Patent Publication Number: US-2004048343-A1

Title: Method and microorganisms for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (hmbpa)

Description:
RELATED APPLICATIONS  
     [0001] The present invention claims the benefit of prior-filed provisional Patent Application Serial No. 60/263,053, filed Jan. 19, 2001 (pending). The present invention is also related to U.S. patent application Ser. No. 09/667,569, filed Sep. 21, 2000 (pending), which is a continuation-in-part of U.S. patent application Ser. No. 09/400,494, filed Sep. 21, 1999 (abandoned). U.S. patent application Ser. No. 09/667,569 also claims the benefit of prior-filed provisional Patent Application Serial No. 60/210,072, filed Jun. 7, 2000, provisional Patent Application Serial No. 60/221,836, filed Jul. 28, 2000, and provisional Patent Application Serial No. 60/227,860, filed Aug. 24, 2000. The entire content of each of the above-referenced applications is incorporated herein by this reference. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] Conventional means of synthesizing chemical compounds is via synthesis from bulk chemicals, a process which is limited by factors such as substrate availability and/or cost, difficulty in resolving complex mixtures of products, complexities in synthesizing large quantities of compounds in purified form, and difficulty in producing chiral compounds. Accordingly, researchers have recently looked to bacterial or microbial systems that express enzymes useful for various biosynthetic processes, for example, in the synthesis of pharmaceutical compounds, research reagents, nutriceuticals, vitamins, nutritional supplements, antibiotic compounds and the like. In particular, bioconversion processes have been evaluated as a means of favoring production of preferred compounds and recently methods of direct microbial synthesis have been the focus of much research in the areas of pharmaceuticals and agriculture.  
       SUMMARY OF THE INVENTION  
       [0003] The present invention relates to a processes for the direct microbial synthesis of [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”), referred to interchangeably herein as “β-alanine 2-(R)-hydroxyisolvalerate”, “β-alanine 2-hydroxyisolvalerate”, “β-alanyl-α-hydroxyisovalarate”, N-(2-hydroxy-3-methyl-1-oxobutyl)-β-alanine (“HMOBA”) and/or “fantothenate”. In particular, it has been discovered that in microorganisms engineered to overexpress certain enzymes conventionally associated with pantothenate and/or isoleucine-valine (ilv) biosynthesis, an alternative biosynthetic pathway is present that competes for key precursors of pantothenate biosynthesis, namely α-ketoisovalerate (α-KIV) and β-alanine. α-KIV is converted to α-hydroxyisovalerate (α-HIV) catalyzed by various reductase enzymes and α-HIV is subsequently condensed with β-alanine to produce HMBPA.  
       [0004] In one embodiment, the invention features a process for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includes culturing a microorganism having increased keto reductase activity or increased pantothenate synthetase activity in the presence of excess α-ketoisovalerate and excess β-alanine, such that HMBPA is produced. In another embodiment, the invention features a process for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includes culturing a microorganism having increased keto reductase activity and increased pantothenate synthetase activity in the presence of excess α-ketoisovalerate and excess β-alanine, such that HMBPA is produced. In one embodiment, the microorganism has a modified panE gene, for example, a modified panel gene and/or a modified panE2 gene (e.g., the panE gene is overexpressed, deregulated or present in multiple copies). In another embodiment, the microorganism has a modified panC gene (e.g., the panC gene is overexpressed, deregulated or present in multiple copies). In another embodiment, the microorganism further has increased acetohydroxyacid isomeroreductase activity. In another embodiment, the microorganism is cultured under conditions of increased acetohydroxyacid isomeroreductase activity in the presence of excess α-ketoisovalerate and excess β-alanine, such that HMBPA is produced. In yet another embodiment, the microorganism comprises a modified ilvC gene (e.g., the ilvC gene is overexpressed, deregulated or present in multiple copies). In yet another embodiment, the microorganism further has reduced ketopantoate hydroxymethyltransferase activity (e.g., has a modified panB gene, for example a panB gene that has been deleted.  
       [0005] In another aspect, the invention features a process for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includes culturing a microorganism having reduced ketopantoate hydroxymethyltransferase activity in the presence of excess α-ketoisovalerate and excess β-alanine, such that HMBPA is produced. In another aspect, the invention features a method for enhancing production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) relative to pantothenate that includes culturing a recombinant microorganism under conditions such that the HMBPA production is enhanced relative to pantothenate production. In another aspect, the invention features a process for the production of 2-hydroxyisovaleric acid (α-HIV) that includes culturing a microorganism which overexpresses PanE1 or PanE2 and which further has reduced PanC or PanD activity under conditions such that α-HIV is produced. In another aspect, the invention features a process for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includes culturing a recombinant microorganism having decreased expression or activity of serA or glyA under conditions such that HMBPA is produced. In another aspect, the invention features a process for the production of 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) that includes culturing a recombinant microorganism having decreased expression or activity of serA and glyA under conditions such that HMBPA is produced. Conditions for culturing the above described microorganisms include, for example, conditions of increased steady state glucose, conditions of decreased steady state dissolved oxygen, and/or cultured under conditions of decreased serine. Products produced according to the above described processes and/or methods are also featured. Also featured are recombinant microorganisms utilized in the above-described methods.  
       [0006] Compounds produced according to the methodologies of the present invention have a variety of uses. For example, HMBPA can be used to synthesize inhibitors of HMG CoA Reductase (II) (Gordon et al.  Bio. Med. Chem. Lett.  1(3):161 (1991). Inhibitors of HMG CoA Reductase (II) have been studied for use as in the treatment of hypercholesterolaemia and coronary atherosclerosis progression. Inhibitors of HMG CoA Reductase also have been used to reduce risk of cardiovascular events in patients at risk. Moreover, the HMBPA precursor 2-hydroxyisovalerate (α-HIV) has been demonstrated to have nutriceutical properties, for example, in the prevention of aging of the skin. In particular, α-hydroxy acids, such as α-HIV (or 2-hydroxyvaline), can be used to synthesize α-hydroxy esters which have been found to induce increased skin thickness by increasing biosyntheses of glycosaminoglycans, proteoglycans, collagen, elastin, and other dermal components. The compounds can be used to treat skin disorders such as age spots, skin lines, wrinkles, photoaging and aging.  
       [0007] Other features and advantages of the invention will be apparent from the following detailed description and claims.  
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0008]FIG. 1 is a schematic representation of the pantothenate and isoleucine-valine (ilv) biosynthetic pathways. Pantothenate biosynthetic enzymes are depicted in bold and their corresponding genes indicated in italics. Isoleucine-valine (ilv) biosynthetic enzymes are depicted in bold italics and their corresponding genes indicated in italics.  
     [0009]FIG. 2 is a schematic representation of the biosynthetic pathway leading to [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”) in  B. subtilis.    
     [0010]FIG. 3 is a schematic depiction of the structure of [R]-3-(2-hydroxy-3methyl-butyrylamino)-propionic acid (“HMBPA”).  
     [0011]FIG. 4 is a HPLC chromatogram of a sample of medium from a 14 L fermentation of PA824.  
     [0012]FIG. 5 is a mass spectrum depicting the relative monoisotopic mass of [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid.  
     [0013]FIG. 6 depicts an alignment of the C-terminal amino acids from known or suspected PanB proteins.  
     [0014]FIG. 7 is a schematic representation of the construction of the plasmid pAN624.  
     [0015]FIG. 8 is a schematic representation of the construction of the plasmid pAN620.  
     [0016]FIG. 9 is a schematic representation of the construction of the plasmid pAN636.  
     [0017]FIG. 10 is a schematic representation of the construction of the plasmid pAN637 which allows selection for single or multiple copies using chloramphenicol.  
     [0018]FIG. 11 is a schematic representation of the construction of the plasmid pAN238, a plasmid for overexpressing  B. subtilis  panE2 from the P 26  promoter. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0019] The present invention is based, at least in part, on the discovery of a novel biosynthetic pathway in bacteria, namely the [R]-3-(2-hydroxy-3-methyl-butyrylamino)propionic acid (“HMBPA”) biosynthetic pathway. In particular, it has been discovered that bacteria are capable of generating HMBPA from α-ketoisovalerate (α-KIV), a key product of the isoleucine-valine (ilv) biosynthetic pathway and precursor of the pantothenate biosynthetic pathway. Production of HMBPA in bacteria involves at least the pantothenate biosynthetic enzymes ketopantoate reductase (the panE1 gene product) and/or acetohydroxyacid isomeroreductase (the ilvC gene product) and results from the condensation of 2-hydroxyisovaleric acid (α-HIV), formed by reduction of α-KIV, and β-alanine, the latter reaction being catalyzed by the pantothenate biosynthetic enzyme pantothenate synthetase (the panC gene product). Production of HMBPA is achieved by increasing ketopantoate reductase (e.g., PanE1) and/or PanE2 and/or acetohydroxyacid isomeroreductase activities (e.g., IlvC) in microorganisms, for example, by overexpressing or deregulating the genes encoding said enzymes. Optimal production of HMBPA is achieved by decreasing or deleting ketopantoate hydroxymethyltransferase activity (the panB gene product) in microorganisms, for example, by modifying or deleting the panB gene which encodes ketopantoate hydroxymethyltransferase (e.g., PanB), optionally in addition to increasing ketopantoate reductase and/or PanE2 and/or acetohydroxyacid isomeroreductase activities in said microorganisms. The substrates α-KIV and β-alanine are required for HMBPA production, the latter provided, for example, by β-alanine feeding and/or increased aspartate-α-decarboxylate activity (the panD gene product). Increasing substrate concentration (i.e., α-KIV and/or β-alanine) further enhances production of HMBPA. α-KIV production can be increased by overexpressing ilvBNCD genes and/or alsS. HMBPA production can further be increased by limiting serine availability or synthesis in appropriately engineered microorganisms.  
     [0020] In order that the present invention may be more readily understood, certain terms are first defined herein.  
     [0021] The term “pantothenate biosynthetic pathway” includes the biosynthetic pathway involving pantothenate biosynthetic enzymes (e.g., polypeptides encoded by biosynthetic enzyme-encoding genes), compounds (e.g., precursors, substrates, intermediates or products), cofactors and the like utilized in the formation or synthesis of pantothenate. The term “pantothenate biosynthetic pathway” includes the biosynthetic pathway leading to the synthesis of pantothenate in microorganisms (e.g., in vivo) as well as the biosynthetic pathway leading to the synthesis of pantothenate in vitro.  
     [0022] The term “pantothenate biosynthetic enzyme” includes any enzyme utilized in the formation of a compound (e.g., intermediate or product) of the pantothenate biosynthetic pathway. For example, synthesis of pantoate from α-ketoisovalerate (α-KIV) proceeds via the intermediate, ketopantoate. Formation of ketopantoate is catalyzed by the pantothenate biosynthetic enzyme ketopantoate hydroxymethyltransferase (the panB gene product). Formation of pantoate is catalyzed by the pantothenate biosynthetic enzyme ketopantoate reductase (the panE gene product). Synthesis of β-alanine from aspartate is catalyzed by the pantothenate biosynthetic enzyme aspartate-α-decarboxylase (the panD gene product). Formation of pantothenate from pantoate and β-alanine (e.g., condensation) is catalyzed by the pantothenate biosynthetic enzyme pantothenate synthetase (the panC gene product). Based on the newly discovered HMBPA biosynthesis pathway, pantothenate biosynthetic enzymes may also perform an alternative function as enzymes in the HMBPA biosynthetic pathway described herein.  
     [0023] The term “pantothenate” includes the free acid form of pantothenate, also referred to as “pantothenic acid” as well as any salt thereof (e.g., derived by replacing the acidic hydrogen of pantothenate or pantothenic acid with a cation, for example, calcium, sodium, potassium, ammonium), also referred to as a “pantothenate salt”. The term “pantothenate” also includes alcohol derivatives of pantothenate. Preferred pantothenate salts are calcium pantothenate or sodium pantothenate. A preferred alcohol derivative is pantothenol. Pantothenate salts and/or alcohols of the present invention include salts and/or alcohols prepared via conventional methods from the free acids described herein. In another embodiment, calcium pantothenate is synthesized directly by a microorganism of the present invention. A pantothenate salt of the present invention can likewise be converted to a free acid form of pantothenate or pantothenic acid by conventional methodology.  
     [0024] The term “isoleucine-valine biosynthetic pathway” includes the biosynthetic pathway involving isoleucine-valine biosynthetic enzymes (e.g., polypeptides encoded by biosynthetic enzyme-encoding genes), compounds (e.g., precursors, substrates, intermediates or products), cofactors and the like utilized in the formation or synthesis of conversion of pyruvate to valine or isoleucine. The term “isoleucine-valine biosynthetic pathway” includes the biosynthetic pathway leading to the synthesis of valine or isoleucine in microorganisms (e.g., in vivo) as well as the biosynthetic pathway leading to the synthesis of valine or isoleucine in vitro. FIG. 1 includes a schematic representation of the isoleucine-valine biosynthetic pathway. Isoleucine-valine biosynthetic enzymes are depicted in bold italics and their corresponding genes indicated in italics  
     [0025] The term “isoleucine-valine biosynthetic enzyme” includes any enzyme utilized in the formation of a compound (e.g., intermediate or product) of the isoleucine-valine biosynthetic pathway. According to FIG. 1, synthesis of valine from pyruvate proceeds via the intermediates, acetolactate, α,β-dihydroxyisovalerate (α,β-DHIV) and α-ketoisovalerate (α-KIV). Formation of acetolactate from pyruvate is catalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxyacid synthetase (the ilvBN gene product, or alternatively, the alsS gene product). Formation of α,β-DHIV from acetolactate is catalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxyacidisomero reductase (the ilvC gene product). Synthesis of α-KIV from α,β-DHIV is catalyzed by the isoleucine-valine biosynthetic enzyme dihydroxyacid dehydratase (the ilvD gene product). Moreover, valine and isoleucine can be interconverted with their respective α-keto compounds by branched chain amino acid transaminases. Based on the newly discovered HMBPA biosynthesis pathway, isoleucine-valine biosynthetic enzymes may also perform an alternative function as enzymes in the HMBPA biosynthetic pathway described herein.  
     [0026] The term “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”) biosynthetic pathway” includes the alternative biosynthetic pathway involving biosynthetic enzymes and compounds (e.g., substrates and the like) traditionally associated with the pantothenate biosynthetic pathway utilized in the formation or synthesis of HMBPA. The term “HMBPA biosynthetic pathway” includes the biosynthetic pathway leading to the synthesis of HMBPA in microorganisms (e.g., in vivo) as well as the biosynthetic pathway leading to the synthesis of HMBPA in vitro.  
     [0027] The term “HMBPA biosynthetic enzyme” includes any enzyme utilized in the formation of a compound (e.g., intermediate or product) of the HMBPA biosynthetic pathway. For example, synthesis of 2-hydroxyisovaleric acid (α-HIV) from α-ketoisovalerate (α-KIV) is catalyzed by the panE1 or panE2 gene product (PanE1, alternatively referred to herein ketopantoate reductase or PanE2, a α-ketoacid reductase that does not significantly contribute to pantothenate biosynthesis) and/or is catalyzed by the ilvC gene product (alternatively referred to herein as acetohydroxyacid isomeroreductase). Formation of HMBPA from β-alanine and α-HIV is catalyzed by the panC gene product (alternatively referred to herein as pantothenate synthetase).  
     [0028] The term “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”)” includes the free acid form of HMBPA, also referred to as “3-(2-hydroxy-3-methyl-butyrylamino)-propionate” as well as any salt thereof (e.g., derived by replacing the acidic hydrogen of [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionate with a cation, for example, calcium, sodium, potassium, ammonium), also referred to as a “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid salt” or “HMBPA salt”. Preferred HMBPA salts are calcium HMBPA or sodium HMBPA. HMBPA salts of the present invention include salts prepared via conventional methods from the free acids described herein. An HMBPA salt of the present invention can likewise be converted to a free acid form of [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionate by conventional methodology.  
     [0029] Various aspects of the invention are described in further detail in the following subsections.  
     [0030] I. Targeting Genes Encoding Various Pantothenate and/or Isoleucine-Valine(ilv) and/or HMBPA Biosynthetic Enzymes  
     [0031] In one embodiment, the present invention features targeting or modifying various biosynthetic enzymes of the pantothenate and/or isoleucine-valine(ilv) and/or HMBPA biosynthetic pathways. In particular, the invention features modifying various enzymatic activities associated with said pathways by modifying or altering the genes encoding said biosynthetic enzymes.  
     [0032] The term “gene”, as used herein, includes a nucleic acid molecule (e.g., a DNA molecule or segment thereof) that, in an organism, can be separated from another gene or other genes, by intergenic DNA (i.e., intervening or spacer DNA which naturally flanks the gene and/or separates genes in the chromosomal DNA of the organism). Alternatively, a gene may slightly overlap another gene (e.g., the 3′ end of a first gene overlapping the 5′ end of a second gene), said overlapping genes separated from other genes by intergenic DNA. A gene may direct synthesis of an enzyme or other protein molecule (e.g., may comprise coding seqeunces, for example, a contiguous open reading frame (ORF) which encodes a protein) or may itself be functional in the organism. A gene in an organism, may be clustered in an operon, as defined herein, said operon being separated from other genes and/or operons by the intergenic DNA. An “isolated gene”, as used herein, includes a gene which is essentially free of sequences which naturally flank the gene in the chromosomal DNA of the organism from which the gene is derived (i.e., is free of adjacent coding sequences which encode a second or distinct protein, adjacent structural sequences or the like) and optionally includes 5′ and 3′ regulatory sequences, for example promoter sequences and/or terminator sequences. In one embodiment, an isolated gene includes predominantly coding sequences for a protein (e.g., sequences which encode Bacillus proteins). In another embodiment, an isolated gene includes coding sequences for a protein (e.g., for a Bacillus protein) and adjacent 5′ and/or 3′ regulatory sequences from the chromosomal DNA of the organism from which the gene is derived (e.g., adjacent 5′ and/or 3′ Bacillus regulatory sequences). Preferably, an isolated gene contains less than about 10 kb, 5 kb, 2 kb, 1 kb, 0.5 kb, 0.2 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences that naturally flank the gene in the chromosomal DNA of the organism from which the gene is derived.  
     [0033] The term “operon” includes at least two adjacent genes or ORFs, optionally overlapping in sequence at either the 5′ or 3′ end of at least one gene or ORF. The term “operon” includes a coordinated unit of gene expression that contains a promoter and possibly a regulatory element associated with one or more adjacent genes or ORFs (e.g., structural genes encoding enzymes, for example, biosynthetic enzymes). Expression of the genes (e.g., structural genes) can be coordinately regulated, for example, by regulatory proteins binding to the regulatory element or by anti-termination of transcription. The genes of an operon (e.g., structural genes) can be transcribed to give a single mRNA that encodes all of the proteins.  
     [0034] A “gene having a mutation” or “mutant gene” as used herein, includes a gene having a nucleotide sequence which includes at least one alteration (e.g., substitution, insertion, deletion) such that the polypeptide or protein encoded by said mutant exhibits an activity that differs from the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene. In one embodiment, a gene having a mutation or mutant gene encodes a polypeptide or protein having an increased activity as compared to the polypeptide or protein encoded by the wild-type gene, for example, when assayed under similar conditions (e.g., assayed in microorganisms cultured at the same temperature). As used herein, an “increased activity” or “increased enzymatic activity” is one that is at least 5% greater than that of the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene, preferably at least 5-10% greater, more preferably at least 10-25% greater and even more preferably at least 25-50%, 50-75% or 75-100% greater than that of the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene. Ranges intermediate to the above-recited values, e.g., 75-85%, 85-90%, 90-95%, are also intended to be encompassed by the present invention. As used herein, an “increased activity” or “increased enzymatic activity” can also include an activity that is at least 1.25-fold greater than the activity of the polypeptide or protein encoded by the wild-type gene, preferably at least 1.5-fold greater, more preferably at least 2-fold greater and even more preferably at least 3-fold, 4-fold, 5-fold, 10-fold, 20fold, 50-fold, 100-fold or greater than the activity of the polypeptide or protein encoded by the wild-type gene.  
