxylase Lysinibacillus sphaericus C3-41 >gi289522732|ref ZP 06439586.1 aspartate 1-decarboxy iI299134421 refZP 07027614.1 aspartate 1-decarboxy lase Anaerobaculum hydrogeniformans ATCC BAA lase Afipia sp. 1 NLS2 1850 iI242372243 refZP 04817817.1 aspartate alpha-decar >gi315605842|ref ZP 07880874.1 aspartate 1-decarboxy 25 boxylase Staphylococcus epidermidis M23864:W1 lase Actinomyces sp. oral taxon 180 str. F0310 iI226325379|refZP 038.00897.1) hypothetical protein >gi255994.546|ref ZP 05427681.1 aspartate 1-decarboxy COPCOM 03181 Coprococcus comes ATCC 27758 lase Eubacterium saphenum ATCC 49989 >gi284029184|refYP 003379115.1 aspartate 1-decar >gi251796457|refYP 00301 1188.1 aspartate 1-decar boxylase Kribbella flavida DSM 17836 boxylase Paenibacillus sp. JDR-2 30 >gi31965.1315|ref ZP 08005445.1 aspartate 1-decarboxy >gil 54295766|refYP 128181.1 aspartate alpha-decar lase Bacillus sp. 2. A 57 CT2 boxylase Legionella pneumophila Str. Lens >gi254496676|refZP 05109539.1 aspartate alpha-decar >gi258654511|refYP 003203.667.1 aspartate 1-decar boxylase Legionella drancourtii LLAP12) boxylase Nakamurella multipartita DSM 44233 >gi256.833308|refYP 003.162035.1 aspartate 1-decar >gi311068755 refYP 003973678.1 aspartate alpha-de 35 boxylase Jonesia denitrificans DSM 20603 carboxylase Bacillus atrophaeus 1942 >gi261417985 refYP 003251667.1 aspartate 1-decar iI229544.027 refZP 04433086.1 aspartate 1-decarboxy boxylase Geobacillus sp. Y412MC61 lase Bacillus coagulans 36D1 >gi302876569|refYP 00384.520211 aspartate 1-decar iI257068435 refYP 003154690.1 L-aspartate 1-decar boxylase Clostridium cellulovorans 743B boxylase Brachybacterium faecium DSM 4810 40 >gi21263.8969|refYP 002315489.1 aspartate alpha-de >gi256753469|ref ZP 054.94262.1 aspartate 1-decarboxy carboxylase Anoxybacillus flavithermus WK1 lase Clostridium papyrosolvens DSM 2782 >gil 15896.169|refNP 349518.1 aspartate alpha-decar >gi3249804081gb|EGC16074.1 aspartate 1-decarboxylase boxylase Clostridium acetobutyllicum ATCC 824 Kingella denitrificans ATCC 33394 >gi304406905 refZP 07388559.1 aspartate 1-decarboxy >gi225075174|ref ZP 03718373.1 hypothetical protein 45 lase Paenibacillus curdlanolyticus YK9 NEIFLAOT 00174 Neisseria flavescens NRL30031/ >gi3171286.16|refYP 004094898.1 aspartate 1-decar H210 boxylase Bacillus cellulosilyticus DSM 2522 >gi258510312|refYP 0.03183746.1 aspartate 1-decar >gil 149183103 |ref ZP 01861554.1 aspartate 1-decarboxy boxylase Alicyclobacillus acidocaldarius Subsp. aci lase precursor Bacillus sp. SG-1 docaldarius DSM 446 50 >gil 169335507|ref ZP 02862700.1 hypothetical protein >gi309813243 refZP 07706964.1 aspartate 1-decarboxy ANASTE 01921 Anaerofustis stercorihominis DSM lase Dermacoccus sp. Ellin185 17244 >gil 77919424|refYP 357239.1 aspartate alpha-decar >gil 197303353 refZP 03168393.1 hypothetical protein boxylase Pelobacter carbinolicus DSM 2380 RUMLAC 02076 Ruminococcus lactaris ATCC 291.76 >gil 124009556|ref ZP 01694230.1 aspartate 1-decarboxy 55 >gi268608082|ref ZP 06141811.1 aspartate alpha-decar lase Microscilla marina ATCC 23134 boxylase Ruminococcus flavefaciens FD-1 >gi320007101gb|ADW01951.1|aspartate 1-decarboxylase >gi294789798 |ref ZP 06755027.1 aspartate 1-decarboxy Streptomyces flavogriseus ATCC 33331 lase Simonsiella muelleri ATCC 2.9453 >gi299538597 refZP 07051880.1 aspartate 1-decarboxy >gi49484793 refYP 042017.1 aspartate alpha-decar lase precursor Lysinibacillus fusiformis ZC1 60 boxylase Staphylococcus aureus Subsp. aureus >gi225017697 refZP 03706889.1 hypothetical protein MRSA252 CLOSTMETH 01626 Clostridium methylpentosum >gi28377460|refNP 784352.1 aspartate alpha-decar DSM 5476 boxylase Lactobacillus plantarum WCFS1 >gi256397482|refYP 003119046.1 aspartate 1-decar >gil 182418494|ref ZP 02949787.1 aspartate 1-decarboxy boxylase Catenulispora acidiphila DSM 44928 65 lase Clostridium butyricum 5521 >gi3200067021gb|ADW01552.1 aspartate 1-decarboxylase >gi302553297 refZP 07305639.1 aspartate 1-decarboxy Streptomyces flavogriseus ATCC 33331 lase Streptomyces viridochromogenes DSM 40736 US 8,765,426 B2 53 54 >gil 1598.96918 |refYP 001543.165.1 aspartate 1-decar >gi70725184|refYP 252098.1 aspartate alpha-decar boxylase Herpetosiphon aurantiacus ATCC 23779 boxylase Staphylococcus haemolyticus JCSC 1435 >gil 114570673|refYP 757353.1 aspartate alpha-decar >gi238924848 |refYP 0029383.64.1 aspartate 1-decar boxylase Maricaulis maris MCS10 boxylase Eubacterium rectale ATCC 33.656 >gi239827468|refYP 002950092.1| aspartate 1-decar >gil 1677483.67|ref ZP 02420494.1 hypothetical protein boxylase Geobacillus sp. WCH70 ANACAC 03111 Anaerostipes caccae DSM 14662 >gi323465432gb|ADX77585.1 aspartate 1-decarboxylase >gi294498.158 |refYP 003561858.1 aspartate 1-decar Staphylococcus pseudintermedius ED99 boxylase Bacillus megaterium QMB.1551 >gil 182434568 |refYP 001822287.1 aspartate alpha-de >gi229172278 |ref ZP 04299842.1 Aspartate 1-decar 10 boxylase alpha chain Bacillus cereus MM3 carboxylase Streptomyces griseus Subsp. griseus NBRC >gi89892958|refYP 516445.1 aspartate alpha-decar 13350 boxylase Desulfitobacterium hafniense Y51 >gil 149178749|ref ZP 01857332.1 aspartate 1-decarboxy >gil 158312064 refYP 001504572.1 aspartate alpha-de lase Planctomyces maris DSM 8797 carboxylase Frankia sp. EAN1 pec >gi297616344|refYP 003701503.1 aspartate 1-decar 15 >gi303257894|ref ZP 07343903.1 aspartate 1-decarboxy boxylase Syntrophothermus lipocalidus DSM 12680 lase Burkholderiales bacterium 1 1 47 >gi83816527|refYP 445536.1 aspartate 1-decarboxylase >gi296131758 |refYP 003639005.1 aspartate 1-decar Salinibacter ruber DSM 13855 boxylase Thermincola sp. JR >gi288556128 |refYP 003428063.1 aspartate alpha-de >gi288817983|refYP 003432330.1 aspartate 1-decar carboxylase Bacillus pseudofirmus OF4 boxylase Hydrogenobacter thermophilus TK-6 >gi239635857|ref ZP 04676881.1 aspartate 1-decarboxy >gi291246356|refYP 003505742.1 putative aspartate lase Staphylococcus warneri L37603 alpha-decarboxylase PanD Staphylococcus simulans bv. >gi302342373|refYP 003806902.1 aspartate 1-decar staphylolyticus/ boxylase Desulfarculus baarsii DSM 2075 >gil 188996356|refYP 001930607.1 aspartate 1-decar >gil 146297.697 refYP 001181468.1 aspartate 1-decar 25 boxylase Sulfurihydrogenibium sp.YO3AOP1 boxylase Caldicellulosiruptor saccharolyticus DSM >gil 160894.028 |ref ZP 02074807.1 hypothetical protein 8903 CLOL250 01583 Clostridium sp. L2-50 >gil 147676557|refYP 0012.10772.1 aspartate alpha-de >gi228475771|ref ZP 04060489.1 aspartate 1-decarboxy carboxylase Pelotomaculum thermopropionicum SI lase Staphylococcus hominis SK119 >gil 138895755 refYP 001 126208.1 aspartate alpha-de 30 >gi56963830|refYP 175561.11 aspartate alpha-decar carboxylase Geobacillus thermodenitrificans NG80-2 boxylase Bacillus clausii KSM-K16 >gil 193215499 refYP 001996698.11 aspartate alpha-de >gi82548277Igb|ABB82968.1 putative aspartate decar carboxylase Chloroherpeton thalassium ATCC 35110 boxylase uncultured organism HF70 19B12 >gi270158276|ref ZP 06186933.1 aspartate 1-decarboxy >gil 148244908 |refYP 0012 19602.1 aspartate alpha-de lase Legionella longbeachae D-4968 35 carboxylase Candidatus Vesicomyosocius Okutani HA >gil 154483470|ref ZP 02025918.1 hypothetical protein >gil 153855966|ref ZP 01996.907.1 hypothetical protein EUBVEN 0.1174 Eubacterium ventriosum ATCC DORLON 02932 Dorea longicatena DSM 13814 27560 >gi22694.7534|refYP 002802625.1 aspartate 1-decar >gil 19551386|refNP 599388.1 aspartate alpha-decar boxylase Clostridium botulinum A2 str. Kyoto boxylase Corynebacterium glutamicum ATCC 13032 40 >gi297568701 |refYP 003690045.1 aspartate 1-decar >gi269956437|refYP 003326226.1 aspartate 1-decar boxylase Desulfurivibrio alkaliphilus AHT2 boxylase LXvlanimonas cellulosilytica DSM 15894 >gi289548459|refYP 0.03473447.1 aspartate 1-decar >gil 154491071|ref ZP 02031012.1 hypothetical protein boxylase Thermocrinis albus DSM 14484 PARMER 00990 Parabacteroides merdae ATCC >gi225180974|ref ZP 03734422.1 aspartate 1-decarboxy 43184) 45 lase Dethiobacter alkaliphilus AHT 1 >gil 16451 1440lemb|CAN89641.1 putative aspartate-1-de >gil 117165150|emb|CAJ88706.1 putative L-aspartate-al carboxylase precursor Streptomyces collinus/ pha-decarboxylase Streptomyces ambofaciens ATCC >gi284044344|refYP 003394.684.1 