     [0035] In another embodiment, a gene having a mutation or mutant gene encodes a polypeptide or protein having a reduced activity as compared to the polypeptide or protein encoded by the wild-type gene, for example, when assayed under similar conditions (e.g., assayed in microorganisms cultured at the same temperature). A mutant gene also can encode no polypeptide or have a reduced level of production of the wild-type polypeptide. As used herein, a “reduced activity” or “reduced enzymatic activity” is one that is at least 5% less than that of the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene, preferably at least 5-10% less, more preferably at least 10-25% less and even more preferably at least 25-50%, 50-75% or 75-100% less than that of the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene. Ranges intermediate to the above-recited values, e.g., 75-85%, 85-90%, 90-95%, are also intended to be encompassed by the present invention. As used herein, a “reduced activity” or “reduced enzymatic activity” can also include an activity that has been deleted or “knocked out” (e.g., approximately 100% less activity than that of the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene).  
     [0036] Activity can be determined according to any well accepted assay for measuring activity of a particular protein of interest. Activity can be measured or assayed directly, for example, measuring an activity of a protein isolated or purified from a cell or mocroorganism. Alternatively, an activity can be measured or assayed within a cell or mocroorganism or in an extracellular medium. For example, assaying for a mutant gene (i.e., said mutant encoding a reduced enzymatic activity) can be accomplished by expressing the mutated gene in a microorganism, for example, a mutant microorganism in which the enzyme is temperature-sensitive, and assaying the mutant gene for the ability to complement a temperature sensitive (Ts) mutant for enzymatic activity. A mutant gene that encodes an “increased enzymatic activity” can be one that complements the Ts mutant more effectively than, for example, a corresponding wild-type gene. A mutant gene that encodes a “reduced enzymatic activity” is one that complements the Ts mutant less effectively than, for example, a corresponding wild-type gene.  
     [0037] It will be appreciated by the skilled artisan that even a single substitution in a nucleic acid or gene sequence (e.g., a base substitution that encodes an amino acid change in the corresponding amino acid sequence) can dramatically affect the activity of an encoded polypeptide or protein as compared to the corresponding wild-type polypeptide or protein. A mutant gene (e.g., encoding a mutant polypeptide or protein), as defined herein, is readily distinguishable from a nucleic acid or gene encoding a protein homologue in that a mutant gene encodes a protein or polypeptide having an altered activity, optionally observable as a different or distinct phenotype in a microorganism expressing said mutant gene or producing said mutant protein or polypeptide (i.e., a mutant microorganism) as compared to a corresponding microorganism expressing the wild-type gene. By contrast, a protein homologue has an identical or substantially similar activity, optionally phenotypically indiscernable when produced in a microorganism, as compared to a corresponding microorganism expressing the wild-type gene. Accordingly it is not, for example, the degree of sequence identity between nucleic acid molecules, genes, protein or polypeptides that serves to distinguish between homologues and mutants, rather it is the activity of the encoded protein or polypeptide that distinguishes between homologues and mutants: homologues having, for example, low (e.g., 30-50% sequence identity) sequence identity yet having substantially equivalent functional activities, and mutants, for example sharing 99% sequence identity yet having dramatically different or altered functional activities.  
     [0038] It will also be appreciated by the skilled artisan that nucleic acid molecules, genes, protein or polypeptides for use in the instant invention can be derived from any microorganisms having a HMBPA biosynthetic pathway, an ilv biosynthetic pathway or a pantothenate biosynthetic pathway. Such nucleic acid molecules, genes, protein or polypeptides can be identified by the skilled artisan using Blown techniques such as homology screening, sequence comparison and the like, and can be modified by the skilled artisan in such a way that expression or production of these nucleic acid molecules, genes, protein or polypeptides occurs in a recombinant microorganism (e.g., by using appropriate promoters, ribosomal binding sites, expression or integration vectors, modifying the sequence of the genes such that the transcription is increased (taking into account the preferable codon usage), etc., according to techniques described herein and those known in the art).  
     [0039] In one embodiment, the genes of the present invention are derived from a Gram positive microorganism organism (e.g., a microorganism which retains basic dye, for example, crystal violet, due to the presence of a Gram-positive wall surrounding the microorganism). The term “derived from” (e.g., “derived from” a Gram positive microorganism) refers to a gene which is naturally found in the microorganism (e.g., is naturally found in a Gram positive microorganism). In a preferred embodiment, the genes of the present invention are derived from a microorganism belonging to a genus selected from the group consisting of Bacillus, Cornyebacterium (e.g.,  Cornyebacterium glutamicum ), Lactobacillus, Lactococci and Streptomyces. In a more preferred embodiment, the genes of the present invention are derived from a microorganism is of the genus Bacillus. In another preferred embodiment, the genes of the present invention are derived from a microorganism selected from the group consisting of  Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus firmus, Bacillus pantothenticus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurans , and other Group 1 Bacillus species, for example, as characterized by 16S rRNA type. In another preferred embodiment, the gene is derived from  Bacillus brevis  or  Bacillus stearothermophilus . In another preferred embodiment, the genes of the present invention are derived from a microorganism selected from the group consisting of  Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis , and  Bacillus pumilus . In a particularly preferred embodiment, the gene is derived from  Bacillus subtilis  (e.g., is  Bacillus subtilis -derived). The term “derived from  Bacillus subtilis ” or “Bacillus&#39;s subtilis-derived” includes a gene which is naturally found in the microorganism  Bacillus subtilis . Included within the scope of the present invention are Bacillus-derived genes (e.g.,  B. subtilis -derived genes), for example, Bacillus or  B. subtilis  coaX genes, serA genes, glyA genes, coaA genes, pan genes and/or ilv genes.  
     [0040] In another embodiment, the genes of the present invention are derived from a Gram negative (excludes basic dye) microorganism. In a preferred embodiment, the genes of the present invention are derived from a microorganism belonging to a genus selected from the group consisting of Salmonella (e.g.,  Salmonella typhimurium ), Escherichia, Klebsiella, Serratia, and Proteus. In a more preferred embodiment, the genes of the present invention are derived from a microorganism of the genus Escherichia. In an even more preferred embodiment, the genes of the present invention are derived from  Escherichia coli.  In another embodiment, the genes of the present invention are derived from Saccharomyces (e.g.,  Saccharomyces cerevisiae ).  
     [0041] II. Recombinant Nucleic Acid Molecules and Vectors  
     [0042] The present invention further features recombinant nucleic acid molecules (e.g., recombinant DNA molecules) that include genes described herein (e.g., isolated genes), preferably Bacillus genes, more preferably  Bacillus subtilis  genes, even more preferably  Bacillus subtilis  pantothenate biosynthetic genes and/or isoleucine-valine (ilv) biosynthetic genes and/or HMBPA biosynthetic genes. The term “recombinant nucleic acid molecule” includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides). Preferably, a recombinant nucleic acid molecule (e.g., a recombinant DNA molecule) includes an isolated gene of the present invention operably linked to regulatory sequences. The phrase “operably liked to regulatory sequence(s)” means that the nucleotide sequence of the gene of interest is linked to the regulatory sequence(s) in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, attenuated, decreased or repressed expression) of the gene, preferably expression of a gene product encoded by the gene (e.g., when the recombinant nucleic acid molecule is included in a recombinant vector, as defined herein, and is introduced into a microorganism).  
     [0043] The term “regulatory sequence” includes nucleic acid sequences which affect (e.g., modulate or regulate) expression of other nucleic acid sequences (i.e., genes). In one embodiment, a regulatory sequence is included in a recombinant nucleic acid molecule in a similar or identical position and/or orientation relative to a particular gene of interest as is observed for the regulatory sequence and gene of interest as it appears in nature, e.g., in a native position and/or orientation. For example, a gene of interest can be included in a recombinant nucleic acid molecule operably linked to a regulatory sequence which accompanies or is adjacent to the gene of interest in the natural organism (e.g., operably linked to “native” regulatory sequences (e.g., to the “native” promoter). Alternatively, a gene of interest can be included in a recombinant nucleic acid molecule operably linked to a regulatory sequence which accompanies or is adjacent to another (e.g., a different) gene in the natural organism. Alternatively, a gene of interest can be included in a recombinant nucleic acid molecule operably linked to a regulatory sequence from another organism. For example, regulatory sequences from other microbes (e.g., other bacterial regulatory sequences, bacteriophage regulatory sequences and the like) can be operably linked to a particular gene of interest.  
     [0044] In one embodiment, a regulatory sequence is a non-native or non-naturally-occurring sequence (e.g., a sequence which has been modified, mutated, substituted, derivatized, deleted including sequences which are chemically synthesized). Preferred regulatory sequences include promoters, enhancers, termination signals, anti-termination signals and other expression control elements (e.g., sequences to which repressors or inducers bind and/or binding sites for transcriptional and/or translational regulatory proteins, for example, in the transcribed mRNA). Such regulatory sequences are described, for example, in Sambrook, J., Fritsh, E. F., and Maniatis, T.  Molecular Cloning: A Laboratory Manual.  2 nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in a microorganism (e.g., constitutive promoters and strong constitutive promoters), those which direct inducible expression of a nucleotide sequence in a microorganism (e.g., inducible promoters, for example, xylose inducible promoters) and those which attenuate or repress expression of a nucleotide sequence in a microorganism (e.g., attenuation signals or repressor sequences). It is also within the scope of the present invention to regulate expression of a gene of interest by removing or deleting regulatory sequences. For example, sequences involved in the negative regulation of transcription can be removed such that expression of a gene of interest is enhanced.  
     [0045] In one embodiment, a recombinant nucleic acid molecule of the present invention includes a nucleic acid sequence or gene that encode at least one bacterial gene product (e.g., a pantothenate biosynthetic enzyme, an isoleucine-valine biosynthetic enzyme and/or a HMBPA biosynthetic enzyme) operably linked to a promoter or promoter sequence. Preferred promoters of the present invention include Bacillus promoters and/or bacteriophage promoters (e.g., bacteriophage which infect Bacillus). In one embodiment, a promoter is a Bacillus promoter, preferably a strong Bacillus promoter (e.g., a promoter associated with a biochemical housekeeping gene in Bacillus or a promoter associated with a glycolytic pathway gene in Bacillus). In another embodiment, a promoter is a bacteriophage promoter. In a preferred embodiment, the promoter is from the bacteriophage SP01. In a particularly preferred embodiment, a promoter is selected from the group consisting of P 15 , P 26  or P veg , having for example, the following respective seqeunces: GCTATTGACGACAGCTATGGTTCACTGTCCACCAACCAAAACTGTGCTCAGT ACCGCCAATATTTCTCCCTTGAGGGGTACAAAGAGGTGTCCCTAGAAGAGAT CCACGCTGTGTAAAAATTTTACAAAAAGGTATTGACTTTCCCTACAGGGTGT GTAATAATTTAATTACAGGCGGGGGCAACCCCGCCTGT(SEQ ID NO:1), GCCTACCTAGCTTCCAAGAAAGATATCCTAACAGCACAAGAGCGGAAAGAT GTTTTGTTCTACATCCAGAACAACCTCTGCTAAAATTCCTGAAAAATTTTGCA AAAAGTTGTTGACTTTATCTACAAGGTGTGGTATAATAATCTTAACAACAGC AGGACGC (SEQ ID NO:2), and GAGGAATCATAGAATTTTGTCAAAATAATTTTATTGACAACGTCTTATTAAC GTTGATATAATTTAAATTTTATTTGACAAAAATGGGCTCGTGTTGTACAATA AATGTAGTGAGGTGGATGCAATG (SEQ ID NO:3). Additional preferred promoters include tef (the translational elongation factor (TEF) promoter) and pyc (the pyruvate carboxylase (PYC) promoter), which promote high level expression in Bacillus (e.g.,  Bacillus subtilis ). Additional preferred promoters, for example, for use in Gram positive microorganisms include, but are not limited to, amy and SPO2 promoters. Additional preferred promoters, for example, for use in Gram negative microorganisms include, but are not limited to, cos, tac, trp, tei, trp-tet, lpp, lac, lpp-lac, lacIQ, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL.  
     [0046] In another embodiment, a recombinant nucleic acid molecule of the present invention includes a terminator sequence or terminator sequences (e.g., transcription terminator sequences). The term “terminator sequences” includes regulatory sequences that serve to terminate transcription of mRNA. Terminator sequences (or tandem transcription terminators) can further serve to stabilize mRNA (e.g., by adding structure to mRNA), for example, against nucleases.  
     [0047] In yet another embodiment, a recombinant nucleic acid molecule of the present invention includes sequences which allow for detection of the vector containing said sequences (i.e., detectable and/or selectable markers), for example, genes that encode antibiotic resistance or sequences that overcome auxotrophic mutations, for example, trpC, fluorescent markers, drug markers, and/or calorimetric markers (e.g., lacZ/β-galactosidase). In yet another embodiment, a recombinant nucleic acid molecule of the present invention includes an artificial ribosome binding site (RBS) or a sequence that becomes transcribed into an artificial RBS. The term “artificial ribosome binding site (RBS)” includes a site within an mRNA molecule (e.g., coded within DNA) to which a ribosome binds (e.g., to initiate translation) which differs from a native RBS (e.g., a RBS found in a naturally-occurring gene) by at least one nucleotide. Preferred artificial RBSs include about 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28, 29-30 or more nucleotides of which about 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-15 or more differ from the native RBS (e.g., the native RBS of a gene of interest, for example, the native panB RBS TAAACATGAGGAGGAGAAAACATG (SEQ ID NO:4) or the native panD RBS ATTCGAGAAATGGAGAGAATATAATATG (SEQ ID NO:5)).  
     [0048] Preferably, nucleotides that differ are substituted such that they are identical to one or more nucleotides of an ideal RBS when optimally aligned for comparisons. Ideal RBSs include, but are not limited to, AGAAAGGAGGTGA (SEQ ID NO:6), TTAAGAAAGGAGGTGANNNNATG (SEQ ID NO:7), TTAGAAAGGAGGTGANNNNNATG (SEQ ID NO:8), AGAAAGGAGGTGANNNNNNNATG (SEQ ID NO:9), and AGAAAGGAGGTGANNNNNNATG (SEQ ID NO:10). Artificial RBSs can be used to replace the naturally-occurring or native RBSs associated with a particular gene. Artificial RBSs preferably increase translation of a particular gene. Preferred artificial RBSs (e.g., RBSs for increasing the translation of panB, for example, of  B. subtilis  panB) include CCCTCTAGAAGGAGGAGAAAACATG (SEQ ID NO:11) and CCCTCTAGAGGAGGAGAAAACATG (SEQ ID NO:12). Preferred artificial RBSs (e.g., RBSs for increasing the translation of panD, for example, of  B. subtilis  panD) include TTAGAAAGGAGGATTTAAATATG (SEQ ID NO:13), TTAGAAAGGAGGTTTAATTAATG (SEQ ID NO:14), TTAGAAAGGAGGTGATTTAAATG (SEQ ID NO:15), TTAGAAAGGAGGTGTTTAAAATG (SEQ ID NO:16), ATTCGAGAAAGGAGG TGAATATAATATG (SEQ ID NO:17), ATTCGAGAAAGGAGGTGAATAATAATG (SEQ ID NO:18), and ATTCGTAGAAAGGAGGTGAATTAATATG (SEQ ID NO:19).  
     [0049] The present invention further features vectors (e.g., recombinant vectors) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules comprising said genes) as described herein. The term “recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived. Preferably, the recombinant vector includes a biosynythetic enzyme-encoding gene or recombinant nucleic acid molecule including said gene, operably linked to regulatory sequences, for example, promoter sequences, terminator sequences and/or artificial ribosome binding sites (RBSs), as defined herein. In another embodiment, a recombinant vector of the present invention includes sequences that enhance replication in bacteria (e.g., replication-enhancing sequences). In one embodiment, replication-enhancing sequences are derived from  E. coli . In another embodiment, replication-enhancing sequences are derived from pBR322.  
     [0050] In yet another embodiment, a recombinant vector of the present invention includes antibiotic resistance sequences. The term “antibiotic resistance sequences” includes sequences which promote or confer resistance to antibiotics on the host organism (e.g., Bacillus). In one embodiment, the antibiotic resistance sequences are selected from the group consisting of cat (chloramphenicol resistance) sequences, tet (tetracycline resistance) sequences, erm (erythromycin resistance) sequences, neo (neomycin resistance) sequences, kan (kanamycin resistance) and spec (spectinomycin resistance) sequences. Recombinant vectors of the present invention can further include homologous recombination sequences (e.g., sequences designed to allow recombination of the gene of interest into the chromosome of the host organism). For example, bpr, vpr, and/or amyE sequences can be used as homology targets for recombination into the host chromosome. It will further be appreciated by one of skill in the art that the design of a vector can be tailored depending on such factors as the choice of microorganism to be genetically engineered, the level of expression of gene product desired and the like.  
     [0051] IV. Recombinant Microorganisms  
     [0052] The present invention further features microorganisms, i.e., recombinant microorganisms, that include vectors or genes (e.g., wild-type and/or mutated genes) as described herein. As used herein, the term “recombinant microorganism” includes a microorganism (e.g., bacteria, yeast cell, fungal cell, etc.) that has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype and/or phenotype (e.g., when the genetic modification affects coding nucleic acid sequences of the microorganism) as compared to the naturally-occurring microorganism from which it was derived.  
     [0053] In one embodiment, a recombinant microorganism of the present invention is a Gram positive organism (e.g., a microorganism which retains basic dye, for example, crystal violet, due to the presence of a Gram-positive wall surrounding the microorganism). In a preferred embodiment, the recombinant microorganism is a microorganism belonging to a genus selected from the group consisting of Bacillus, Cornyebacterium, Lactobacillus, Lactococci and Streptomyces. In a more preferred embodiment, the recombinant microorganism is of the genus Bacillus. In another preferred embodiment, the recombinant microorganism is selected from the group consisting of  Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus firmus, Bacillus pantothenticus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurans , and other Group 1 Bacillus species, for example, as characterized by 16S rRNA type. In another preferred embodiment, the recombinant microorganism is  Bacillus brevis  or  Bacillus stearothermophilis . In another preferred embodiment, the recombinant microorganism is selected from the group consisting of  Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis , and  Bacillus pumilus.    
     [0054] In another embodiment, the recombinant microorganism is a Gram negative (excludes basic dye) organism. In a preferred embodiment, the recombinant microorganism is a microorganism belonging to a genus selected from the group consisting of Salmonella, Escherichia, Klebsiella, Serratia, and Proteus. In a more preferred embodiment, the recombinant microorganism is of the genus Escherichia. In an even more preferred embodiment, the recombinant microorganism is  Escherichia coli . In another embodiment, the recombinant microorganism is Saccharomyces (e.g.,  S. cerevisiae ).  
     [0055] A preferred “recombinant” microorganism of the present invention is a microorganism having a deregulated pantothenate biosynthesis pathway or enzyme, a deregulated isoleucine-valine (ilv) biosynthetic pathway or enzyme and/or a deregulated HMBPA biosynthetic pathway or enzyme. The term “deregulated” or “deregulation” includes the alteration or modification of at least one gene in a microorganism that encodes an enzyme in a biosynthetic pathway, such that the level or activity of the biosynthetic enzyme in the microorganism is altered or modified. Preferably, at least one gene that encodes an enzyme in a biosynthetic pathway is altered or modified such that the gene product is enhanced or increased. The phrase “deregulated pathway” can also include a biosynthetic pathway in which more than one gene that encodes an enzyme in a biosynthetic pathway is altered or modified such that the level or activity of more than one biosynthetic enzyme is altered or modified. The ability to “deregulate” a pathway (e.g., to simultaneously deregulate more than one gene in a given biosynthetic pathway) in a microorganism in some cases arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed an “operon” (defined herein). Due to the coordinated regulation of genes included in an operon, alteration or modification of the single promoter and/or regulatory element can result in alteration or modification of the expression of each gene product encoded by the operon. Alteration or modification of the regulatory element can include, but is not limited to removing the endogenous promoter and/or regulatory element(s), adding strong promoters, inducible promoters or multiple promoters or removing regulatory sequences such that expression of the gene products is modified, modifying the chromosomal location of the operon, altering nucleic acid sequences adjacent to the operon or within the operon such as a ribosome binding site, increasing the copy number of the operon, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of the operon and/or translation of the gene products of the operon, or any other conventional means of deregulating expression of genes routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). Deregulation can also involve altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity.  
     [0056] In another preferred embodiment, a recombinant microorganism is designed or engineered such that at least one pantothenate biosynthetic enzyme, at least one isoleucine-valine biosynthetic enzyme, and/or at least one HMBPA biosynthetic enzyme is overexpressed. The term “overexpressed” or “overexpression” includes expression of a gene product (e.g., a biosynthetic enzyme) at a level greater than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. In one embodiment, the microorganism can be genetically designed or engineered to overexpress a level of gene product greater than that expressed in a comparable microorganism which has not been engineered.  
     [0057] Genetic engineering can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). Genetic engineering can also include deletion of a gene, for example, to block a pathway or to remove a repressor. In embodiments featuring microorganisms having deleted genes, the skilled artisan will appreciate that at least low levels of certain compounds may be required to be present in or added to the culture medium in order that the viability of the microorganism is not compromised. Often, such low levels are present in complex culture media as routinely formulated. Moreover, in processes featuring culturing microorganisms having deleted genes cultured under conditions such that commercially or industrially attractive quantities of product are produced, it may be necessary to supplement culture media with slightly increased levels of certain compounds. For example, in processes featuring culturing a microorganism having a deleted panB gene, at least low levels of pantothenate must be present in the media, e.g., levels such as those found in routinely formulated complex media, whereas slightly increased levels of pantothenate may be added to the media in order to produce commercially or industrially attractive amounts of, for example, HMBPA. For example, 10-30 mg/L pantothenate can be added to the media in order to produce commercially or industrially attractive amounts of HMBPA.  
     [0058] In another embodiment, the microorganism can be physically or environmentally manipulated to overexpress a level of gene product greater than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. For example, a microorganism can be treated with or cultured in the presence of an agent known or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.  
     [0059] V. Culturing and Fermenting Recombinant Microorganisms  
     [0060] The term “culturing” includes maintaining and/or growing a living microorganism of the present invention (e.g., maintaining and/or growing a culture or strain). In one embodiment, a microorganism of the invention is cultured in liquid media. In another embodiment, a microorganism of the invention is cultured in solid media or semi-solid media. In a preferred embodiment, a microorganism of the invention is cultured in media (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism (e.g., carbon sources or carbon substrate, for example carbohydrate, hydrocarbons, oils, fats, fatty acids, organic acids, and alcohols; nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, phosphoric acid, sodium and potassium salts thereof, trace elements, for example, magnesium, iron, manganese, calcium, copper, zinc, boron, molybdenum, and/or cobalt salts; as well as growth factors such as amino acids, vitamins, growth promoters and the like).  
     [0061] Preferably, microorganisms of the present invention are cultured under controlled pH. The term “controlled pH” includes any pH which results in production of the desired product (e.g., HMBPA). In one embodiment microorganisms are cultured at a pH of about 7. In another embodiment, microorganisms are cultured at a pH of between 6.0 and 8.5. The desired pH may be maintained by any number of methods known to those skilled in the art.  
     [0062] Also preferably, microorganisms of the present invention are cultured under controlled aeration. The term “controlled aeration” includes sufficient aeration (e.g., oxygen) to result in production of the desired product (e.g., HMBPA). In one embodiment, aeration is controlled by regulating oxygen levels in the culture, for example, by regulating the amount of oxygen dissolved in culture media. Preferably, aeration of the culture is controlled by agitating the culture. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the cuture vessel (e.g., tube or flask) or by various pumping equipment. Aeration may be further controlled by the passage of sterile air or oxygen through the medium (e.g., through the fermentation mixture). Also preferably, microorganisms of the present invention are cultured without excess foaming (e.g., via addition of antifoaming agents).  
     [0063] Moreover, microorganisms of the present invention can be cultured under controlled temperatures. The term “controlled temperature” includes any temperature which results in production of the desired product (e.g., HMBPA). In one embodiment, controlled temperatures include temperatures between 15° C. and 95° C. In another embodiment, controlled temperatures include temperatures between 15° C. and 70° C. Preferred temperatures are between 20° C. and 55° C., more preferably between 30° C. and 50° C.  
     [0064] Microorganisms can be cultured (e.g., maintained and/or grown) in liquid media and preferably are cultured, either continuously or intermittently, by conventional culturing methods such as standing culture, test tube culture, shaking culture (e.g., rotary shaking culture, shake flask culture, etc.), aeration spinner culture, or fermentation. In a preferred embodiment, the microorganisms are cultured in shake flasks. In a more preferred embodiment, the microorganisms are cultured in a fermentor (e.g., a fermentation process). Fermentation processes of the present invention include, but are not limited to, batch, fed-batch and continuous processes or methods of fermentation. The phrase “batch process” or “batch fermentation” refers to a closed system in which the composition of media, nutrients, supplemental additives and the like is set at the beginning of the fermentation and not subject to alteration during the fermentation, however, attempts may be made to control such factors as pH and oxygen concentration to prevent excess media acidification and/or microorganism death. The phrase “fed-batch process” or “fed-batch” fermentation refers to a batch fermentation with the exception that one or more substrates or supplements are added (e.g., added in increments or continuously) as the fermentation progresses. The phrase “continuous process” or “continuous fermentation” refers to a system in which a defined fermentation media is added continuously to a fermentor and an equal amount of used or “conditioned” media is simultaneously removed, preferably for recovery of the desired product (e.g., HMBPA). A variety of such processes have been developed and are well-known in the art.  
     [0065] The phrase “culturing under conditions such that a desired compound is produced” includes maintaining and/or growing microorganisms under conditions (e.g., temperature, pressure, pH, duration, etc.) appropriate or sufficient to obtain production of the desired compound or to obtain desired yields of the particular compound being produced. For example, culturing is continued for a time sufficient to produce the desired amount of a compound (e.g., HMBPA). Preferably, culturing is continued for a time sufficient to substantially reach suitable production of the compound (e.g., a time sufficient to reach a suitable concentration of HMBPA or suitable ratio of HMBPA:pantothenate). In one embodiment, culturing is continued for about 12 to 24 hours. In another embodiment, culturing is continued for about 24 to 36 hours, 36 to 48 hours, 48 to 72 hours, 72 to 96 hours, 96 to 120 hours, 120 to 144 hours, or greater than 144 hours. In yet another embodiment, microorganisms are cultured under conditions such that at least about 5 to 10 g/L of compound are produced in about 36 hours, at least about 10 to 20 g/L compound are produced in about 48 hours, or at least about 20 to 30 g/L compound in about 72 hours. In yet another embodiment, microorganisms are cultured under conditions such that at least a ratio of HMBPA:HMBPA+pantothenate of 1:10 is achieved (i.e., 10% HMBPA versus 90% pantothenate, for example, as determined by comparing the peaks when a sample of product is analyzed be HPLC), preferably such that at least a ratio of 2:10 is achieved (20% HMBPA versus 90% pantotheante), more preferably such that a ratio of at least 2.5:10 is achieved (25% HMBPA versus 75% pantotheante), more preferably at least 3:10 (30% HMBPA versus 70% pantotheante), 4:10 (40% HMBPA versus 60% pantotheante), 5:10 (50% HMBPA versus 50% pantotheante), 6:10 (60% HMBPA versus 40% pantotheante), 7:10 (70% HMBPA versus 30% pantotheante), 8:10 (80% HMBPA versus 20% pantotheante), 9:10 (90% HMBPA versus 10% pantotheante) or greater.  
     [0066] The methodology of the present invention can further include a step of recovering a desired compound (e.g., HMBPA). The term “recovering” a desired compound includes extracting, harvesting, isolating or purifying the compound from culture media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non/ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. For example, a compound can be recovered from culture media by first removing the microorganisms from the culture. Media are then passed through or over a cation exchange resin to remove cations and then through or over an anion exchange resin to remove inorganic anions and organic acids having stronger acidities than the compound of interest. The resulting compound can subsequently be converted to a salt (e.g., a calcium salt) as described herein.  
     [0067] Preferably, a desired compound of the present invention is “extracted”, “isolated” or “purified” such that the resulting preparation is substantially free of other media components (e.g., free of media components and/or fermentation byproducts). The language “substantially free of other media components” includes preparations of the desired compound in which the compound is separated from media components or fermentation byproducts of the culture from which it is produced. In one embodiment, the preparation has greater than about 80% (by dry weight) of the desired compound (e.g., less than about 20% of other media components or fermentation byproducts), more preferably greater than about 90% of the desired compound (e.g., less than about 10% of other media components or fermentation byproducts), still more preferably greater than about 95% of the desired compound (e.g., less than about 5% of other media components or fermentation byproducts), and most preferably greater than about 98-99% desired compound (e.g., less than about 1-2% other media components or fermentation byproducts). When the desired compound has been derivatized to a salt, the compound is preferably further free of chemical contaminants associated with the formation of the salt. When the desired compound has been derivatized to an alcohol, the compound is preferably further free of chemical contaminants associated with the formation of the alcohol.  
     [0068] In an alternative embodiment, the desired compound is not purified from the microorganism, for example, when the microorganism is biologically non-hazardous (e.g., safe). For example, the entire culture (or culture supernatant) can be used as a source of product (e.g., crude product). In one embodiment, the culture (or culture supernatant) is used without modification. In another embodiment, the culture (or culture supernatant) is concentrated. In yet another embodiment, the culture (or culture supernatant) is dried or lyophilized.  
     [0069] Preferably, a production method of the present invention results in production of the desired compound at a significantly high yield. The phrase “significantly high yield” includes a level of production or yield which is sufficiently elevated or above what is usual for comparable production methods, for example, which is elevated to a level sufficient for commercial production of the desired product (e.g., production of the product at a commercially feasible cost). In one embodiment, the invention features a production method that includes culturing a recombinant microorganism under conditions such that the desired product (e.g., HMBPA) is produced at a level greater than 2 g/L. In another embodiment, the invention features a production method that includes culturing a recombinant microorganism under conditions such that the desired product (e.g., HMBPA) is produced at a level greater than 10 g/L. In another embodiment, the invention features a production method that includes culturing a recombinant microorganism under conditions such that the desired product (e.g., HMBPA) is produced at a level greater than 20 g/L. In yet another embodiment, the invention features a production method that includes culturing a recombinant microorganism under conditions such that the desired product (e.g., HMBPA) is produced at a level greater than 30 g/L. In yet another embodiment, the invention features a production method that includes culturing a recombinant microorganism under conditions such that the desired product (e.g., HMBPA) is produced at a level greater than 40 g/L. The invention further features a production method for producing the desired compound that involves culturing a recombinant microorganism under conditions such that a sufficiently elevated level of compound is produced within a commercially desirable period of time.  
     [0070] Depending on the biosynthetic enzyme or combination of biosynthetic enzymes manipulated, it may be desirable or necessary to provide (e.g., feed) microorganisms of the present invention at least one biosynthetic precursor such that the desired compound or compounds are produced. The term “biosynthetic precursor” or “precursor” includes an agent or compound which, when provided to, brought into contact with, or included in the culture medium of a microorganism, serves to enhance or increase biosynthesis of the desired product. In one embodiment, the biosynthetic precursor or precursor is aspartate. In another embodiment, the biosynthetic precursor or precursor is β-alanine. The amount of aspartate or β-alanine added is preferably an amount that results in a concentration in the culture medium sufficient to enhance productivity of the microorganism (e.g., a concentration sufficient to enhance production of HMBPA. The term “excess β-alanine” includes β-alanine levels increased or higher that those routinely utilized for culturing the microorganism in question. For example, culturing the Bacillus microorganisms described in the instant Examples is routinely done in the presence of about 0-5 g/L β-alanine. Accordingly, excess β-alanine levels can include levels of about 5-10 g/L or more preferably about 5-20 g/L β-alanine. Biosynthetic precursors of the present invention can be added in the form of a concentrated solution or suspension (e.g., in a suitable solvent such as water or buffer) or in the form of a solid (e.g., in the form of a powder). Moreover, biosynthetic precursors of the present invention can be added as a single aliquot, continuously or intermittently over a given period of time.  
     [0071] In yet another embodiment, the biosynthetic precursor is valine. In yet another embodiment, the biosynthetic precursor is α-ketoisovalerate. Preferably, valine or α-ketoisovalerate is added in an amount that results in a concentration in the medium sufficient for production of the desired product (e.g., HMBPA) to occur. The term “excess α-KIV” includes α-KIV levels increased or higher that those routinely utilized for culturing the microorganism in question. For example, culturing the Bacillus microorganisms described in the instant Examples can be done in the presence of about 0-5 g/L α-KIV. Accordingly, excess α-KIV levels can include levels of about 5-10 g/L, and more preferably about 5-20 g/L. The term “excess valine” includes valine levels increased or higher that those routinely utilized for culturing the microorganism in question. For example, culturing the Bacillus microorganisms described in the instant Examples is routinely done in the presence of about 0-0.5 g/L valine. Accordingly, excess valine levels can include levels of about 0.5-5 g/L, preferably about 5-20 g/L valine. Biosynthetic precursors are also referred to herein as “supplemental biosynthetic substrates”.  
     [0072] Moreover, certain aspects of the present invention include culturing microorganisms (e.g., recombinant microorganisms) under conditions of increased steady state glucose, decreased steady state dissolved oxygen and/or decreased serine. The term “increased steady state glucose” includes steady state glucose levels increased or higher that those routinely utilized for culturing the microorganism in question. For example, culturing the Bacillus microorganisms described in the instant Examples is routinely done in the presence of about 0.2-1.0 g/L steady state glucose. Accordingly, increased steady state glucose levels can include levels of about 1-2 g/l, about 2-5 g/l, and preferably about 5-20 g/L steady state glucose. The term “decreased steady state dissolved oxygen” includes steady state dissolved oxygen levels less or lower that those routinely utilized for culturing the microorganism in question and, for example, inversely correlates with increased steady state glucose levels. For example, culturing the Bacillus microorganisms described in the instant Examples is routinely done in the presence of about 10-30% dissolved oxygen. Accordingly, decreased steady state dissolved oxygen can include levels of about 0-10%, and preferably about 0-5% steady state dissolved oxygen. The term “reduced serine” includes serine levels within the lower range of those routinely utilized for culturing the microorganism in question. For example, culturing the Bacillus microorganisms described in the instant Examples is routinely done in the presence of about 0-0.5 g/L serine. Accordingly, reduced serine levels can include, for example, levels of 0-0.1 g/L serine.  
     [0073] Another aspect of the present invention includes biotransformation processes which feature the recombinant microorganisms described herein. The term “biotransformation process”, also referred to herein as “bioconversion processes”, includes biological processes which results in the production (e.g., transformation or conversion) of appropriate substrates and/or intermediate compounds into a desired product (e.g., HMBPA).  
     [0074] In one embodiment, the invention features a biotransformation process for the production of HMBPA comprising contacting a microorganism which overexpresses a reductase (e.g., overexpresses PanE1, PanE2 and/or IlvC) with appropriate substrates or precursors under conditions such that HMBPA is produced and recovering said HMBPA. In another embodiment, the invention features a biotransformation process for the production of HMBPA comprising contacting a microorganism which has a reduced or deleted PanB activity with appropriate substrates or precursors under conditions such that HMBPA is produced and recovering said HMBPA. In yet another embodiment, the invention features a biotransformation process for the production of HMBPA comprising contacting a microorganism which overexpresses at least one reductase and has a reduced or deleted PanB activity with appropriate substrates or precursors under conditions such that HMBPA is produced and recovering said HMBPA. Preferred recombinant microorganisms for carrying out the above-described biotransformations include the recombinant microorganisms described herein. In yet another embodiment, the invention features a biotransformation reaction that includes contacting αHIV and β-alanine with isolated or purified PanC under conditions such that HMBPA is produced. α-HIV can optionally be obtained by contacting α-KIV with purified or isolated reductase (e.g., PanE1, PanE2 and/or IlvC) and a source of reducing equivalent, for example, NADH. Conditions under which α-HIV or HMBPA are produced can include any conditions which result in the desired production of α-HIV or HMBPA, respectively. In yet another embodiment, the present invention includes a method of producing α-HIV that includes culturing a microorganism that overexpresses PanE1 and/or PanE2, and/or IlvC and has a reduced or deleted PanC or PanD (to reduce HMBPA or β-alanine sunthesis, respectively) under conditions such that α-HIV is produced.  
     [0075] The microorganism(s) and/or enzymes used in the biotransformation reactions are in a form allowing them to perform their intended function (e.g., producing a desired compound). The microorganisms can be whole cells, or can be only those portions of the cells necessary to obtain the desired end result. The microorganisms can be suspended (e.g., in an appropriate solution such as buffered solutions or media), rinsed (e.g., rinsed free of media from culturing the microorganism), acetone-dried, immobilized (e.g., with polyacrylamide gel or k-carrageenan or on synthetic supports, for example, beads, matrices and the like), fixed, cross-linked or permeablized (e.g., have permeablized membranes and/or walls such that compounds, for example, substrates, intermediates or products can more easily pass through said membrane or wall).  
     [0076] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.  
     EXAMPLES  
     Example I  
     Discovery and Characterization of the [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) Biosynthetic Pathway  
     [0077] In developing Bacillus strains for the production of pantothenate, various genetic manipulations were made to enzymes involved in the pantothenate biosynthetic pathway and the isoleucine-valine (ilv) pathway (FIG. 1) as described in U.S. patent application Ser. No. 09/400,494 and U.S. patent application Ser. No. 09/667,569. For example, strains having a deregulated panBCD operon and/or having deregulated panE1 exhibited enhanced pantothenate production (when cultured in the presence of β-alanine and α-KIV). Strains further deregulated for ilvBNC and ilvD exhibited enhanced pantothenate production in the presence of only β-alanine. Moreover, it was possible to achieve β-alanine independence by further deregulating panD.  
     [0078] An exemplary strain is PA824, a tryptophan prototroph, Spec and Tet resistant, deregulated for panBCD at the panBCD locus, deregulated for panE1 at the panE1 locus (two genes in the  B. subtilis  genome are homologous to  E. coli  panE, panE1 and panE2, the former encoding the major ketopantoate reductase involved in pantothenate production, while panE2 does not contribute to pantothenate synthesis (U.S. patent application Ser. No. 09/400,494), deregulated for ilvD at the ilvD locus, overexpressing an ilvBNC cassette at the amyE locus, and overexpressing panD at the bpr locus.  
     [0079] The production of pantothenic acid by PA824 was investigated in 14 L fermentor vessels. The composition of the batch and feed media are as follows.  
                              BATCH                             MATERIAL   g/L (final)                                     1   Yeast extract   10       2   Na Glutamate   5       3   (NH 4 ) 2 SO 4     8       4   KH 2 PO 4     5       5   K 2 HPO 4     7.6                  
 