aspartate 1-decar 23877 boxylase Conexibacter woesei DSM 14684 >gi317123772|refYP 004097884.1 L-aspartate 1-decar >gi300786909|refYP 003767200.11 aspartate 1-decar 50 boxylase Intrasporangium calvum DSM 43043) boxylase Amycolatopsis mediterranei U32 >gi2.94055934|refYP 003549592.1 aspartate 1-decar >gil 118443940|refYP 877.157.1 aspartate alpha-decar boxylase Coraliomargarita akajimensis DSM 45221 boxylase Clostridium novyi NT >gil 15614252|refNP 242555.1 aspartate alpha-decar >gi294671143 refZP 06735998.1 hypothetical protein boxylase Bacillus halodurans C-125 NEIELOOT 02851 Neisseria elongata subsp. glyco 55 >gi297560480|refYP 003679454.1 aspartate 1-decar lytica ATCC 29315 boxylase Nocardiopsis dassonvillei Subsp. dassonvillei >gi3174992.17|ref ZP 07957492.1 aspartate decarboxy DSM 43111 lase Lachnospiraceae bacterium 5 1 63FAA >gi295094090|emb|CBK83181.1 L-aspartate 1-decar >gi300087723|refYP 003758245.11 aspartate 1-decar boxylase Coprococcus sp. ART55/1 boxylase Dehalogenimonas lykanthroporepellens 60 >gi1312197107 refYP 004.017168.1| aspartate 1-decar BL-DC-9 boxylase Frankia sp. Eullc >gi 312792944|refYP 004025867.1 aspartate 1-decar >gi302038222|refYP 003798544.1 aspartate 1-decar boxylase Caldicellulosiruptor kristianssonii 177R1B boxylase Candidatus Nitrospira defluvii/ >gi253577525 refZP 04854838.1 aspartate 1-decarboxy >gi294640899|ref ZP 06718851.1 aspartate 1-decarboxy lase Paenibacillus sp. oral taxon 786 str. D14 65 lase Ruminococcus albus 8 >gi304364119|ref ZP 0736.1364.1 aspartate 1-decarboxy >gi227548075 refZP 03978124.1 aspartate alpha-decar lase Actinomyces viscosus C505 boxylase Corynebacterium lipophiloflavum DSM 44291 US 8,765,426 B2 55 56 >gi314934646|ref ZP 07842005.1 aspartate 1-decarboxy >gi57237347|refYP 1783.60.11 aspartate alpha-decar lase Staphylococcus Caprae C87 boxylase Campylobacter jejuni RM1221 >gi258405137|refYP 003197879.11 aspartate 1-decar >gil 1105975.96 |ref ZP 01385881.1 aspartate 1-decarboxy boxylase Desulfohalobium retbaense DSM5692 lase Chlorobium ferrooxidans DSM 13031 >gi302538161|ref ZP 07290503.1 aspartate 1-decarboxy >gi.224477558 |refYP 002635164.1 putative aspartate lase Streptomyces sp. C 1-decarboxylase Staphylococcus carnosus Subsp. carno >gil 87308237 refZP 01090378.1 aspartate 1-decarboxy sus TM300 lase precursor Blastopirellula marina DSM 3645 >gi31 1900398ldb|BAJ32806.1 putative L-aspartate 1-de >gi269797992|refYP 00331 1892.1 aspartate 1-decar carboxylase Kitasatospora setae KM-6054 10 >gi228472221|ref ZP 04056987.1 aspartate 1-decarboxy boxylase Veillonella parvula DSM 2008 lase Capnocytophaga gingivalis ATCC 33624 >gi21673091|refNP 661156.1 aspartate alpha-decar >gi212696954|ref ZP 03305082.1 hypothetical protein boxylase Chlorobium tepidum TLS) ANHYDRO 01517 Anaerococcus hydrogenalis DSM >gi225851045 refYP 002731279.1 aspartate alpha-de 7454) carboxylase Persephonella marina EX-H1 15 >gil 152975064|refYP 0013745.81.1 aspartate alpha-de >gil 119356218 |refYP 910862.1 aspartate alpha-decar carboxylase Bacillus cereus subsp. cytotoxis NVH 391 boxylase Chlorobium phaeobacteroides DSM 266 98) >gi323141842|ref ZP 08076706.1 aspartate 1-decarboxy >gi31 1030322|ref ZP 07708412.1 aspartate alpha-decar lase Phascolarctobacterium sp. YIT 12067 boxylase Bacillus sp. m3-13 >gil 15605956|refNP 213333.11 aspartate alpha-decar >gi23099218|refNP 692684.1 aspartate alpha-decar boxylase Aquifex aeolicus VF5 boxylase Oceanobacillus iheyensis HTE831 >gi317052380|refYP 0041 13496.1 aspartate 1-decar >gil 183983171 refYP 001851462.1 aspartate 1-decar boxylase Desulfurispirillum indicum S5 boxylase precursor PanD 2 Mycobacterium marinum >gil 163782147|ref ZP 02177146.1 aspartate 1-decarboxy M lase precursor Hydrogenivirga sp. 128-5-R1-1) 25 >gi306820308|ref ZP 07453947.1 aspartate 1-decarboxy >gil 125973417 refYP 001037327.1 aspartate alpha-de lase Eubacterium yurii subsp. margaretiae ATCC 43715 carboxylase Clostridium thermocellum ATCC 27405 >gi312621811|refYP 004023424.1 aspartate 1-decar >gi315647107 refZP 07900220.1 aspartate 1-decarboxy boxylase Caldicellulosiruptor kronotskyensis 2002 lase Paenibacillus vortex V453 >gi2399273.16|ref ZP 04684269.1 aspartate alpha-decar >gil 152993855|refYP 001359576.1|aspartate decarboxy 30 boxylase Streptomyces ghanaensis ATCC 14672 lase Sulfurovum sp. NBC37-1 >gi295696300|refYP 003589538.11 aspartate 1-decar >gil 16803939|refNP 465424.1 aspartate alpha-decar boxylase Bacillus tusciae DSM 2912 boxylase Listeria monocytogenes EGD-e >gi303238877|ref ZP 07325408.1 aspartate 1-decarboxy >gi29833787|refNP 828421.1 aspartate alpha-decar lase Acetivibrio cellulolyticus CD2 boxylase Streptomyces avermitilis MA-4680 35 >gi289435249|refYP 003465121.1 hypothetical protein >gil 167629397 refYP 001679896.1 aspartate 1-decar Ise 1886 Listeria seeligeri serovar 1/2b str. SLCC3954 boxylase Heliobacterium modesticaldum Icel >gi302555661|ref ZP 07308003.1 aspartate 1-decarboxy >gil 157692740|refYP 001487202.1 aspartate alpha-de lase Streptomyces viridochromogenes DSM 40736 carboxylase Bacillus pumilus SAFR-032 >gi297584390|refYP 003700170.11 aspartate 1-decar >gi258513596 |refYP 0.031898.18.1 aspartate 1-decar 40 boxylase Bacillus selenitireducens MLS10) boxylase Desulfotomaculum acetoxidans DSM 771 >gi291532722emb|CBL05835.1 L-aspartate 1-decarboxy >gi320105461|refYP 004181051.1 aspartate 1-decar lase Megamonas hypermegale ART12/1 boxylase Terriglobus saanensis SP1 PR4 >gil 167041215 lgb|ABZ05972.1 putative aspartate decar >gi238019295 refZP 04599721.1 hypothetical protein boxylase uncultured marine microorganism HF 4000 VEIDISOL 01159 Veillonella dispar ATCC 17748 45 001NO2 >gi261406085 refYP 003242326.1 aspartate 1-decar >gil 168181292|ref ZP 02615956.1 aspartate 1-decarboxy boxylase Paenibacillus sp. Y412MC10 lase Clostridium botulinum Bf >gi323703659|ref ZP 08115301.1 aspartate 1-decarboxy >gil 134298040|refYP 001111536.1 aspartate 1-decar lase Desulfotomaculum nigrificans DSM574 boxylase Desulfotomaculum reducens MI-1 >gi254389556|ref ZP 05004782.1 aspartate 1-decarboxy 50 il 124514996 lgb|EAY56507.1|Aspartate 1-decarboxylase lase Streptomyces clavuligerus ATCC 27064 Leptospirillum rubarum/ >gil 163813891 |ref ZP 02205285.1 hypothetical protein >gil 195953149|refYP 002121439.1 aspartate 1-decar COPEUT 00044 Coprococcus eutactus ATCC 27759. boxylase Hydrogenobaculum sp.YO4AAS1 >gil 118473676|refYP 884.441.1 aspartate alpha-decar >gi22.9102244|ref ZP 04232953.11 Aspartate 1-decar boxylase Mycobacterium Smegmatis str. MC2 155 55 boxylase alpha chain Bacillus cereus Rock3-28 >gi242243653|ref ZP 04798097.1 aspartate alpha-decar >gi319955894 refYP 004167157.1 1-aspartate 1-decar boxylase Staphylococcus epidermidis W23144 boxylase Nitratifractor salsuginis DSM 16511 iI29584.0549|ref ZP 06827482.1 aspartate 1-decarboxy >gi227819486 |refYP 002823457.1 aspartate alpha-de lase Streptomyces sp. SPB74 carboxylase Sinorhizobium fredii NGR234 >gi229084647|ref ZP 04216915.1 Aspartate 1-decar 60 >gil 134299644|refYP 001113140.1 aspartate 1-decar boxylase alpha chain Bacillus cereus Rock3-44 boxylase Desulfotomaculum reducens MI-1 >gil 193213421|refYP 001999374.1 aspartate alpha-de >gi 78187623 refYP 375666.1 aspartate alpha-decar carboxylase Chlorobaculum parvum NCIB 8327 boxylase Chlorobium luteolum DSM 273 >gi 7818941 1|refYP 379749.1 aspartate alpha-decar >gi294632508 |ref ZP 0671 1068.1 aspartate 1-decarboxy boxylase Chlorobium chlorochromatii CalD3 65 lase Streptomyces sp. e14 >gi262203861|refYP 003275069.1 aspartate 1-decar >gi218246666|refYP 002372037.1| aspartate 1-decar boxylase Gordonia bronchialis DSM 43247 boxylase Cyanothece sp. PCC 8801 US 8,765,426 B2 57 58 >gil 153954248 |refYP 0013950 13.1 aspartate alpha-de >gi34557994|refNP 907809.1 aspartate alpha-decar carboxylase Clostridium kluyveri DSM 555 boxylase Wolinella succinogenes DSM 1740 >gi2401.45726|ref ZP 04744327.1 aspartate 1-decarboxy >gi83589014|refYP 429023.1 aspartate alpha-decar lase Roseburia intestinalis L1-82 boxylase Moorella thermoacetica ATCC 39073 >gil 187777515 refZP 02993.988.1 hypothetical protein 5 >gi288904380|refYP 00342.96.01.1 aspartate 1-decar CLOSPO 01106 Clostridium sporogenes ATCC 15579 boxylase Streptococcus gallolyticus UCN34 >gil 189345854|refYP 001942383.1 aspartate 1-decar >gi256546079|ref ZP 05473432.1 aspartate 1-decarboxy boxylase Chlorobium limicola DSM 245 lase Anaerococcus vaginalis ATCC 51170 >gi22584.9174|refYP 002729338.1 aspartate alpha-de >gi84687580|refZP 01 015455.1 aspartate 1-decarboxy carboxylase Sulfurihydrogenibium azorense AZ-Ful 10 lase precursor Maritimibacter alkaliphilus HTCC2654 >gi42524914|refNP 970294.1 aspartate alpha-decar >gi319440712|ref ZP 07989868.1 aspartate alpha-decar boxylase Baello vibrio bacteriovorus HD100 boxylase Corynebacterium variabile DSM 44702 >gi296.124338|refYP 003632116.1 aspartate 1-decar >gil 152990004|refYP 001355726.1 aspartate alpha-de boxylase Planctomyces limnophilus DSM 3776 carboxylase Nitratiruptor sp. SB155-2 >gi260592486 |ref ZP 05857944.1 aspartate 1-decarboxy 15 >gil 196229549|ref ZP 031284.14.1 aspartate 1-decarboxy lase Prevotella veroralis F0319 lase Chthoniobacter flavus Ellin428 >gi.224373697 refYP 002608069.1 aspartate alpha-de >gi310778937|refYP 003967270.1| L-aspartate 1-decar carboxylase Nautilia profundicola AmH boxylase Ilvobacter polytropus DSM 2926 >gi290955740|refYP 003486922.1 L-aspartate-alpha >gil 194333223 refYP 002015083.1 aspartate alpha-de decarboxylase Streptomyces scabiei 87.22 carboxylase Prosthecochloris aestuarii DSM 271 >gil 169830320|refYP 001716302.1 aspartate 1-decar >gi307720220|refYP 003891360.1 L-aspartate 1-decar boxylase Candidatus Desulforudis audaxviator boxylase Sulfurimonas autotrophica DSM 16294 MP104C >gi317057177|refYP 004.105644.1 aspartate 1-decar >gil 154246282|refYP 001417240.1 aspartate alpha-de boxylase Ruminococcus albus 7 carboxylase Xanthobacter autotrophics Py2 25 >gi281358405 refZP 06244887.1 aspartate 1-decarboxy >gil 194335559|refYP 002017353.11 aspartate 1-decar lase Victivallis vadensis ATCC BAA-548 boxylase Pelodictyon phaeoclathratiforme BU-1 >gil 13474851 |refNP 106421.1 aspartate alpha-decar >gi31518753.11gb|EFU21287.1 L-aspartate 1-decarboxy boxylase Mesorhizobium loti MAFF303099 lase Spirochaeta thermophila DSM 6578 >gi270308113|refYP 003330171.1 aspartate 1-decar >gil 145220354|refYP 001131063.1 aspartate alpha-de 30 boxylase Dehalococcoides sp. VS carboxylase Prosthecochloris vibrioformis DSM 265 >gi324963812gb|ADY54591.1 L-aspartate 1-decarboxy >gi206890877|refYP 002249664.11 aspartate 1-decar lase Syntrophobotulus glycolicus DSM 8271 boxylase Thermodesulfovibrio yellowstonii DSM 11347 >gil 13488064|refNP 085658.1 aspartate alpha-decar >gi315303837|ref ZP 07874325.1 aspartate 1-decarboxy boxylase Mesorhizobium loti MAFF303099 lase Listeria ivanovii FSL F6-596 35 >gi315638262|ref ZP 07893443.1 aspartate 1-decarboxy >gi257065635 refYP 003 151891.1 aspartate 1-decar lase Campylobacter upsaliensis JV21 boxylase Anaerococcus previotii DSM 20548 >gil 171778858 |ref ZP 02919920.1 hypothetical protein >gi299821666|ref ZP 07053554.1 aspartate 1-decarboxy STRINF 00779 Streptococcus infantarius subsp. infan lase Listeria grayi DSM 20601 tarius ATCC BAA-102 >gi254458768 |ref ZP 05072192.1 aspartate 1-decarboxy 40 >gil 17231061|refNP 487609.1| aspartate alpha-decar lase Campylobacterales bacterium GD 1 boxylase Nostoc sp. PCC 7120 >gi209526587|ref ZP 032751 12.1 aspartate 1-decarboxy >gi317121598 |refYP 0041.01601.1 L-aspartate 1-decar lase Arthrospira maxima CS-328 boxylase Thermaerobacter marianensis DSM 12885 >gi2423.10629|refZP 04809784.1 aspartate 1-decarboxy >gi225163545 refZP 03725856.1 Aspartate 1-decar lase Helicobacter pullorum MIT 98-5489 45 boxylase Opitutaceae bacterium TAV2 >gi260888452|ref ZP 05899715.1 aspartate 1-decarboxy >gi.224437987|ref ZP 03658926.1 aspartate alpha-decar lase Selenomonas sputigena ATCC 35185) boxylase Helicobacter cinaedi CCUG 18818 il 1167478.16|refYP 844503.1 aspartate 1-decarboxy >gi237752532|ref ZP 04583012.1 aspartate alpha-decar lase Syntrophobacter fitmaroxidans MPOB boxylase Helicobacter winghamensis ATCC BAA-430 >gil 149195160|ref ZP 01872251.1 aspartate alpha-decar 50 >gil 15773.6689|refYP 001489372.1 aspartate alpha-de boxylase Caminibacter mediatlanticus TB-2 carboxylase Arcobacter butzleri RM4018 >gi237750942|ref ZP 04581422.1 aspartate alpha-decar >gi317152682|refYP 004120730.1 aspartate 1-decar boxylase Helicobacter bilis ATCC 43879 boxylase Desulfovibrio aespoeensis Aspo-2 >gil 189499388 |refYP 001958858.1 aspartate alpha-de >gi298491519 refYP 003721696.1 aspartate 1-decar carboxylase Chlorobium phaeobacteroides BS1 55 boxylase "Nostoc azolae 0708 >gi313142475 refZP 07804668.1 aspartate 1-decarboxy >gi268680556 refYP 0.03304987.1 aspartate 1-decar lase Helicobacter canadensis MIT 98-5491 boxylase Sulfurospirillum delevianum DSM 6946 >gi 78776535|refYP 3928.50.1 aspartate alpha-decar >gi269792079|refYP 003316983.1 aspartate 1-decar boxylase Sulfurimonas denitrificans DSM 1251 boxylase Thermanaerovibrio acidaminovorans DSM >gi312879261|ref ZP 07739061.1 L-aspartate 1-decar 60 6589 boxylase Aminomonas paucivorans DSM 12260 >gil 158335510|refYP 001516682.1 aspartate alpha-de >gi313683409|refYP 004061147.1 1-aspartate 1-decar carboxylase Acaryochloris marina MBIC 11017 boxylase Sulfuricurvum kujiense DSM 16994 >gil 91204562emb|CAJ70790.1 strongly similar to aspar >gi290968108 |ref ZP 06559653.1 aspartate 1-decarboxy tate 1-decarboxylase precursor (aspartate alpha-decar lase Megasphaera genomosp. type 1 str. 28L 65 boxylase) Candidatus Kuenenia Stuttgartiensis| >gil.94269421|refZP 01291457.1|Aspartate 1-decarboxy >gi288573810|ref ZP 06392167.1 aspartate 1-decarboxy lase delta proteobacterium MLMS-1 lase Dethiosulfovibrio peptidovorans DSM 11002 US 8,765,426 B2 59 60 >gi289432636|refYP 003462509.1 aspartate 1-decar- >gil 119872877|refYP 93.0884.1 aspartate 1-decarboxy boxylase Dehalococcoides sp. GTI lase Pyrobaculum islandicum DSM 4184 >gil 182677509|refYP 00183.1655.1 aspartate alpha-de- >gi226312675IrefYP 002772569.1 aspartate 1-decar carboxylase Beijerinckia indica subsp. indica ATCC boxylase precursor Brevibacillus brevis NBRC 100599 9039 5 -g 297, ref ZE: SS decarboxy >gi229004643 retzP 04162381.1 aspartate alpha-decar- is:aSC 3.T.C.T.'607IFOFOSDeFFODSIS FOCIOPSill 20.1 aspartateS 1-decar boxylase Bacillus mycoides Rock 1-4 boxylase Cyanothece sp. PCC 7822 >g1 119512431 ref ZP 01631513.1 aspartate 1-decarboxy- >gi 2221 OO611 ref YP 0025351.79.1 Aspartate 1-decar lase precursor Nodularia spumigena CCY9414 to boxylase precursor Thermotoga neapolitana DSM 4359 >gi2698.39249 refYP 003323941.1 aspartate 1-decar- " >gi32.0102432|refYP 004178023.1 L-aspartate 1-decar. boxylase Thermobaculum terrenum ATCC BAA-798 boxylase Isosphaera pallida ATCC 43644 >gil 186682855 refYP 001866051.1 aspartate alpha-de- >gi322378504|ref ZP 08052954.1 Aspartate 1-decar carboxylase Nostoc punctiforme PCC 73102 boxylase precursor Helicobacter suis HS1 >giI25851 1926|refYP 0031853.60.11 aspartate 1-decar- is >gi217076689|refYP 002334405.1 aspartate alpha-de boxylase Alicyclobacillus acidocaldarius Subsp. aci- carboxylase Thermosipho africanus TCF52B docaldarius DSM 446 >gi315497331|refYP 004086135.1 aspartate 1-decar >gi218288596 |ref ZP 03492873.1 aspartate 1-decarboxy- boxylase Asticcacaulis excentricus CB 48 lase Alicyclobacillus acidocaldarius LAA1 >gil 166366461|refYP 001658734.1 aspartate alpha-de >gi282896795|ref ZP 06304801.1 Aspartate decarboxy- carboxylase Microcystis aeruginosa NIES-843) lase Raphidiopsis brookii D9 >gi220904255 refYP 002479567.1| Aspartate 1-decar >gil 206896.045 refYP 00224.6959.1 aspartate 1-decar- boxylase Desulfovibrio desulfuricans subsp. desulfuri boxylase Coprothermobacter proteolyticus DSM 5265 cans str. ATCC 27774 >gi320012833 lgb|ADW07683.1 aspartate 1-decarboxylase >gi57339838gb|AAW49906.1 hypothetical protein Streptomyces flavogriseus ATCC 33331 as FTT1391 synthetic construct >gi323700614|ref ZP 08112526.1 aspartate 1-decarboxy- >gil 16331671|refNP 442399.1 aspartate alpha-decar lase Desulfo vibrio sp. ND132 boxylase Synechocystis sp. PCC 6803 37523797 refNP 9271 74.1 aspartate alpha-decar 303327013 refZP 07357455.1|aspartate 1-decarboxy boxylase Gloeobacter violaceus PCC 7421 lase Desulfovibrio sp. 3 1 syn3 296272234 refYP 003654865.1 aspartate 1-decar 67460926 sp|Q7VJB2.2|PAND HELHP RecName: boxylase Arcobacter nitrofigilis DSM 7299 Full=Aspartate 1-decarboxylase;