     [0080] Addded After Sterilization and Cool Down  
                                              1   Glucose   2.5           2   CaCl 2     0.1           3   MgCl 2     1           4   Sodium Citrate   1           5   FeSO 4 .7 H 2 O   0.01           5   SM-1000X   1   ml                  
 
     [0081] The final volume of the batch medium is 6 L. The trace element solution Sm-1000X has following composition: 0.15 g Na 2 MoO 4 .2H 2 O, 2.5 g H 3 BO 3 , 0.7 g CoCl 2 .6 H 2 O, 0.25 g CuSO 4 .5H 2 O, 1.6 g MnCl 2 .4H 2 O, 0.3 g ZnSO 4 .7H 2 O are dissolved in water final volume 1L).  
     [0082] The batch medium was inoculated with 60 ml of shake flask PA824 culture (OD=10 in SVY medium: Difco Veal Infusion broth 25 g, Difco Yeast extract 5 g, Sodium Glutamate 5 g, (NH 4 ) 2 SO 4  2.7 g in 740 ml H 2 O, autoclave; add 200 ml sterile 1 M K 2 HPO 4  (pH 7) and 60 ml sterile 50% Glucose solution (final volume 1L)). The fermentation was run at 43° C. at an air flow rate of 12 L/min as a glucose limited fed batch. The initial batched glucose (2.5 g/L) was consumed during exponential growth). Afterwards glucose concentrations were maintained between 0.2-1 g/L by continuous feeding of FEED solution as follows.  
                              FEED                             MATERIAL   g/L (final)                                         1   Glucose   550           2   CaCl 2     0.1           3   SM-1000X   3   ml                  
 
     [0083] The variable feed rate pump was computer controlled and linked to the glucose concentration in the tank by an algorithm. In this example the total feeding was 6 L.  
     [0084] During fermentation the pH was set at 7.2. Control was achieved by pH measurements linked to computer control. The pH value was maintained by feeding either a 5% NH 3 -solution or a 20% H 3 PO 4 -solution. NH 3  acts simultaneousely as a N-source for the fermentation. The dissolved oxygen concentration [pO 2 ] was set at 30% by regulation of the agitation and aeration rate. Foaming was controlled by addition of silicone oil. After the stop of the addition of the feed solution, in this example after 48 h, the fermentation was continued until the [pO 2 ] value reached 95%. Then the fermentation was stopped by killing the microorganism through sterilization for 30 min. The successful sterilization was proven by plating a sample of the fermentation broth on agar plates. The pantothenate titer in the fermentation broth was 21.7 g/L after sterilization and removal of the cells by centrifugation (determined by HPLC analysis).  
     [0085] For HPLC analysis the fermentation broth sample was diluted with sterile water (1:40). 5 μl of this dilution was injected into a HPLC column (Aqua C18, 5 μm, 150*2.0 mm, Phenomenex™). Temperature of the column was held at 40° C. Mobile phase A was 14.8 mM H 3 PO 3 , mobile phase B 100% Acetonitrile. Flow rate was constant at 0.5 mL/min. A gradient was applied:  
                                                      start:   2% mobile phase B           0-3 min   linear increase to 3% mobile phase B           3-3.5 min   linear increase to 20% mobile phase B                      
 
     [0086] The detection was carried out by an UV-detector (210 nm). Run time was 7 min with an additional 3 min posttime. The retention time for pantothenic acid is 3.9 minutes. The HPLC chromatogram for the above mentioned sample is given in FIG. 4.  
     [0087] Identification of Compound Related to Peak with Retention Time 4.7 Minutes  
     [0088] Under the described fermentation conditions, PA824 routinely yields approximately 20-30 g/L pantothenate. In addition to producing significant quantities of pantothenate, it was discovered a second compound eluted with an approximate retention time of 4.7 minutes in this system. The second prominent product formed in the fermentation was shown to be [R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) (also referred to herein as “β-alanine 2-(R)-iydroxyisolvalerate”, “β-alanine 2-hydroxyisolvalerate”, and/or “β-alanyl-α-hydroxyisovalarate). It was identified by its mass spectrum (FIG. 5; relative monoisotopic mass 189),  1 H- and 13C-NMR (data not shown) after chromatographic purification by reverse phase flash chromatography (mobile phase 10 mM KH 2 PO 4 , with increasing contents of acetonitrile (1-50%)).  
     [0089] In order to verify the identity of the compound, deliberate synthesis of racemic β-alanine 2-hydroxyisolvalerate was performed as follows. β-alanine (2.73 g/30 mmol) and sodium methoxide (5.67 g of a 30% solution in methanol/31.5 mmol) were dissolved in methanol (40 mL). Methyl 2-hydroxyisovalerate (2-hydroxy-3-methylbutyric acid methyl ester) (3.96 g/30 mmol) was added and refluxed for 18 hours. Methanol was then removed by rotavap and replaced by tert-butanol (50 mL). Potassium tert-butoxide was added (50 mg) and refluxed for 26 hours. The solvent was removed in vacuo, the residue dissolved in water (50 mL) and passed through a strongly acidic ion-exchange resin (H+-form Lewatite™ S 100 G1; 100 mL). More water is used to rinse the ion exchanger. The aqueous eluates are combined and the water removed in vacuo. The residue is subjected to flash chromatography (silica gel; 2% acetic acid in ethyl acetate as eluent) and the product fractions evaporated to give a solid residue. The residue was recrystallized from ethyl acetate/toluene (10 mL /20 mL, respectively) and analytically pure HMBPA (β-alanine 2-hydroxyisolvalerate) was obtained, which showed a relative monoisotopic mass of 190 (189+H + ) in the mass spec and the same  1 H-NMR resonances as the product obtained from fermentation.  
     [0090] The biosynthetic pathway resulting in HMBPA production is set forth in FIG. 2. The chemical structure of [R]-3-(2-hydroxy-3-methyl-butyrylamino)propionic acid (HMBPA) is depicted in FIG. 3. As depicted in FIG. 2, HMBPA is the condensation product of α-hydroxyisovaleric acid (α-HIV) and β-alanine, catalyzed by the PanC enzyme. α-HIV is generated by reduction of α-KIV, a reaction which is catalyzed by the reductases PanE (e.g., PanE1 and/or PanE2) and/or IlvC.  
     [0091] Based on the chemical structure and biosynthetic pathway leading to HMBPA production, the present inventors formulated the following model to describe the interaction between the-previously known pantothenate and isoleucine-valine (ilv) pathways and the newly characterized HMBPA biosynthetic pathway. In at least one aspect, the model states that there exist at least two pathways in microorganisms that compete for α-KIV, the substrate for the biosynthetic enzyme PanB, namely the pantothenate biosynthetic pathway and the HMBPA biosynthetic pathway. (A third and fourth pathway competing for α-KIV are those resulting in the production of valine or leucine from α-KIV, see e.g., FIG. 1). At least the pantothenate biosynthetic pathway and the HMBPA biosynthetic pathway further produce competitive substrates for the enzyme PanC, namely α-HIV and pantoate. The model predicts that reducing PanB activity will increase α-KIV availability for α-HIV synthesis (and ultimately, HMBPA synthesis) and decrease the amount of pantoate and/or pantothenate synthesized by a microorganism. Conversely, increasing PanB activity will increase pantoate and ketopantoate availability for pantoate/pantothenate synthesis. The following examples provide experimental support for the model and further exemplify processes for increasing the production of HMBPA based on the model.  
     Examples II-VI  
     [0092] For Examples II-VI, quanitation of pantothenate and/or HMBPA was performed as follows. Aliquots of fermentation media were diluted 1:100 and aliquots of test tube cultures were diluted 1:10 in water or 5% acetonitrile prior to injection on a Phenomenex Aqua™ 5μ C18 HPLC column (250×4.60 mm, 125A). Mobile phases were A=5% acetonitrile, 50 mM monosodium phosphate buffer adjusted to pH 2.5 with phosphoric acid; and B=95% acetonitrile, 5% H 2 O.  
     [0093] Linear gradients were as follows.  
                                       Minutes   Solvent A   Solvent B                                            0   100%   0%       16   100%   0%       17   0%   100%       20   0%   100%       21   100%   0%                  
 
     [0094] Additional parameters and apparatus were as follows: Flow rate=1.0 ml/min; Injection volume=20 μl; Detector=Hewlett Packard 1090 series DAD UV detector-3014, Signal A=197 nm, ref.=450 nm, Firmware revision E; Column heater=Oven tempature 40° C.; Hardware=Hewlett Packard Kayak™ XA; and Software=Hewlett Packard Chemstation Plus™ family revision A.06.03[509].  
     [0095] Under these fermentation conditions, PA824 routinely yields approximately 30-40 g/L pantothenate. HMBPA elutes at approximately 13 minutes in this system.  
     Example II  
     Ketopantoate Reductase Contributes to the Production of HMBPA and Increasing Ketopantoate Reductase Activity in Bacillus Results in Enhanced HMBPA Production  
     [0096] As described in Example I, a novel HPLC peak corresponding to HMBPA was observed in microorganisms overexpressing panE1 indicating that increased ketopantoate reductase contributes to the production of HMBPA (in addition to production of pantothenate). As mentioned previously, two genes in the  B. subtilis  genome are homologous to the  E. coli  panE gene encoding ketopantoate reductase and have been named panE1 and panE2. In Bacillus, the panE1 gene encodes the major ketopantoate reductase involved in pantothenate production, while panE2 does not contribute to pantothenate synthesis. In fact, overexpression of panE2 from a P 26  promoter leads to a reduction in pantothenate titer (see e.g., U.S. patent application Ser. No. 09/400,494).  
     [0097] Accordingly, it was tested whether, beside being produced by the panE1 gene product, it was possible that a significant portion of the α-HIV necessary to make HMBPA was being produced by the panE2 gene product. It was hypothesized that the panE2 gene product is an enzyme that can reduce α-KIV to α-HIV, but that can not significantly reduce ketopantoate to pantoate.  
     [0098] To test the hypothesis, panE2 was deleted from pantotheniate production strain PA824 (described in Example I) by transforming with a ΔpanE2::cat cassette from chromosomal DNA of strain PA248 (ΔpanE2::cat) (set forth as SEQ ID NO:24, for construction see e.g., U.S. patent application Ser. No. 09/400,494) to give strain PA919. Three isolates of PA919 were compared to PA824 for pantothenate and HMBPA production in test tube cultures grown in SVY plus β-alanine.  
               TABLE 1                          Production of pantothenate and HMBPA by derivatives of PA824 and       PA880 grown at 43° C. in 48 hour test tube cultures       of SVY glucose + β-alanine 5 .                                     Strain   new trait   parent   OD 600     [pan ] g/l   [HMBPA] g/l               PA824   —       13.9   4.3   0.64       PA919-1   ΔpanE2::cat   PA824   13.2   4.2   0.15       PA919-2   ″   ″   14.8   3.8   0.13       PA919-3   ″   ″   18.0   5.5   0.14                  
 
     [0099] As indicated by the data in Table 1, all three isolates of PA919 produced about four-fold lower HMBPA than PA824 demonstrating that the panE2 gene product is a potent contributor to HMBPA synthesis. Moreover, significant increases in HMBPA production can be achieved simply by overexpression of panE2. An exemplary plasmid for the overexpression of panE2, named pAN238, is set forth as SEQ ID NO:25 (FIG. 10).  
     Example III  
     Increasing Production of HMBPA by Reducing PanB Activity in Microorganisms  
     [0100] Strains derived from PA365 (the RL-1 lineage equivalent of PA377, described in U.S. patent application Ser. No. 09/667,569) which are deleted for the P 26  panBCD cassette and which contain a P 26 panC*D cassette amplified at the vpr locus and either the wild type P 26 panB cassette (PA666) or a P26 ΔpanB cassette (PA664) amplified at the bpr locus were constructed as follows. An alignment of the C-terminal amino acids of known or suspected PanB proteins is shown in FIG. 6. Three regions called 1, 2 and 3, that were identified having conserved or semi-conserved amino acid residues, are indicated by arrows at the top of the figure. The  B. subtilis  PanB protein (RBS02239) is underlined. Two of the PanB proteins (RCY14036 and CAB56202.1) are missing region 3 while the latter PanB protein is also missing region 2 and has non-conserved amino acid residues occupying region 1.  
     [0101] B. subtilis  PanB variants were created that were missing regions 1, 2 and 3. The desired variants were created by designing 3′ PCR primers to amplify the  B. subtilis  pan B gene such that region 3, regions 2 and 3, or all three regions would be missing from the final product. The PCR products were generated and cloned into  E. coli  expression vector pASK-1BA3, creating plasmids pAN446, pAN447, and pAN448, respectively. The plasmids were then transformed into  E. coli  strain SJ2 that contains the panB6 mutation to test for complementation. Only pAN446, which is missing region 3, was able to complement. This indicates that region 3 is not essential for  B. subtilis  PanB activity but that region 2 is required for activity or stability.  
     [0102] The next step in this analysis was to transfer the panB gene from pAN446 to a  B. subtilis  expression vector and then introduce it into a strain appropriate for testing activity of the encoded PanB protein in  B. subtilis . To do this, a strain that is deleted for the P 26  panBCD operon was first created. This was accomplished by first inserting a cat gene between the BseRI site located just upstream of the panB RBS and the Bg/II site located in panD, creating plasmid pAN624 (FIG. 7). The sequence of pAN624 is set forth as SEQ ID NO:20. The resulting deletion-substitution mutation (ΔpanBCD::cat624), which removes all of panB and panC, was crossed into PA354 by transformation, with selection for resistance to chloramphenicol on plates supplemented with 1 mM pantothenate. One of the transformants was saved and named PA644. Chromosomal DNA isolated from PA644 was analyzed by PCR and was shown to contain the deletion-substitution mutation. As expected, PA644 requires pantothenate for growth but retains the engineered ilv genes (P 26 ilvBNC P 26 ilvD) as well as the P 26 pan E1 gene originally present in PA354. Thus, it has all the enzymes involved in pantoate synthesis overproduced except PanB. The gene containing the shortest panB deletion was inserted into  B. subtilis  expression vector pOTP61 (described in U.S. patent application Ser. No. 09/667,569), creating plasmid pAN627. At the same time, a wild-type panB control gene was inserted into pOTP61, creating plasmid pAN630. The NotI fragments of each plasmid, lacking  E. coli  vector sequences, were ligated and transformed into PA644, with selection for resistance to tetracycline.  
     [0103] One transformant from each transformation was saved and further transformed with chromosomal DNA from PA628 with selection for Pan*. PA628 contains a multicopy P 26 panC*D expression plasmid (pAN620) integrated at the vpr locus. In order to determine the effects of the panB gene mutation directly on pantothenate production, plasmid pAN620, set forth as SEQ ID NO:21 and illustrated schematically in FIG. 8, provides the remaining two enzymes required for pantothenate synthesis (PanC and PanD). Four transformants from each transformation were isolated, grown in SVY medium containing 10 g/L aspartate for 48 hours, then assayed for pantothenate production. Transformants with the 3′deleted panB gene were named PA664 and those containing the wild-type gene were called PA666. The data showed that the 3′ deleted panB gene in PA664 encodes a PanB protein with greatly reduced activity. To test for HMBPA production, test tube cultures of PA365, PA666, and PA664 were grown in SVY+aspartate medium with and without added α-KIV or pantoate for 48 hours and then assayed for HMBPA and pantothenate as described previously.  
               TABLE 2                          Effect of PanB activity and addition of precursors on HMBPA and pantothenate production, 48 hour       test tube culture data, SVY + aspartate (10 g/L) medium.                                                             +α-KIV   +pantoate                       no additions   (5 g/L)   (5 g/L)                                                             pan C*D   pauB   [pan]   HMBPA   [pan]   HMBPA   [pan]   HMBPA       Strain   pan operon   plasmid   plasmid   (g/L)   peak*   (g/L)   peak   (g/L)   peak               PA365   P 26 panBCD   NONE   NONE   3.0   0.71   3.2   1.28   4.8   0.38       PA666   ΔpanBCD::cat   pAN620   pAN630   3.7   0.55   3.3   1.70   5.2   0.26       PA664   ΔpanBCD::cat   pAN620   pAN627   0.3   1.39   0.6   1.76   2.5   0.74                          
 