SEQUENCE LISTING

16 Os NUMBER OF SEO ID NOS: 19

<21 Os SEQ ID NO 1 &211s LENGTH: 162O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic operon with GI promoter, E. coli panE and panD codign regions

<4 OOs SEQUENCE: 1 to act catcc atggggc.cgg cc act agtcg atctgtgctg tttgcc acgg tatgcagcac 60 cagcgc.gaga titatgggctic gcacgctica citgtcggacg ggggcactgg aacgagaagt 12O

cagg.cgagcc gt cacgc.cct tdacaatgcc acatcct gag caaataattic aaccactaaa 18O

caaat caac C gogtttc.ccg gagg talacca taaaattac C9tattggga tigcggtgc ct 24 O tagggcaatt atggcttaca gCactttgca aa cagggtca taagttcag ggctggctgc 3 OO

gcgtaccgca acctt attgt agcgtgaat C togttgaga C agatggttcg at atttalacg 360 aatcgctgac cqccaacgat cocq attitt c togccaccag cqatctgctic ct ggtgacgc 42O

tgaaagcatg gCaggitttcc gatgc.cgt.ca aaagcct cqc gtCc acactg. CCt9talacta 48O cgc.caatact gttaatt cac aacggcatgg gcac catcga agagttgcaa aa catt cago 54 O

agc catt act gatgggcacc accacccatg cagc.ccgc.cg cgacggcaat gt catt attc 6 OO atgtggcaaa cqgtat cacg catattggcc cq9cacggca acaggacggg gattacagtt 660

atctggcgga tattittgcaa accgtgttgc ctgacgttgc ctgg cataac aatatt.cgcg 72O cc.gagctgtg gCdCaagctg gCagt caact gcgtgattaa to cactgact gc Catctgga 78O

attgc.ccgala C9gtgaatta cqt catcatC ccaagaaat tatgcagata ticgaagaag 84 O

US 8,765,426 B2 63 - Continued

Ctgtttgaaa tigtaaaaag aaaggaga.gt gaatatgagc gcatcggcac tdgtttgcct 9 OO cgc.ccctggit ag 912

<210s, SEQ ID NO 4 &211s LENGTH: 3O3 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 Met Lys Ile Thr Val Lieu. Gly Cys Gly Ala Lieu. Gly Glin Lieu. Trp Lieu. 1. 5 1O 15 Thir Ala Lieu. Cys Lys Glin Gly His Glu Val Glin Gly Trp Lieu. Arg Val 2O 25 3O Pro Gln Pro Tyr Cys Ser Val Asn Lieu Val Glu Thr Asp Gly Ser Ile 35 4 O 45 Phe Asn. Glu Ser Lieu. Thir Ala Asn Asp Pro Asp Phe Lieu Ala Thir Ser SO 55 6 O Asp Lieu. Lieu. Lieu Val Thr Lieu Lys Ala Trp Glin Val Ser Asp Ala Val 65 70 7s 8O Lys Ser Leu Ala Ser Thr Lieu Pro Val Thir Thr Pro Ile Leu Lieu. Ile 85 90 95 His Asn Gly Met Gly Thr Ile Glu Glu Lieu. Glin Asn Ile Glin Glin Pro 1OO 105 11 O Lieu. Lieu Met Gly. Thir Thir Thr His Ala Ala Arg Arg Asp Gly Asn. Wall 115 12 O 125 Ile Ile His Val Ala ASn Gly Ile Thr His Ile Gly Pro Ala Arg Glin 13 O 135 14 O Glin Asp Gly Asp Tyr Ser Tyr Lieu Ala Asp Ile Lieu. Glin Thr Val Lieu. 145 150 155 160 Pro Asp Wall Ala Trp His Asn. Asn. Ile Arg Ala Glu Lieu. Trp Arg Llys 1.65 17O 17s Lieu Ala Val Asn. Cys Val Ile Asin Pro Lieu. Thir Ala Ile Trp Asn. Cys 18O 185 19 O Pro Asn Gly Glu Lieu. Arg His His Pro Glin Glu Ile Met Glin Ile Cys 195 2OO 2O5 Glu Glu Val Ala Ala Val Ile Glu Arg Glu Gly His His Thir Ser Ala 21 O 215 22O

Glu Asp Lieu. Arg Asp Tyr Val Met Glin Val Ile Asp Ala Thr Ala Glu 225 23 O 235 24 O Asn. Ile Ser Ser Met Lieu. Glin Asp Ile Arg Ala Lieu. Arg His Thr Glu 245 250 255 Ile Asp Tyr Ile Asin Gly Phe Lieu. Lieu. Arg Arg Ala Arg Ala His Gly 26 O 265 27 O