     [0104] The data presented in Table 2 demonstrate that in the absence of supplements, PA664 produced the most HMBPA while PA666 produced the least, indicating an inverse correlation between PanB activity and HMBPA production. This is consistent with the model which predicts that the two pathways compete for α-KIV, the substrate for PanB, and produce competitive substrates for PanC; lowering PanB activity would be expected to increase α-KIV availability for α-HIV synthesis and increase HMBPA production, correspondingly decreasing the amount of pantoate synthesized. When α-KIV is added to the medium, all three strains produced significantly more HMBPA. This result evidences that α-KIV is a precursor to HMBPA, as described in FIG. 2, and that excess α-KIV favors HMBPA production. This result also suggests that synthesis of HMBPA is at least partially due to an overflow effect of excess α-KIV production. When pantoate was added to the medium, HMBPA was reduced by roughly 50 percent in all three strains. Conversely, the strains each produced significantly more pantothenate. This result is also consistent with the model that the two pathways produce competing substrates for PanC (α-HIV and pantoate). Taken together, the above results further indicate that decreasing pantoate synthesis should be beneficial in promoting HMBPA production as well as reducing pantothenate levels.  
     Example IV  
     Methods for Regulating HBPA:Pantothenate Levels  
     [0105] As demonstrated in Examples I and II, PanE1 and/or PanE2 contribute to enhanced HMBPA production as does reduced PanB activity. This Example demonstrates that overexpressing PanE1 increases HMBPA production relative to pantothenate production whereas overexpressing PanB decreases HMBPA production relative to pantothenate production. Furthermore, in strains overexpressing IlvC, HMBPA production is enhanced.  
     [0106] PA668 is a derivative of PA824 that contains extra copies of P 26 panB amplified at the vpr or panB locus. PA668 was constructed using the panB expression vector (pAN636) which allows for selection of multiple copies using chloramphenicol. The sequence of pAN636 is set forth as SEQ ID NO:22 and the vector is depicted schematically in FIG. 9. The pAN636 NotI restriction fragment, missing the  E. coli  vector sequences, was ligated and then used to transform PA824 with selection on plates containing 5 μg/ml chloramphenicol. Transformants resistant to 30 μg/ml chloramphenicol were isolated and screened for pantothenate production in 48 hour test tube cultures. The isolates shown produce less HMBPA that PA824 (conversely producing about 10 percent more pantothenate than PA824). A second strain, called PA669, was constructed which is PA824 with extra copies of P26panE1 amplified at the vpr or panE1 locus. Strain PA669 was constructed by transforming PA824 with the self-ligated NotI fragment of plasmid pAN637 with selection for resistance to chloramphenicol. The sequence of pAN637 is set forth as SEQ ID NO:23 and the vector is depicted schematically in FIG. 10. Two isolates of PA669 were chosen for further study; PA669-5 produces less PanE1 than PA669-7 as judged by SDS-PAGE analysis of total cell extracts made from the two strains.  
     [0107] Test tube cultures of strains PA824, PA668-2, PA668-24, and the two isolates of PA669 (PA669-5 and PA669-7) were grown in three different media (SVY, SVY+aspartate, and SVY+aspartate +pantoate) for 48 hours and then assayed for pantothenate, HMBPA, and β-alanine (Table 3).  
               TABLE 3                          Effect of extra copies of panB and panE1 on pantothenate and HMBPA production       by PA824, 48 hour test tube culture data, SV medium.                         +aspartate (10 g/L) &amp;                                 no additions   +aspartate (10 g/L)   pantoate (5 g/L)                                                                 panB   panE   [pan]   [β-ala]   HMBPA   [pan]   [β-ala]       [pan]   [β-ala]           Strain   plasmid   plasmid   (g/L)   (g/L)   *   (g/L)   (g/L)   HMBPA   (g/L)   (g/L)   HMBPA                                                                     PA824   NONE   NONE   1.8   0.05   &lt;0.1   4.7   2.5   0.53   5.6   2.5   &lt;0.10       PA668-2   pAN636   NONE   1.5   &lt;0.04   &lt;0.1   5.0   1.6   &lt;0.10   4.9   1.2   &lt;0.10       PA668-24   pAN636   NONE   1.8   0.05   &lt;0.1   4.9   2.8   0.34   6.1   2.6   &lt;0.10       PA669-5   NONE   pAN637   1.8   0.04   &lt;0.1   4.2   3.1   0.74   5.8   2.6   0.30       PA669-7   NONE   pAN637   1.8   0.06   &lt;0.1   3.7   3.2   1.41   5.2   2.5   0.75                          
 
     [0108] None of the strains produced detectable quantities of HMBPA in SVY medium. All strains produced roughly equivalent amounts of pantothenate and low amounts of β-alanine indicating that β-alanine is limiting for both pantothenate and HMBPA synthesis in these cultures and that β-alanine is a precursor for both compounds. When grown in SVY+aspartate medium, the two PA669 isolates produced more HMBPA than PA824 whereas both PA668 isolates produced less HMBPA than PA824. It is noteworthy that the strain that produces the most PanE1 (PA669-7) produced the most HMBPA (and the least pantothenate). This suggests that high levels of PanE1 favor the production of HMBPA at the expense of lower pantothenate synthesis. It is also interesting that PA668-24 produced more HMBPA than PA668-2, even though SDS-PAGE analysis of extracts from the two strains showed that they produce roughly equivalent levels of PanB. The SDS-PAGE analysis also showed that PA668-24 makes much more IlvC than PA668-2. Based on these data, it is proposed that IlvC influences HMBPA synthesis by increasing steady state levels of α-KIV and/or by catalyzing α-HIV formation from α-KIV, thereby accounting for the observed shift towards production of HMBPA.  
     [0109] The final set of data in Table 3 shows that adding pantoate to the growth medium decreased HMBPA production by all strains that had previously produced detectable levels, e.g., by shifting synthesis towards pantothenate. This further supports the model that α-HIV and pantoate are competitive substrates for PanC.  
     Example V  
     Increasing HMBPA Production by Limiting Serine Availability  
     [0110] It was hypothesized that the ratio of pantothenate to HMBPA production could also be controlled by regulating the availability of serine or methylene tetrahydrofolate in the microorganism cultures. In particular, it is proposed that decreasing the availability of serine could increase HMBPA production relative to pantothenate production, whereas increasing the availability of serine would decrease the production of HMBPA relative to pantothenate production. This method is based on the understanding that the PanB substrate, methylenetetrahydrofolate is derived from serine. Thus, regulating serine levels should effectively regulate PanB substrate levels. To test this hypothesis, PA824 was grown in test tube cultures of SVY glucose plus 5 g/L β-alanine and ±5 g/L serine for 48 hours at 43° C.  
               TABLE 4                          Production of HMBPA and pantothenate by PA824 with and without       the addition of serine                             serine added                                         at 5 g/L   OD 600     [pan] g/L   [HMBPA] g/L                       −   16.3   4.9   0.84           −   14.0   4.5   0.80           +   13.1   6.4   0.56           +   12.9   6.0   0.62                      
 
     [0111] As demonstrated in Table 4, addition of serine decreases the level of production while conversely increasing pantothenate production. At least one method of decreasing methylene tetrahydrofolate levels in order to regulate HMBPA production levels is to decrease the activity of serine hydroxymethyl transferase (the glyA gene product), thereby decreasing methylene tetrahydrofolate biosynthesis in appropriately engineered microorganisms. At least one method of decreasing serine levels in order to regulate HMBPA production is to decrease the activity of 3-phosphoglycerate dehydrogenase (the serA gene product).  
     Example VI  
     Increasing HMBPA Production by Modifying Culture Conditions for Recombinant Microorganisms  
     [0112] In at least one fermentation (Fermentation P162), levels of HMBPA n reached 35 g/L. Briefly, fermentation of strain PA824 was carried out as in Example I but utilizing PFM-155 medium formulated as follows.  
                              BATCH                             MATERIAL   g/L (final)                                     1   Amberex 1003   5       2   Cargill 200/20 (soy flour)   40       3   Na Glutamate   5       4   (NH 4 ) 2 SO 4     8       5   MgSO 4 .7H 2 O   1       6   MAZU DF204C   1       7   H 2 O   qs to 4 L                 Added After Sterilization and Cool Down                         1   KH 2 PO 4     10       2   K 2 HPO 4 .3H 2 O   20       3   H 2 O   qs to 400 ml       1   80% Glucose   20       2   CaCl 2 .2H 2 O   0.1       1   Sodium Citrate   1       2   FeSO 4 .7H 2 O   0.01       3   SM-1000X   1 X                 FEED                         1   80% Glucose   800       2   CaCl 2 .2H 2 O   0.8       3   H 2 O   qs to 3500 ml                 Added After Sterilization and Cool Down                         1   Sodium Citrate   2.0       2   FeSO 4 .7H 2 O   0.02       3   SM-1000X   2 X       4   Glutamate Na   5.0       5   H 2 O   qs to 500 ml                  
 
     [0113] However, as a result of loss of process control during the fermentation, the dissolved oxygen became limiting between 16 and 17 hours and glucose began to accumulate after 16 hours.  
     [0114] These changes in fermentation conditions produced the following significant results at or after 16 hours. Namely, synthesis of HMBPA began to increase with a corresponding decrease in pantothenate synthesis. In the four hour interval before 16 hours the culture produced 7 g/l HMBPA, four hours afterwards, 9.0 g/l. Pantothenate was the reverse with 10 g/l and 6.0 g/l produced between 12-16 hours and 16-20 hours, respectively. Between 20 and 36 hours the average rate of HMBPA synthesis was 1.0 gal hr. Overall, fermentation P162 produced 35 g/l of HMBPA in 36 hours.  
     [0115] Thus, it appears that overfeeding of glucose, and/or limitation of dissolved oxygen (e.g., beginning at about 16 hours) leads to an increase in HMBPA production. Accordingly, two methods for increasing HMBPA production (relative to pantothenate production) are to increase steady state glucose levels and/or decrease steady state dissolved oxygen levels.  
     [0116] Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.  
    
     
       
         1 
         
           
             25  
           
           
             1  
             194  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequencepromoter 
      sequence  
             
           
            1 

gctattgacg acagctatgg ttcactgtcc accaaccaaa actgtgctca gtaccgccaa     60 

tatttctccc ttgaggggta caaagaggtg tccctagaag agatccacgc tgtgtaaaaa    120 

ttttacaaaa aggtattgac tttccctaca gggtgtgtaa taatttaatt acaggcgggg    180 

gcaaccccgc ctgt                                                      194 

 
           
             2  
             163  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequencepromoter 
      sequence  
             
           
            2 

gcctacctag cttccaagaa agatatccta acagcacaag agcggaaaga tgttttgttc     60 

tacatccaga acaacctctg ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt    120 

atctacaagg tgtggtataa taatcttaac aacagcagga cgc                      163 

 
           
             3  
             127  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequencepromoter 
      sequence  
             
           
            3 

gaggaatcat agaattttgt caaaataatt ttattgacaa cgtcttatta acgttgatat     60 

aatttaaatt ttatttgaca aaaatgggct cgtgttgtac aataaatgta gtgaggtgga    120 

tgcaatg                                                              127 

 
           
             4  
             24  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            4 

taaacatgag gaggagaaaa catg                                            24 

 
           
             5  
             28  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            5 

attcgagaaa tggagagaat ataatatg                                        28 

 
           
             6  
             13  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            6 

agaaaggagg tga                                                        13 

 
           
             7  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            7 

ttaagaaagg aggtgannnn atg                                             23 

 
           
             8  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            8 

ttagaaagga ggtgannnnn atg                                             23 

 
           
             9  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            9 

agaaaggagg tgannnnnnn atg                                             23 

 
           
             10  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            10 

agaaaggagg tgannnnnna tg                                              22 

 
           
             11  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            11 

ccctctagaa ggaggagaaa acatg                                           25 

 
           
             12  
             24  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            12 

ccctctagag gaggagaaaa catg                                            24 

 
           
             13  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            13 

ttagaaagga ggatttaaat atg                                             23 

 
           
             14  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            14 

ttagaaagga ggtttaatta atg                                             23 

 
           
             15  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            15 

ttagaaagga ggtgatttaa atg                                             23 

 
           
             16  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            16 

ttagaaagga ggtgtttaaa atg                                             23 

 
           
             17  
             28  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            17 

attcgagaaa ggaggtgaat ataatatg                                        28 

 
           
             18  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            18 

attcgagaaa ggaggtgaat aataatg                                         27 

 
           
             19  
             28  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequenceribosome 
      binding site  
             
           
            19 

attcgtagaa aggaggtgaa ttaatatg                                        28 

 
           