Ile Ala Val Pro Glu Asn. Thir Arg Lieu. Phe Glu Met Val Lys Arg Llys 27s 28O 285

Glu Ser Glu Tyr Glu Arg Ile Gly Thr Gly Lieu Pro Arg Pro Trp 29 O 295 3 OO

<210s, SEQ ID NO 5 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Zymomonas mobilis

<4 OOs, SEQUENCE: 5 taagttagga gaataaac 18 US 8,765,426 B2 65 - Continued

<210s, SEQ ID NO 6 &211s LENGTH: 381 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 6 atgatt.cgca catgctgca gggcaaactic caccgcgtga aagtgactica toggacctg 6 O cactatgaag gttcttgcgc cattgaccag gattittctitg acgcago cqg tatt citcgaa 12 O aacgaa.gc.ca ttgat atctg gaatgtcacc aacggcaa.gc gtttct c cac titatgccatc 18O gcggcagaac gcggttcgag aattatttct gttaacggtg cggcggcc.ca citgcgc.cagt 24 O gtcggcgata ttgtcatcat cqc.ca.gct tc gttaccatgc Cagatgaaga agct cqc acc 3OO tggcgaccca acgt.cgc.cta ttittgaaggc gacaatgaaa taaacgtac cqcgaaag.cg 360 attic.cgg tac aggttgcttg a 381

<210s, SEQ ID NO 7 &211s LENGTH: 126 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 7 Met Ile Arg Thr Met Lieu. Glin Gly Llys Lieu. His Arg Val Llys Val Thr 1. 5 1O 15 His Ala Asp Lieu. His Tyr Glu Gly Ser Cys Ala Ile Asp Glin Asp Phe 2O 25 3O Lieu. Asp Ala Ala Gly Ile Lieu. Glu Asn. Glu Ala Ile Asp Ile Trp Asn 35 4 O 45 Val Thr Asn Gly Lys Arg Phe Ser Thr Tyr Ala Ile Ala Ala Glu Arg SO 55 6 O Gly Ser Arg Ile Ile Ser Val Asn Gly Ala Ala Ala His Cys Ala Ser 65 70 7s 8O Val Gly Asp Ile Val Ile Ile Ala Ser Phe Val Thr Met Pro Asp Glu 85 90 95 Glu Ala Arg Thir Trp Arg Pro Asn. Wall Ala Tyr Phe Glu Gly Asp Asn 1OO 105 11 O Glu Met Lys Arg Thr Ala Lys Ala Ile Pro Val Glin Val Ala 115 12 O 125

<210s, SEQ ID NO 8 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 8 gcc.cggittat cqgtagcgat accgggcatt tttitt 35

<210s, SEQ ID NO 9 &211s LENGTH: 312 212. TYPE: PRT <213> ORGANISM: Methylococcus capsulatus

<4 OOs, SEQUENCE: 9 Met Asin Ile Leu Val Ile Gly Thr Gly Ala Ile Gly Ser Phe Tyr Gly 1. 5 1O 15

Ala Lieu. Lieu Ala Lys Thr Gly His Cys Val Ser Val Val Ser Arg Ser 2O 25 3O

Asp Tyr Glu Thr Val Lys Ala Lys Gly Ile Arg Ile Arg Ser Ala Thr 35 4 O 45 US 8,765,426 B2 67 68 - Continued

Lell Gly Asp Thir Phe Arg Pro Ala Ala Wall Val Arg Ser Ala Ala SO 55 6 O

Glu Luell Glu Thir Lys Pro Asp Thir Luell Luell Cys Ile Lys Wall Wall 65 70

Glu Gly Ala Asp Arg Wall Gly Luell Luell Arg Asp Ala Wall Ala Pro Asp 85 90 95

Thir Gly Ile Wall Lell Ile Ser Asn Gly Ile Asp Ile Glu Pro Glu Wall 105 11 O

Ala Ala Ala Phe Pro Asp Asn Glu Wall Ile Ser Gly Lell Ala Phe Ile 115 12 O 125

Gly Wall Thir Arg Thir Ala Pro Gly Glu Ile Trp His Glin Ala Gly 13 O 135 14 O

Arg Luell Met Luell Gly Asn Pro Gly Gly Wall Ser Glu Arg Wall Lys 145 150 155 160

Thir Luell Ala Ala Ala Phe Glu Glu Ala Gly Ile Asp Gly Ile Ala Thir 1.65 17O

Glu Asn Ile Thir Thir Ala Arg Trp Glin Wall Trp Asn Ala Ala 18O 185 19 O

Phe Asn Pro Luell Ser Wall Lell Ser Gly Gly Luell Asp Thir Luell Asp Ile 195

Lell Ser Thir Glin Glu Gly Phe Wall Arg Ala Ile Met Glin Glu Ile Arg 21 O 215

Ala Wall Ala Ala Ala Asn Gly His Pro Luell Pro Glu Asp Ile Wall Glu 225 23 O 235 24 O

Asn Wall Ala Ser Thr Met Pro Pro Thr Ser Met 245 250 255

Lell Wall Asp Phe Glu Ala Gly Glin Pro Met Glu Thir Glu Wall Ile Luell 26 O 265 27 O

Gly Asn Ala Wall Arg Ala Gly Arg Arg Thir Arg Wall Ala Ile Pro His 28O 285

Lell Glu Ser Wall Tyr Ala Lell Met Luell Luell Glu Lell Arg Thir Ser 29 O 295 3 OO

Lys Ser Luell Trp Gly Asn Pro Pro 3. OS 310

<210s, SEQ ID NO 10 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Geobacter metallireducens

<4 OOs, SEQUENCE: 10

Met Arg Ile Ala Ile Wall Gly Ala Gly Ala Luell Gly Lell Tyr Gly 1. 5 1O 15

Ala Luell Luell Glin Arg Ser Gly Glu Asp Wall His Phe Lell Luell Arg Arg 2O 25 3O

Asp Glu Ala Ile Ala Gly Asn Gly Luell Lys Wall Phe Ser Ile Asn 35 4 O 45

Gly Asp Phe Thir Lell Pro His Wall Gly Tyr Arg Ala Pro Glu Glu SO 55 6 O

Ile Gly Pro Met Asp Lell Wall Luell Wall Gly Luell Thir Phe Ala 65 70

Ser Arg Glu Glu Lell Ile Arg Pro Luell Wall Glu Glu Gly Thir Glin 85 90 95

Ile Luell Thir Luell Glin Asn Gly Luell Gly Asn Glu Glu Ala Luell Ala Thir 1OO 105 11 O US 8,765,426 B2 69 70 - Continued

Lell Phe Gly Ala Glu Arg Ile Ile Gly Gly Val Ala Phe Luell Cys Ser 115 12 O 125

Asn Arg Gly Glu Pro Gly Glu Wall His His Lieu. Gly Ala Gly Arg Ile 13 O 135 14 O

Ile Luell Gly Glu Phe Lell Pro Arg Asp Thr Gly Arg Ile Glu Glu Luell 145 150 155 160

Ala Ala Met Phe Arg Glin Ala Gly Wall Asp Cys Arg Thir Thir Asp Asp 1.65 17O 17s

Lell Arg Ala Arg Trp Glu Luell Val Trp Asn Ile Pro Phe Asn 18O 185 19 O

Gly Luell Cys Ala Lell Lell Glin Glin Pro Wall Asn Lell Ile Luell Ala Arg 195

Asp Wall Ser Arg Lell Wall Arg Gly Ile Met Lell Glu Wall Ile Ala 21 O 215

Gly Ala Asn Ala Glin Gly Lell Ala Thir Phe Ile Ala Asp Gly Tyr Wall 225 23 O 235 24 O

Asp Asp Met Luell Glu Phe Thir Asp Ala Met Gly Glu Tyr Pro Ser 245 250 255

Met Glu Ile Asp Arg Glu Glu Gly Arg Pro Leu. Glu Ile Ala Ala Ile 26 O 265 27 O

Phe Arg Thir Pro Lell Ala Gly Ala Arg Glu Gly Ile Ala Met Pro 27s 285

Arg Wall Glu Met Lell Ala Thir Luell Luell Glu Glin Ala Thir Gly 29 O 295 3 OO

SEQ ID NO 11 LENGTH: 350 TYPE : PRT ORGANISM: Schizosaccharomyces pombe

< 4 OOs SEQUENCE: 11

Met Asn. Asn. Thir Ile Ile Luell Gly Ala Gly Ser Ile Gly Ser Luell 1. 5 1O 15

Lell Ala Tyr Glu Lell Ala Ser Luell Lys Ser Ile Asn Asn Arg Wall Ile 2O 25

Lell Luell Luell Arg Asp Ser Arg Wall Asn. Ser Phe Lys Asp Asn 35 4 O 45

Ser Thir Luell Ile Asp Arg Luell Phe Glu Glu Asn Wall Pro His Luell SO 55 6 O

Cys Glin Wall Thir Ala Ser Glu Pro Ser Glin Lell Asn Wall Glin Ser 65 70 7s

Ile Glu Asn Met Ile Wall Thir Thir Ala Gly Glin Thir Glu Asn Ala 85 90 95

Lell Ser Tyr Lell Pro Luell Ser Lys Asn Ser Asn Ile Luell Phe 105 11 O

Wall Glin Asn Gly Met Gly Ala Wall Glu Asn. Wall Gly Lys Luell Trp 115 12 O 125

Pro Glu Glu Glin Asn Pro Ser Ile Tyr Glin Gly Wall Ile Ser His 13 O 135 14 O

Gly Cys Phe Glin Thir Ala Pro Phe His Phe Ser His Ala Gly Luell Gly 145 150 155 160

Asp Luell Ile Ser Wall Pro Asn. Pro Ile Luell Pro 1.65 17O 17s

Asp Glu Ala Ala Glu Thir Pro Glu Met Ile Ser Luell Gly 18O 185 19 O US 8,765,426 B2 71 - Continued Ser Glu Lieu. Lieu. Arg Lieu. Arg Tyr Met Asn Tyr Pro Glu Lieu. Lieu Val 195 2OO 2O5 Asn Glin Cys Glu Lys Lieu Val Ile Asn Ala Cys Ile Asn. Pro Thir Thr 21 O 215 22O Ala Thr Lieu. Asp Cys Val Asn Gly Glu Lieu. Tyr Asn Asp Glu Ser Ala 225 23 O 235 24 O Lys Glu Lieu. Phe Arg Cys Ile Ile Lys Glu. Cys Val Asp Ile Phe Phe 245 250 255 Lys Cys Ile Pro Lieu. Phe Lys Asn. Asn. Glu Glu Ala Glu Lys Ile Lieu. 26 O 265 27 O Asn Val Asn Arg Lieu. Lieu. Asp Arg Val Met Phe Val Gly Thr Llys Val 27s 28O 285 Asn Gly Ala Asn. Ser Ser Ser Thr Arg Glin Asp Cys Lieu. Lieu. Lieu. Arg 29 O 295 3 OO Glu Thr Glu Ile Asp Ala Ile Asin Gly Tyr Val Val Llys Lieu Ala Glu 3. OS 310 315 32O Asn Asn Gly Phe Glin Ala Thr Val Asn Llys Thr Met Met Leu Lieu. Thr 3.25 330 335 Llys Ser Arg Lieu. Gly Lieu. Asn Arg Cys Arg Ala His Ala Arg 34 O 345 35. O

<210s, SEQ ID NO 12 &211s LENGTH: 379 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae < 4 OO SEQUENCE: 12 Met Thr Ala Pro His Arg Ser Thr Ile His Ile Leu Gly Lieu. Gly Ala 1. 5 1O 15 Met Gly. Thr Val Lieu Ala Val Asp Lieu. Lieu. Arg Phe Thr Asn Ala Lieu. 2O 25 3O Val Val Pro Lieu. Phe Arg Ser Glin Glu Arg Lieu Ala Glin Phe Glin Lys 35 4 O 45 Thir Asn Gly Asn. Asn. Ile Ser Ile Arg Llys Lieu. Tyr Lieu. Glu Gly Ser SO 55 6 O Pro Ile Phe Ser Tyr Pro Val Glu Lys Cys Glu. Cys Pro Glu. Thir Phe 65 70 7s 8O Ser Lys Llys Pro Ile Asp Asn Lieu Val Val Thr Thr Lys Thr Tyr Glin 85 90 95 Thir Lys Glu Ala Lieu Ala Pro Tyr Lieu Pro Tyr Ile Asn Lys Asn Thr 1OO 105 11 O Asn Lieu. Ile Lieu. Ile Glin Asn Gly Lieu. Gly Val Lieu. Glu Lieu. Lieu. Arg 115 12 O 125 Glu Glu Ile Phe Thr Asp Ser Lys Asn Arg Pro His Leu Phe Glin Gly 13 O 135 14 O Val Ile Ser His Gly Val Tyr Glin Asp Lys Ala Gly Val Phe Asn His 145 150 155 160

Ala Gly Trp Ala Gly Met Lys Ile Ala Lys Lieu Pro Trp Thr Glu Glu 1.65 17O 17s

Glu Met Ile Glin Llys Llys Ser Val Val Glu Asp Asp Ala Ala Asn. Asn 18O 185 19 O

Ser Lieu Val Lys Lieu. Lieu. Thr Glu Pro Llys Phe Ala Lys Glu Phe Gly 195 2OO 2O5

Ile Glu. His Ser Thr Tyr Glin Glu Met Leu Phe Gly Glin Leu Phe Lys 21 O 215 22O US 8,765,426 B2 73 74 - Continued

Phe Luell Wall Asn Ala Cys Met Asn Pro Wall Thir Ala Ile Luell Asp Cys 225 23 O 235 24 O

Wall Asn Gly Glu Met Ala Ser Gly Pro Wall Phe Thir Ser Ile 245 250 255

Ile Asp Glu Cys Lell Glin Ile Luell Arg Wall Ala Arg Pro Luell Phe 26 O 265 27 O

Glin Tyr His Glu Ser Gly Asn Glu Glu Tyr Pro Glu Met Asp 27s 285

Wall Asn Ala Wall Lell Thir Thir Asp Asn Met Wall Ser Glu Wall Thir Arg 29 O 295 3 OO

Ile Gly Asp Ile Asn Ser Arg Asn Ser Ser Ser Met Arg Glin Asp 3. OS 310 315

Thir Luell Phe Luell Arg Asp Ile Glu Ile Glu Tyr Ile Asn Gly Tyr Wall 3.25 330 335

Wall Luell Ala Asp Asn Lell Asn Luell Asp Pro Asn Cys Wall 34 O 345 35. O

Asn Thir Ile Gly Glu Lell Ala Thir Met Arg Lell Ala Luell Asn Arg 355 360 365

Ser Arg Ser Ile Asn Gly Asp Trp Arg Asp 37 O 375

SEQ ID NO 13 LENGTH: 299 TYPE : PRT ORGANISM: Ralstonia eutropha < 4 OO SEQUENCE: 13

Met Llys Val Ala Ile Met Gly Ala Gly Ala Wall Gly Tyr Gly 1. 5 1O 15

Gly Met Luell Arg Ala Gly His Glu Wall Ile Lell Ile Ala Arg Pro 25

Glin His Wall Ala Ile Glu Ala Thir Gly Luell Arg Lell Glu Thir Glin 35 4 O 45

Ser Phe Asp Glin Wall Lys Wall Ser Ala Ser Ser Asp Pro Ser Ala SO 55 6 O

Wall Glin Gly Asp Lell Wall Luell Phe Wall Ser Thir Asp Thir 65 70

Glin Ser Ala Lell Ala Met Pro Ala Luell Ala Ser Ala Luell 85 90 95

Wall Luell Ser Luell Glin Asn Gly Wall Glu Asn Ala Asp Thir Luell Arg Ser 1OO 105 11 O

Lell Luell Glu Glin Glu Wall Ala Ala Ala Wall Wall Wall Ala Thir Glu 115 12 O 125

Met Ala Gly Pro Gly His Wall Arg His His Gly Arg Gly Glu Luell Wall 13 O 135 14 O

Ile Glu Pro Thir Ser His Gly Ala Asn Luell Ala Ala Ile Phe Ala Ala 145 150 155 160

Ala Gly Wall Pro Wall Glu Thir Ser Asp Asn Wall Arg Gly Ala Luell Trp 1.65 17O 17s

Ala Luell Ile Lell Asn Ala Tyr Asn Ala Lell Ser Ala Ile Thir 18O 185 19 O

Glin Luell Pro Gly Arg Lell Wall Arg Gly Glu Gly Wall Glu Ala Wall 195 2OO

Met Arg Asp Wall Met Glu Glu Phe Ala Wall Ala Arg Ala Glu Gly 21 O 215 22O US 8,765,426 B2 75 76 - Continued

Wall Luell Pro Asp Asp Wall Ala Luell Ala Ile Arg Arg Ile Ala Glu 225 23 O 235 24 O

Thir Met Pro Gly Glin Ser Ser Ser Thir Ala Glin Asp Lell Ala Arg Gly 245 250 255

Arg Ser Glu Ile Asp His Luell Asn Gly Luell Ile Wall Arg Arg Gly 26 O 265 27 O

Asp Ala Luell Gly Ile Pro Wall Pro Ala Asn Arg Wall Lell His Ala Luell 285

Wall Arg Luell Ile Glu Asp Lys Glin Glin His Gly 29 O 295

<210s, SEQ ID NO 14 &211s LENGTH: 3.25 212. TYPE : PRT &213s ORGANISM: Ralstonia Solanacearum

<4 OOs, SEQUENCE: 14

Met Thr Arg Ile Cys Ile Wall Gly Ala Gly Ala Wall Gly Gly Tyr Luell 1. 5 15

Gly Ala Arg Luell Wall Lell Ala Gly Glu Ala Ile Asn Wall Luell Ala Arg 25

Gly Ala Thir Luell Glin Ala Lell Glin Thir Ala Gly Lell Arg Luell Thir Glu 35 4 O 45

Asp Gly Ala Thir His Thir Lell Pro Wall Arg Ala Thir His Asp Ala Ala SO 55 6 O

Ala Luell Gly Glu Asp Wall Wall Ile Wall Ala Wall Ala Pro Ala 65 70 75

Lell Glu Ser Wall Ala Gly Ile Ala Pro Luell Ile Gly Pro Gly Thir 90 95

Arg Wall Wall Wall Met Asn Gly Wall Pro Trp Trp Phe Phe Asp Arg 105 11 O

Pro Gly Pro Luell Gly Glin Arg Luell Glin Ala Wall Asp Pro His Gly 115 12 O 125

Arg Ile Ala Glin Ile Pro Thir Arg His Wall Lell Gly Gly Wall Wall 13 O 135 14 O

His Luell Thir Thir Wall Ser Pro Gly His Ile Arg His Gly Asn 145 150 155 160

Gly Arg Arg Luell Lell Gly Glu Pro Ala Gly Gly Ala Ser Pro Arg 17O 17s

Lell Ala Ser Ile Ala Lell Phe Gly Arg Ala Gly Lell Glin Ala Glu 18O 185 19 O

Ser Glu Ala Glin Arg Asp Ile Trp Phe Lell Trp Gly Asn 195

Met Thir Met Asn Pro Wall Ser Wall Luell Thir Gly Ala Thir Asp Arg 21 O 215 22O

Ile Luell Asp Asp Pro Lell Wall Ser Ala Phe Cys Lell Ala Wall Met Ala 225 23 O 235 24 O