             20  
             6886  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            20 

aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct     60 

tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca    120 

aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg    180 

attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg    240 

atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg    300 

ctaacaggag gaattaacca tggatccgag ctcgacagta tcaagcactt cacaatctgg    360 

gagctgaaag cccgccttat gtagctcata cttgacaaat ccaaggtcaa aatggatatt    420 

gtgggcgaca aaataagcgc cgtcaagcaa ttggaatact tcttcagcaa ctgcttcaaa    480 

tggctgttca ttctcgacca tttgattaga gattccagta agctgctcaa taaaagcagg    540 

gattgattta tttggattaa tgtattttga aaaccgctca gtaatttgtc cattttcgat    600 

tacaaccgct gcgatttgta tgattttatc gcctttcttc ggcgaattcc ctgttgtctc    660 

tacatctata acaacgaacc gttgcttatt cattaaaatg gacacctcaa ttcttgcata    720 

cgacaaaagt gtaacacgtt ttgtacggaa atggagcggc aaaaccgttt tactctcaaa    780 

atcttaaaag aaaacccccg ataaaggggg cttttcttct acaaaattgt acgggctggt    840 

tcgttcccca gcatttgttc aattttgttt tgatcattca gaacagccac tttcggctca    900 

tggcttgccg cttcttgatc agacatcatt ttgtaggaaa taataatgac cttatctcct    960 

tcctgcacaa ggcgtgcggc tgcaccgttt aagcatatga cgccgcttcc ccgtttacca   1020 

ggaataatat acgtttcaag acgtgctcca ttattattat tcacaatttg tactttttca   1080 

ttaggaagca ttcccacagc atcaatgaga tcctctagag tcgacctgca ggcatgcaag   1140 

cttccgtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt   1200 

gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag   1260 

tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa   1320 

gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc   1380 

tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcaatc ttcatccatt   1440 

ccaaggtaaa tcccccttcg ccgtttctgt taccattata caccttttga accttaacgt   1500 

aaacgttaag ttttaaaaaa caataaaaaa gacgagcagc atacagcacc cgtctttcac   1560 

tttcctgttt aagctaaact tcccgccact gacagagact ctttttgaag gctttcagaa   1620 

agcactcgat acgcgatctg gagctgtaat ataaaaacct tcttcaacta acggggcagg   1680 

ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa   1740 

gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat   1800 

aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt   1860 

tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat   1920 

ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag   1980 

gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt   2040 

ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt   2100 

tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc   2160 

cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga   2220 

aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa   2280 

tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct   2340 

cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa   2400 

tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc   2460 

ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat   2520 

ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa   2580 

aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct   2640 

tatcttgata ataagggtaa ctattgcatg ataagctgtc aaacatgaga attcccgttt   2700 

tcttctgcaa gccaaaaaac cttccgttac aacgagaagg attcttcact ttctaaagtt   2760 

cggcgagttt catccctctg tcccagtcct tttttggatc aaggcagact gctgcaatgt   2820 

ctatctattt taataatagg tgcagttcgc aggcgatact gcccaatgga agtataccaa   2880 

aatcaacggg cttgtaccaa cacattagcc caattcgata tcggcagaat agattttttt   2940 

aatgccttcg ttcgtttcta aaagcagaac gccttcatca tctataccta acgccttacc   3000 

gtaaaaggtt ccgtttaacg ttctggctct catattagtg ccaataccga gcgcatagct   3060 

ttcccataaa agcttaatcg gcgtaaatcc gtgcgtcata taatcccggt accgtttctc   3120 

aaagcatagt aaaatatgct ggatgacgcc ggcccgatca attttttccc cagcagcttg   3180 

gctgaggctt gtcgcgatgt ccttcaattc atctggaaaa tcattaggct gctggttaaa   3240 

cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa   3300 

atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt   3360 

cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg   3420 

gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga   3480 

aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa   3540 

atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac   3600 

gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt   3660 

ttgcgtttct acaaactctt tttgtttatt tttctaaata cattcaaata tgtatccgct   3720 

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat   3780 

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc   3840 

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg   3900 

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg   3960 

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga   4020 

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta   4080 

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc   4140 

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc   4200 

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg   4260 

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc   4320 

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca   4380 

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct   4440 

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat   4500 

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg   4560 

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat   4620 

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact   4680 

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat   4740 

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc   4800 

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct   4860 

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg   4920 

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca   4980 

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc   5040 

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga   5100 

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac   5160 

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga   5220 

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag   5280 

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg   5340 

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag   5400 

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc   5460 

tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc   5520 

tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct   5580 

gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct   5640 

cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt   5700 

gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct   5760 

tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt   5820 

cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag   5880 

gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta   5940 

tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct   6000 

acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat   6060 

tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg   6120 

gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg   6180 

cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc   6240 

gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg   6300 

ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta   6360 

atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa   6420 

tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc   6480 

tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc   6540 

cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc   6600 

tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt   6660 

cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg   6720 

agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc   6780 

tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga   6840 

tcattttgcg cttcagccat acttttcata ctcccgccat tcagag                  6886 

 
           
             21  
             7140  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            21 

tcggcggccg cttcgtcgac cgaaacagca gttataaggc atgaagctgt ccggtttttg     60 

caaaagtggc tgtgactgta aaaagaaatc gaaaaagacc gttttgtgtg aaaacggtct    120 

ttttgtttcc ttttaaccaa ctgccataac tcgaggccta cctagcttcc aagaaagata    180 

tcctaacagc acaagagcgg aaagatgttt tgttctacat ccagaacaac ctctgctaaa    240 

attcctgaaa aattttgcaa aaagttgttg actttatcta caaggtgtgg tataataatc    300 

ttaacaacag caggacgctc tagattagaa aggaggattt aaatatgaga cagattactg    360 

atatttcaca gctgaaagaa gccataaaac aataccattc agagggcaag tcaatcggat    420 

ttgttccgac gatggggttt ctgcatgagg ggcatttaac cttagcagac aaagcaagac    480 

aagaaaacga cgccgttatt atgagtattt ttgtgaatcc tgcacaattc ggccctaatg    540 

aagattttga agcatatccg cgcgatattg agcgggatgc agctcttgca gaaaacgccg    600 

gagtcgatat tctttttacg ccagatgctc atgatatgta tcccggtgaa aagaatgtca    660 

cgattcatgt agaaagacgc acagacgtgt tatgcgggcg ctcaagagaa ggacattttg    720 

acggggtcgc gatcgtactg acgaagcttt tcaatctagt caagccgact cgtgcctatt    780 

tcggtttaaa agatgcgcag caggtagctg ttgttgatgg gttaatcagc gacttcttca    840 

tggatattga attggttcct gtcgatacgg tcagagagga agacggctta gccaaaagct    900 

ctcgcaatgt atacttaaca gctgaggaaa gaaaagaagc gcctaagctg tatcgggccc    960 

ttcaaacaag tgcggaactt gtccaagccg gtgaaagaga tcctgaagcg gtgataaaag   1020 

ctgcaaaaga tatcattgaa acgactagcg gaaccataga ctatgtagag ctttattcct   1080 

atccggaact cgagcctgtg aatgaaattg ctggaaagat gattctcgct gttgcagttg   1140 

ctttttcaaa agcgcgttta atagataata tcattattga tattcgtaga aaggaggtga   1200 

attaatatgt atcgtacgat gatgagcggc aaacttcaca gggcaactgt tacggaagca   1260 

aacctgaact atgtgggaag cattacaatt gatgaagatc tcattgatgc tgtgggaatg   1320 

cttcctaatg aaaaagtaca aattgtgaat aataataatg gagcacgtct tgaaacgtat   1380 

attattcctg gtaaacgggg aagcggcgtc atatgcttaa acggtgcagc cgcacgcctt   1440 

gtgcaggaag gagataaggt cattattatt tcctacaaaa tgatgtctga tcaagaagcg   1500 

gcaagccatg agccgaaagt ggctgttctg aatgatcaaa acaaaattga acaaatgctg   1560 

gggaacgaac cagcccgtac aattttgtaa aggatcctgt tttggcggat gagagaagat   1620 

tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc   1680 

tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg   1740 

tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa   1800 

taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga   1860 

acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc   1920 

ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg   1980 

ccatcctgac ggatggcctt tttgcgtttc tacaaactct tggtaccgag acgatcgtcc   2040 

tctttgttgt agcccatcac ttttgctgaa gagtaggagc cgaaagtgac ggcgtattca   2100 

ttgagcggca gctgagtcgc accgacagaa atcgcttctc ttgatgtgcc cggcgatccg   2160 

actgtccagc cgttcggtcc gctgttgccg tttgaggtaa cagcgacaac gccttctgac   2220 

atggcccagt caagcgctgt gcttgtcgcc cagtccgggt tgtttaaaga gtttccgaga   2280 

gacaggttca tcacatctgc cccgtcctgc actgcacgtt ccacgcccgc gatgacgttt   2340 

tccgttgtgc cgcttccgcc aggccctaac acacgataag caagaagtgt ggcatcaggc   2400 

gctacgcctt taatcgttcc gtttgcagcc acagttccgg ctacgtgtgt gccatggtca   2460 

gttgcctcgc ccctcggatc gccggttggt gtttcttttg gatcgtaatc attgtccaca   2520 

aaatcgtatc ctttatattg tccaaagttt ttcttcagat ctgggtgatt gtattcaacc   2580 

ccagtgtcaa taatcgccac cttgatgcct tttcctgtgt agcctaaatc ccatgcatcg   2640 

tttgctccga tataaggcgc actgtcatcc atttgcggag atacggcgtc ttcggagatt   2700 

gtggggaatt ctcatgtttg acagcttatc atgcaatagt tacccttatt atcaagataa   2760 

gaaagaaaag gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt   2820 

tttaatgtgg tctttattct tcaactaaag cacccattag ttcaacaaac gaaaattgga   2880 

taaagtggga tatttttaaa atatatattt atgttacagt aatattgact tttaaaaaag   2940 

gattgattct aatgaagaaa gcagacaagt aagcctccta aattcacttt agataaaaat   3000 

ttaggaggca tatcaaatga actttaataa aattgattta gacaattgga agagaaaaga   3060 

gatatttaat cattatttga accaacaaac gacttttagt ataaccacag aaattgatat   3120 

tagtgtttta taccgaaaca taaaacaaga aggatataaa ttttaccctg catttatttt   3180 

cttagtgaca agggtgataa actcaaatac agcttttaga actggttaca atagcgacgg   3240 

agagttaggt tattgggata agttagagcc actttataca atttttgatg gtgtatctaa   3300 

aacattctct ggtatttgga ctcctgtaaa gaatgacttc aaagagtttt atgatttata   3360 

cctttctgat gtagagaaat ataatggttc ggggaaattg tttcccaaaa cacctatacc   3420 

tgaaaatgct ttttctcttt ctattattcc atggacttca tttactgggt ttaacttaaa   3480 

tatcaataat aatagtaatt accttctacc cattattaca gcaggaaaat tcattaataa   3540 

aggtaattca atatatttac cgctatcttt acaggtacat cattctgttt gtgatggtta   3600 

tcatgcagga ttgtttatga actctattca ggaattgtca gataggccta atgactggct   3660 

tttataatat gagataatgc cgactgtact ttttacagtc ggttttctaa tgtcactaac   3720 

ctgccccgtt agttgaagaa cgaagcggcc gcaattcttg aagacgaaag ggcctcgtga   3780 

tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca   3840 

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata   3900 

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga   3960 

gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc   4020 

ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg   4080 

cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc   4140 

ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat   4200 

cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact   4260 

tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat   4320 

tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga   4380 

tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc   4440 

ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga   4500 

tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag   4560 

cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc   4620 

gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt   4680 

ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct   4740 

acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg   4800 

cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg   4860 

atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca   4920 

tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga   4980 

tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa   5040 

aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga   5100 

aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt   5160 

taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt   5220 

taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat   5280 

agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct   5340 

tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca   5400 

cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag   5460 

agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc   5520 

gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga   5580 

aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca   5640 

tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag   5700 

ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg   5760 

aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat   5820 

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct   5880 

atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc   5940 

ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag   6000 

ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag   6060 

ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc   6120 

gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc   6180 

ggttttttcc tgtttggtca cttgatgcct ccgtgtaagg gggaatttct gttcatgggg   6240 

gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat   6300 

gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag   6360 

agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag   6420 

ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc   6480 

cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc   6540 

gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc   6600 

taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg   6660 

cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg   6720 

gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag   6780 

aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca   6840 

ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt   6900 

atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa   6960 

tcgccgtgac gatcagcggt ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc   7020 

cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg   7080 

gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg   7140 

 
           
             22  
             6725  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            22 