Glu Ala Ala Ile Gly Ala Arg Ile Gly Pro Ile Glu Glin Ser 245 250 255

Gly Glu Ala Arg Ser Ala Wall Thir Arg Glin Luell Gly Ala Phe Thir 26 O 265 27 O

Ser Met Luell Glin Asp Ala Glu Ala Gly Arg Gly Pro Lell Glu Ile Asp 27s 285

Ala Luell Wall Ala Ser Wall Arg Glu Ile Gly Luell His Wall Gly Wall Pro 29 O 295 3 OO US 8,765,426 B2 77 - Continued Thr Pro Glin Ile Asp Thir Lieu. Lieu. Gly Lieu Val Arg Lieu. His Ala Glin 3. OS 310 315 32O Thr Arg Gly Lieu. Tyr 3.25

<210s, SEQ ID NO 15 &211s LENGTH: 312 212. TYPE: PRT <213> ORGANISM: Stenotrophomonas maltophilia

<4 OOs, SEQUENCE: 15 Met Arg Ile Lieu. Ile Leu Gly Ala Gly Gly Thr Gly Gly Tyr Phe Gly 1. 5 1O 15 Gly Arg Lieu Ala Glin Ala Gly Val Asp Val Thr Phe Lieu Val Arg Pro 2O 25 3O Ala Arg Ala Ala Glin Lieu. Asp Arg Asp Gly Lieu Val Ile Arg Ser Pro 35 4 O 45 Lieu. Gly Asp Ala Ser Phe Pro Val Glin His Val Thr Ala Asp Ala Lieu. SO 55 6 O Pro Ala Lieu Ala Ala Glin Llys Pro Phe Asp Lieu Val Ile Lieu. Ser Cys 65 70 7s 8O Lys Ala Tyr Asp Lieu. Asp Ser Ser Ile Asp Ala Ile Ala Pro Ala Val 85 90 95 Gly Ala Asn. Thir Thr Val Lieu Pro Ile Lieu. Asn Gly Lieu. His His Tyr 1OO 105 11 O Asn Ala Lieu. Asp Lieu. Arg Phe Gly Arg Asp Ala Val Lieu. Gly Gly Lieu 115 120 125 Cys Phe Ile Ser Ala Thr Lys Ala Pro Asp Gly Ala Val Lieu. His Lieu. 13 O 135 14 O Gly Llys Pro Ala Lys Lieu. Thir Phe Gly Glu Arg Asp Gly Gly Ala Ile 145 150 155 160 Ser Thr Arg Val Arg Ala Phe Ala Ala Ala Cys Ala Glin Ala Asn Lieu. 1.65 17O 17s Asp His Lieu Ala Ser Glu. His Ile Gly Glin Glu Gln Trp Ile Llys Tyr 18O 185 19 O Thir Phe Lieu. Thir Ala Lieu Ala Ala Ala Thr Cys Lieu. Lieu. Arg Ala Asp 195 2OO 2O5 Ile Gly Ser Ile Val Ala Thr Asp Asp Gly Glu Ala Ile Val Arg Gly 21 O 215 22O Lieu. Tyr Asp Glu. Cys Lieu Ala Val Ala Glu Ala Ala Gly Glu Pro Ile 225 23 O 235 24 O Pro Asp Ala Ala Glin Asp Thr Ala Arg Gly Thr Lieu. Thr Glin Ala Gly 245 250 255 Ser Ala Lieu Lys Ala Ser Met Lieu. Arg Asp Lieu. Glu Ala Gly Glin Glin 26 O 265 27 O

Val Glu Ala Ala Glin Ile Val Gly Asp Met Lieu Ala Arg Ala Arg Llys 27s 28O 285

Ala Asp Glin Glu Gly Lieu Lleu Lieu. Glin Val Ala Tyr Ser Ser Lieu. Glin 29 O 295 3 OO

Ala Tyr Glin Ala Lieu. Arg Ala Ala 3. OS 310

<210s, SEQ ID NO 16 &211s LENGTH: 313 212. TYPE: PRT <213> ORGANISM: Enterococcus faecallis US 8,765,426 B2 79 80 - Continued

<4 OOs, SEQUENCE: 16

Met Lys Ile Ala Ile Ala Gly Ala Gly Ala Met Gly Ser Arg Lieu. Gly 1. 15

Ile Met Luell His Glin Gly Gly Asn Asp Wall Thir Lell Ile Asp Glin Trp 25 3O

Pro Ala His Ile Glu Ala Ile Arg Asn Lell Ile Ala Asp Phe 35 4 O 45

Asn Gly Glu Glu Wall Wall Ala Asn Luell Pro Phe Ser Pro Glu Glu SO 55 6 O

Ile Asp His Glin Asn Glu Glin Wall Asp Luell Ile Ala Lieu. Thir Lys 65 70

Ala Glin Glin Luell Asp Ala Met Phe Ala Glin Pro Met Ile Thir 85 90 95

Glu Thir Tyr Wall Lell Luell Luell Asn Lell Gly His Glu. Asp 105 11 O

Wall Luell Glu Tyr Wall Pro Lys Glu Asn Lell Wall Gly Ile Thir 115 12 O 125

Met Trp Thir Ala Gly Lell Glu Gly Pro Gly Arg Wall Luell Luell Gly 13 O 135 14 O

Asp Gly Glu Ile Glu Lell Glu Asn Ile Asp Pro Ser Gly Phe 145 150 155 160

Ala Luell Glu Wall Wall Asp Wall Phe Glin Lys Ala Gly Lell As Pro Ser 1.65 17s

Ser Ser Asn Wall Arg Ser Ile Trp Arg Ala Cys Val Asn 18O 185 19 O

Gly Thir Luell Asn Gly Lell Thir Ile Luell Asp Asn Ile Ala Glu 195

Phe Gly Ala Luell Pro Wall Ser Glu Ser Luell Wall Lys Thir Lieu. Ile Ser 21 O 215 22O

Glu Phe Ala Ala Wall Ala Glu Glu Ala Ile Tyr Lell Asp Glin Ala 225 23 O 235 24 O

Glu Wall Thir His Ile Wall Glin Thir Tyr Asp Pro Asn Gly Ile Gly 245 250 255

Lell His Pro Ser Met Glin Asp Luell Ile Asn His Arg Luell 26 O 265 27 O

Thir Glu Ile Asp Tyr Ile Asn Gly Ala Wall Trp Arg Lys Gly Glin 27s 28O 285

Asn Wall Ala Thir Pro Phe Ala Met Luell Thir Glin Lieu Wall His 29 O 295 3 OO

Gly Lys Glu Glu Lell Lell Gly Ala 3. OS 310

SEO ID NO 17 LENGTH: 298 TYPE : PRT ORGANISM; Bacillus subtilis

< 4 OOs SEQUENCE: 17

Met Lys Ile Gly Ile Ile Gly Gly Gly Ser Val Gly Lell Lieu. Cys Ala 1. 5 15

Tyr Tyr Lieu Ser Lieu. Tyr His Asp Val Thir Wal Wall Thir Arg Arg Glin 25 3O

Glu Glin Ala Ala Ala Ile Glin Ser Glu Gly Ile Arg Lell Tyr Lys Gly 35 4 O 45 US 8,765,426 B2 81 - Continued Gly Glu Glu Phe Arg Ala Asp Cys Ser Ala Asp Thir Ser Ile Asn. Ser SO 55 6 O Asp Phe Asp Lieu. Lieu Val Val Thr Val Lys Glin His Glin Lieu. Glin Ser 65 70 7s 8O Val Phe Ser Ser Lieu. Glu Arg Ile Gly Lys Thr Asn. Ile Lieu. Phe Lieu 85 90 95 Glin Asn Gly Met Gly His Ile His Asp Lieu Lys Asp Trp His Val Gly 1OO 105 11 O His Ser Ile Tyr Val Gly Ile Val Glu. His Gly Ala Val Arg Llys Ser 115 12 O 125 Asp Thir Ala Val Asp His Thr Gly Lieu. Gly Ala Ile Llys Trp Ser Ala 13 O 135 14 O

Phe Asp Asp Ala Glu Pro Asp Arg Lieu. Asn. Ile Lieu. Phe Gln His Asn 145 150 155 160 His Ser Asp Phe Pro Ile Tyr Tyr Glu Thir Asp Trp Tyr Arg Lieu. Leu 1.65 17O 17s

Thr Gly Llys Lieu. Ile Val Asn Ala Cys Ile Asn. Pro Lieu. Thir Ala Lieu. 18O 185 19 O

Lieu. Glin Val Lys Asn Gly Glu Lieu. Lieu. Thir Thr Pro Ala Tyr Lieu Ala 195 2OO 2O5

Phe Met Lys Lieu Val Phe Glin Glu Ala Cys Arg Ile Lieu Lys Lieu. Glu 21 O 215 22O

Asn Glu Glu Lys Ala Trp Glu Arg Val Glin Ala Val Cys Gly Glin Thr 225 23 O 235 24 O

Lys Glu Asn Arg Ser Ser Met Lieu Val Asp Val Ile Gly Gly Arg Glin 245 250 255

Thr Glu Ala Asp Ala Ile Ile Gly Tyr Lieu. Lieu Lys Glu Ala Ser Lieu. 26 O 265 27 O Glin Gly Lieu. Asp Ala Wal His Lieu. Glu Phe Lieu. Tyr Gly Ser Ile Llys 27s 28O 285

Ala Lieu. Glu Arg Asn. Thir Asn Llys Val Phe 29 O 295

<210s, SEQ ID NO 18 &211s LENGTH: 125 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: shortened consensus structure for aspartate 1 decarboxylase 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) ... (8) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (12) ... (21) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (23) . . (21) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (23) . . (23) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid US 8,765,426 B2 83 - Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (26) ... (51) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (53) . . (51) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (53) . . (53) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (55) . . (56) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (59) . . (71) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (74) . . (85) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (87) . . (125) <223> OTHER INFORMATION: X may be any amino acid <4 OOs, SEQUENCE: 18 Xaa Xala Xala Xala Xaa Xaa Xaa Xala Lys Xaa His Xaa Xaa Xala Xala Xala 1. 5 1O 15 Xaa Xala Xala Xala Xaa Tyr Xaa Gly Ser Xaa Xaa Xaa Xaa Xala Xala Xala 2O 25 3O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 35 4 O 45 Xaa Xala Xala Gly Xaa Arg Xaa Xala Thr Tyr Xaa Xaa Xaa Xala Xala Xala SO 55 6 O Xaa Xala Xala Xala Xaa Xaa Xaa Asn Gly Xaa Xaa Xaa Xaa Xala Xala Xala 65 70 7s 8O

Xaa Xala Xala Xala Xaa Ile Xaa Xala Xala Xala Xaa Xaa Xala Xala Xala Xala 85 90 95

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1OO 105 11 O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 115 12 O 125

<210s, SEQ ID NO 19 &211s LENGTH: 270 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conserved structure sequence for 2 dehydropantoate reductase 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (6) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (13) . . (18) <223> OTHER INFORMATION: X may be any amino acid US 8,765,426 B2 85 - Continued

22 Os. FEATURE <221 > NAMEAKEY SC FEATURE <222s. LOCATION: (2O) (63) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY SC FEATURE <222s. LOCATION: (65) . . (89) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY SC FEATURE <222s. LOCATION: (92) ... (162) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (164) (166) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (168) ... (170) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (172) ... (196) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (198) (226) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (228) (230) <223> OTHER INFORMATION: X may be any amino acid 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (232) (238) <223> OTHER INFORMATION: X may be any amino acid & 22 O FEATURE; <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (24 O) ... (270) <223> OTHER INFORMATION: X may be any amino acid <4 OOs, SEQUENCE: 19 Xaa Xala Xala Xala Xaa Xaa Gly Xaa Gly Xaa Xaa Gly Xaa Xala Xala Xala 1. 5 1O 15

Xaa Xala Luell Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 2O 25 3O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 35 4 O 45

Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Lys SO 55 6 O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 65 70 7s 8O

Xaa Xala Xala Xala Xaa Xaa Xaa Xala Xala Asn Gly Xaa Xaa Xala Xala Xala 85 90 95

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 1OO 105 11 O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 115 12 O 125

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 13 O 135 14 O

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 145 150 155 160

Xaa Xala Lys Xaa Xaa Xaa Asn. Xaa Xala Xala Asn. Xaa Xaa Xala Xala Xala 1.65 17O 17s

Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala 18O 185 19 O US 8,765,426 B2 87 88 - Continued

Xaa Xala Xaa Xala Glu Xaa Xala Xaa Xala Xala Xala Xala Xaa Xala Xala Xaa 195 2O5

Xaa Xaa Xala Xala Xala Xala Xaa Xala Xala Xala Xala Xaa Xala Xaa Xaa Xala 21 O 215 22O

Xaa Xala Ser Xala Xala Xaa Asp Xaa Xala Xala Xaa Xala Xala Xaa Glu Xaa 225 23 O 235 24 O

Xaa Xala Xala Xala Xaa Xala Xala Xala Xala Xaa Xala Xala Xala Xaa Xaa Xala 245 250 255

Xaa Xala Xala Xaa Xala Xala Xala Xala Xaa Xala Xala Xala Xala Xaa 26 O 265 27 O

What is claimed is: wherein steps b) and c) may be in either order or simulta 1. A recombinant bacterial Strain of the genus Zymomonas neous, and wherein 2-dehydropantoate reductase activ comprising a heterologous nucleic acid molecule encoding a ity and aspartate 1-decarboxylase activity are both polypeptide having 2-dehydropantoate reductase activity and expressed in the strain produced by steps (a), (b), and (c). a heterologous nucleic acid molecule encoding a polypeptide 10. A method for the producing ethanol comprising: having aspartate 1-decarboxylase activity wherein said a) providing a recombinant bacterial Strain of the genus recombinant bacterial Strain can produce ethanol in the Zymomonas comprising a heterologous nucleic acid absence of externally Supplied pantothenic acid. molecule encoding a polypeptide having 2-dehydropan toate reductase activity and a heterologous nucleic acid 2. A recombinant bacterial strain of claim 1 wherein the 25 polypeptide having 2-dehydropantoate reductase activity is molecule encoding a polypeptide having aspartate 1-de an enzyme belonging to the EC 1.1.1.169 class. carboxylase activity; and 3. A recombinant bacterial strain of claim 2 wherein the b) contacting the strain of (a) with fermentation medium polypeptide having 2-dehydropantoate reductase activity has under conditions whereby the Strain produces ethanol. 11. A method according to claim 10 wherein the polypep at least ten conserved amino acid positions selected from the 30 group consisting of G at position 7, G at position 9, G at tide having 2-dehydropantoate reductase activity is an position 12, K at position 72, Nat position 98, G at position enzyme belonging to the EC 1.1.1.169 class. 99, Kat position 176, Nat position 180, Nat position 184, E 12. A method according to claim 11 wherein the polypep at position 210, Sat position 244, D or Sat position 248, and tide having 2-dehydropantoate reductase activity has at least ten conserved amino acid positions selected from the group E at position 256, as compared to the E. coli 2-dehydropan 35 toate reductase of SEQID NO:4. consisting of Gat position 7. Gat position 9, Gat position 12, 4. A recombinant bacterial strain of claim 1 wherein the Kat position 72, N at position 98, G at position 99, K at polypeptide having aspartate 1-decarboxylase activity is an position 176, N at position 180, N at position 184, E at position 210, Sat position 244, D or Sat position 248, and E enzyme belonging to the EC 4.1.1.11 class. at position 256, as compared to the E. coli 2-dehydropantoate 5. A recombinant bacterial strain of claim 4 wherein the 40 polypeptide having aspartate 1-decarboxylase activity has at reductase of SEQID NO:4. least eight conserved amino acid positions selected from the 13. A method according to claim 10 wherein the polypep group consisting of K at position 9, H at position 11, Y at tide having aspartate 1-decarboxylase activity is an enzyme position 22, G at position 24, Sat position 25, G at position belonging to the EC 4.1.1.11 class. 14. A method according to claim 13 wherein the polypep 52, R at position 54. T at position 57, Yat position 58, Nat 45 position 72, Gat position 73, and I at position 86, as compared tide having aspartate 1-decarboxylase activity has at least to the E. coli aspartate 1-decarboxylase of SEQID NO:7. eight conserved amino acid positions selected from the group 6. A recombinant bacterial strain of claim 4 wherein the consisting of Kat position 9, Hat position 11, Yat position 22, polypeptide having aspartate 1-decarboxylase activity has the G at position 24, S at position 25, G at position 52, R at position 54, Tat position 57, Yat position 58, Nat position 72, five conserved amino acid positions K at position 9, Y at 50 position 22, G at position 24. T at position 57, and Y at G at position 73, and I at position 86, as compared to the E. position 58, as compared to the E. coliaspartate 1-decarboxy coli aspartate 1-decarboxylase of SEQID NO:7. 15. A method according to claim 13 wherein the polypep lase of SEQID NO:7. tide having aspartate 1-decarboxylase activity has the five 7. A recombinant bacterial strain of claim 1 wherein the conserved amino acid positions Kat position 9, Yat position strain grows in media that lacks pantothenic acid. 55 8. The recombinant bacterial strain of claim 1 wherein the 22, Gat position 24, Tat position 57, and Y at position 58, as strain comprises genetic modifications which enhance the compared to the E. coli aspartate 1-decarboxylase of SEQID production of ethanol. NO:7. 9. A process for producing a Zymomonas strain that syn 16. A method according to claim 10 wherein prior to step thesizes pantothenic acid comprising: 60 (b) the bacterial strain of (a) is contacted with medium that a) providing a bacterial strain of the genus Zymomonas, lacks or has a Sub-optimal amount of pantothenic acid b) introducing a heterologous nucleic acid molecule wherein a seed culture is produced to inoculate the fermen encoding a polypeptide having 2-dehydropantoate tation medium of (b). reductase activity; and 17. A method according to claim 10 or 16 wherein the fermentation medium lacks or has a sub-optimal amount of c) introducing a heterologous nucleic acid molecule encod 65 ing a polypeptide having aspartate 1-decarboxylase pantothenic acid. activity;