tcggcggccg cttcgtcgac cgaaacagca gttataaggc atgaagctgt ccggtttttg     60 

caaaagtggc tgtgactgta aaaagaaatc gaaaaagacc gttttgtgtg aaaacggtct    120 

ttttgtttcc ttttaaccaa ctgccataac tcgaggccta cctagcttcc aagaaagata    180 

tcctaacagc acaagagcgg aaagatgttt tgttctacat ccagaacaac ctctgctaaa    240 

attcctgaaa aattttgcaa aaagttgttg actttatcta caaggtgtgg tataataatc    300 

ttaacaacag caggacgctc tagaaggagg agaaaacatg aaaacaaaac tggattttct    360 

aaaaatgaag gagtctgaag aaccgattgt catgctgacc gcttatgatt atccggcagc    420 

taaacttgct gaacaagcgg gagttgacat gattttagtc ggtgattcac ttggaatggt    480 

cgtcctcggc cttgattcaa ctgtcggtgt gacagttgcg gacatgatcc atcatacaaa    540 

agccgttaaa aggggtgcgc cgaatacctt tattgtgaca gatatgccgt ttatgtctta    600 

tcacctgtct aaggaagata cgctgaaaaa tgcagcggct atcgttcagg aaagcggagc    660 

tgacgcactg aagcttgagg gcggagaagg cgtgtttgaa tccattcgcg cattgacgct    720 

tggaggcatt ccagtagtca gtcacttagg tttgacaccg cagtcagtcg gcgtactggg    780 

cggctataaa gtacagggca aagacgaaca aagcgccaaa aaattaatag aagacagtat    840 

aaaatgcgaa gaagcaggag ctatgatgct tgtgctggaa tgtgtgccgg cagaactcac    900 

agccaaaatt gccgagacgc taagcatacc ggtcattgga atcggggctg gtgtgaaagc    960 

ggacggacaa gttctcgttt atcatgatat tatcggccac ggtgttgaga gaacacctaa   1020 

atttgtaaag caatatacgc gcattgatga aaccatcgaa acagcaatca gcggatatgt   1080 

tcaggatgta agacatcgtg ctttccctga acaaaagcat tcctttcaaa tgaaccagac   1140 

agtgcttgac ggcttgtacg ggggaaaata agggggggat cctgttttgg cggatgagag   1200 

aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat   1260 

ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa   1320 

cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca   1380 

tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc   1440 

ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca   1500 

acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca   1560 

gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttggta ccgagacgat   1620 

cgtcctcttt gttgtagccc atcacttttg ctgaagagta ggagccgaaa gtgacggcgt   1680 

attcattgag cggcagctga gtcgcaccga cagaaatcgc ttctcttgat gtgcccggcg   1740 

atccgactgt ccagccgttc ggtccgctgt tgccgtttga ggtaacagcg acaacgcctt   1800 

ctgacatggc ccagtcaagc gctgtgcttg tcgcccagtc cgggttgttt aaagagtttc   1860 

cgagagacag gttcatcaca tctgccccgt cctgcactgc acgttccacg cccgcgatga   1920 

cgttttccgt tgtgccgctt ccgccaggcc ctaacacacg ataagcaaga agtgtggcat   1980 

caggcgctac gcctttaatc gttccgtttg cagccacagt tccggctacg tgtgtgccat   2040 

ggtcagttgc ctcgcccctc ggatcgccgg ttggtgtttc ttttggatcg taatcattgt   2100 

ccacaaaatc gtatccttta tattgtccaa agtttttctt cagatctggg tgattgtatt   2160 

caaccccagt gtcaataatc gccaccttga tgccttttcc tgtgtagcct aaatcccatg   2220 

catcgtttgc tccgatataa ggcgcactgt catccatttg cggagatacg gcgtcttcgg   2280 

agattgtggg gaattctcat gtttgacagc ttatcatgca atagttaccc ttattatcaa   2340 

gataagaaag aaaaggattt ttcgctacgc tcaaatcctt taaaaaaaca caaaagacca   2400 

cattttttaa tgtggtcttt attcttcaac taaagcaccc attagttcaa caaacgaaaa   2460 

ttggataaag tgggatattt ttaaaatata tatttatgtt acagtaatat tgacttttaa   2520 

aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc actttagata   2580 

aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa ttggaagaga   2640 

aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac cacagaaatt   2700 

gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta ccctgcattt   2760 

attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg ttacaatagc   2820 

gacggagagt taggttattg ggataagtta gagccacttt atacaatttt tgatggtgta   2880 

tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga gttttatgat   2940 

ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc caaaacacct   3000 

atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac tgggtttaac   3060 

ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg aaaattcatt   3120 

aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc tgtttgtgat   3180 

ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag gcctaatgac   3240 

tggcttttat aatatgagat aatgccgact gtacttttta cagtcggttt tctaatgtca   3300 

ctaacctgcc ccgttagttg aagaacgaag cggccgcaat tcttgaagac gaaagggcct   3360 

cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg   3420 

tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc   3480 

aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag   3540 

gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg   3600 

ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt   3660 

gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt   3720 

tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt   3780 

attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa   3840 

tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag   3900 

agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac   3960 

aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac   4020 

tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac   4080 

cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac   4140 

tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact   4200 

tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg   4260 

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt   4320 

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat   4380 

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta   4440 

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa   4500 

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga   4560 

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac   4620 

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt   4680 

tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc   4740 

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat   4800 

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag   4860 

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc   4920 

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag   4980 

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac   5040 

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg   5100 

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct   5160 

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc   5220 

tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga   5280 

gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga   5340 

agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg   5400 

catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact   5460 

ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac   5520 

gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc   5580 

gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg   5640 

taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc   5700 

agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta   5760 

agggcggttt tttcctgttt ggtcacttga tgcctccgtg taagggggaa tttctgttca   5820 

tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg   5880 

aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg   5940 

accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc   6000 

cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg   6060 

acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc   6120 

aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat   6180 

tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga   6240 

tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg   6300 

agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc   6360 

gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc   6420 

ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac   6480 

aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc   6540 

ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa gagccgcgag   6600 

cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa   6660 

cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc   6720 

tcgcg                                                               6725 

 
           
             23  
             6806  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            23 

tcggcggccg cttcgtcgac cgaaacagca gttataaggc atgaagctgt ccggtttttg     60 

caaaagtggc tgtgactgta aaaagaaatc gaaaaagacc gttttgtgtg aaaacggtct    120 

ttttgtttcc ttttaaccaa ctgccataac tcgaggccta cctagcttcc aagaaagata    180 

tcctaacagc acaagagcgg aaagatgttt tgttctacat ccagaacaac ctctgctaaa    240 

attcctgaaa aattttgcaa aaagttgttg actttatcta caaggtgtgg tataataatc    300 

ttaacaacag caggacgctc tagacaattg agatcttaag aaaggaggtg ttaattaatg    360 

aagattggaa tcattggcgg aggctccgtt ggtcttttat gcgcctatta tttgtcactt    420 

tatcacgacg tgactgttgt gacgaggcgg caagaacagg ctgcggccat tcagtctgaa    480 

ggaatccggc tttataaagg cggggaggaa ttcagggctg attgcagtgc ggacacgagt    540 

atcaattcgg actttgacct gcttgtcgtg acagtgaagc agcatcagct tcaatctgtt    600 

ttttcgtcgc ttgaacgaat cgggaagacg aatatattat ttttgcaaaa cggcatgggg    660 

catatccacg acctaaaaga ctggcacgtt ggccattcca tttatgttgg aatcgttgag    720 

cacggagctg taagaaaatc ggatacagct gttgatcata caggcctagg tgcgataaaa    780 

tggagcgcgt tcgacgatgc tgaaccagac cggctgaaca tcttgtttca gcataaccat    840 

tcggattttc cgatttatta tgagacggat tggtaccgtc tgctgacggg caagctgatt    900 

gtaaatgcgt gtattaatcc tttaactgcg ttattgcaag tgaaaaatgg agaactgctg    960 

acaacgccag cttatctggc ttttatgaag ctggtatttc aggaggcatg ccgcatttta   1020 

aaacttgaaa atgaagaaaa ggcttgggag cgggttcagg ccgtttgtgg gcaaacgaaa   1080 

gagaatcgtt catcaatgct ggttgacgtc attggaggcc ggcagacgga agctgacgcc   1140 

attatcggat acttattgaa ggaagcaagt cttcaaggtc ttgatgccgt ccacctagag   1200 

tttttatatg gcagcatcaa agcattggag cgaaatacca acaaagtggt ttactaagga   1260 

tcctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga   1320 

agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc   1380 

atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg   1440 

agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt   1500 

tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc   1560 

ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac   1620 

tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca   1680 

aactcttggt accgagacga tcgtcctctt tgttgtagcc catcactttt gctgaagagt   1740 

aggagccgaa agtgacggcg tattcattga gcggcagctg agtcgcaccg acagaaatcg   1800 

cttctcttga tgtgcccggc gatccgactg tccagccgtt cggtccgctg ttgccgtttg   1860 

aggtaacagc gacaacgcct tctgacatgg cccagtcaag cgctgtgctt gtcgcccagt   1920 

ccgggttgtt taaagagttt ccgagagaca ggttcatcac atctgccccg tcctgcactg   1980 

cacgttccac gcccgcgatg acgttttccg ttgtgccgct tccgccaggc cctaacacac   2040 

gataagcaag aagtgtggca tcaggcgcta cgcctttaat cgttccgttt gcagccacag   2100 

ttccggctac gtgtgtgcca tggtcagttg cctcgcccct cggatcgccg gttggtgttt   2160 

cttttggatc gtaatcattg tccacaaaat cgtatccttt atattgtcca aagtttttct   2220 

tcagatctgg gtgattgtat tcaaccccag tgtcaataat cgccaccttg atgccttttc   2280 

ctgtgtagcc taaatcccat gcatcgtttg ctccgatata aggcgcactg tcatccattt   2340 

gcggagatac ggcgtcttcg gagattgtgg ggaattctca tgtttgacag cttatcatgc   2400 

aatagttacc cttattatca agataagaaa gaaaaggatt tttcgctacg ctcaaatcct   2460 

ttaaaaaaac acaaaagacc acatttttta atgtggtctt tattcttcaa ctaaagcacc   2520 

cattagttca acaaacgaaa attggataaa gtgggatatt tttaaaatat atatttatgt   2580 

tacagtaata ttgactttta aaaaaggatt gattctaatg aagaaagcag acaagtaagc   2640 

ctcctaaatt cactttagat aaaaatttag gaggcatatc aaatgaactt taataaaatt   2700 

gatttagaca attggaagag aaaagagata tttaatcatt atttgaacca acaaacgact   2760 

tttagtataa ccacagaaat tgatattagt gttttatacc gaaacataaa acaagaagga   2820 

tataaatttt accctgcatt tattttctta gtgacaaggg tgataaactc aaatacagct   2880 

tttagaactg gttacaatag cgacggagag ttaggttatt gggataagtt agagccactt   2940 

tatacaattt ttgatggtgt atctaaaaca ttctctggta tttggactcc tgtaaagaat   3000 

gacttcaaag agttttatga tttatacctt tctgatgtag agaaatataa tggttcgggg   3060 

aaattgtttc ccaaaacacc tatacctgaa aatgcttttt ctctttctat tattccatgg   3120 

acttcattta ctgggtttaa cttaaatatc aataataata gtaattacct tctacccatt   3180 

attacagcag gaaaattcat taataaaggt aattcaatat atttaccgct atctttacag   3240 

gtacatcatt ctgtttgtga tggttatcat gcaggattgt ttatgaactc tattcaggaa   3300 

ttgtcagata ggcctaatga ctggctttta taatatgaga taatgccgac tgtacttttt   3360 

acagtcggtt ttctaatgtc actaacctgc cccgttagtt gaagaacgaa gcggccgcaa   3420 

ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat   3480 

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg   3540 

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat   3600 

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat   3660 

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt   3720 

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag   3780 

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa   3840 

agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg   3900 

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct   3960 

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac   4020 

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca   4080 

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat   4140 

accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact   4200 

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc   4260 

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga   4320 

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg   4380 

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg   4440 

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca   4500 

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta   4560 

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca   4620 

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg   4680 

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga   4740 

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa   4800 

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc   4860 

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg   4920 

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac   4980 

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct   5040 

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc   5100 

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg   5160 

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg   5220 

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct   5280 

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga   5340 

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg   5400 

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca   5460 

tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc   5520 

atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga   5580 

cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac   5640 

agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg   5700 

aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg   5760 

tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt   5820 

ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcacttg atgcctccgt   5880 

gtaaggggga atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca   5940 

cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac   6000 

tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg   6060 

ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga   6120 

acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga   6180 

agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc   6240 

gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg   6300 

tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga   6360 

tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg   6420 

tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc   6480 

cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg   6540 

acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt   6600 

ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt   6660 

taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg   6720 

cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat   6780 

aatggggaag gccatccagc ctcgcg                                        6806 

 
           
             24  
             3867  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            24 

aagctttctc aagaagcgaa caagaaaaaa gaagagcaga ttaaacagct tcaagagttt     60 

gtcgctagat tcagcgccaa tgcgtctaaa tctaagcagg ctacatcaag aaagaaactt    120 

ctcgaaaaaa tcacgctgga tgatattaaa ccgtcttccc gccgctatcc ttatgttaac    180 

ttcacgccgg aacgggaaat cggaaatgat gttcttcgcg tggaaggctt aacaaaaaca    240 

atcgatggcg taaaggtgct tgacaatgtc agctttatta tgaatcgaga agataaaatt    300 

gctttcactg gccgaaatga acttgctgtt actacgctgt ttaaaatcat ttccggggaa    360 

atggaagctg acagcggaac gtttaaatgg ggtgttacca catctcaagc gtattttcca    420 

aaagacaaca gcgaatattt cgaaggcagt gatctgaacc ttgtagactg gcttcgccaa    480 

tattctccgc acgaccaaag tgagagcttt ttacgcggtt tcttaggacg catgctgttc    540 

tctggagaag aagtccacaa aaaagcaaat gtactttccg ggggagaaaa ggtccgctgt    600 

atgctgtcga aagcgatgct ttctggcgcc aatattttaa ttttggatga gccgaccaac    660 

catttagacc tagagtccat tacagcgctc aataacggct taatcagctt taaaggcgct    720 

atgctgttta cttcccatga ccatcagttt gtgcagacca ttgccaacag aattatagaa    780 

attacaccta acggcatcgt cgataagcaa atgagctatg acgagttcct tgaaaatgct    840 

gatgtgcaga aaaaattgac tgaactatac gccgaataaa aaagcagaga tttctctgct    900 

ttttttgata cctaaatgtg aaaggagatc acaacatgaa atttttggtt gtcggagcag    960 

gtggagtagg cgggtatatt ggcggacggc tttcggagaa aggaaatgat gtgacatttc   1020 

tcgtgcgcca aaaacgagct gagcagctta aaaaaaccgg gcttgtcatc catagtgaaa   1080 

aagggaatgt atcatttcag cccgaactaa tcagtgccgg agaaacaggg caatttgatg   1140 

tcgttatcat tgcttctaaa gcatactcgc ttggtcaagt gatagaggat gtcaaaccat   1200 

ttatccatca agaatctgtc attatccctt ttttaaatgg gtaccgccac tatgagcagc   1260 

tatttgcggc attttcaaaa gaacaggtgc tgggcggcct gtgttttata gaaagtgctt   1320 

tagacaacaa aggagaaatt catcatacga gcgcatcgca tcgttttgta tttggagaat   1380 

ggaacggcga gcgtacggag cggataagag cgcttgaaga ggcattttca ggtgtgaagg   1440 

ctgaagtcat cattagcggg catatcgaga agatcccctg cagcaatagt tacccttatt   1500 

atcaagataa gaaagaaaag gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa   1560 

gaccacattt tttaatgtgg tctttattct tcaactaaag cacccattag ttcaacaaac   1620 

gaaaattgga taaagtggga tatttttaaa atatatattt atgttacagt aatattgact   1680 

tttaaaaaag gattgattct aatgaagaaa gcagacaagt aagcctccta aattcacttt   1740 

agataaaaat ttaggaggca tatcaaatga actttaataa aattgattta gacaattgga   1800 

agagaaaaga gatatttaat cattatttga accaacaaac gacttttagt ataaccacag   1860 

aaattgatat tagtgtttta taccgaaaca taaaacaaga aggatataaa ttttaccctg   1920 

catttatttt cttagtgaca agggtgataa actcaaatac agcttttaga actggttaca   1980 

atagcgacgg agagttaggt tattgggata agttagagcc actttataca atttttgatg   2040 

gtgtatctaa aacattctct ggtatttgga ctcctgtaaa gaatgacttc aaagagtttt   2100 

atgatttata cctttctgat gtagagaaat ataatggttc ggggaaattg tttcccaaaa   2160 

cacctatacc tgaaaatgct ttttctcttt ctattattcc atggacttca tttactgggt   2220 

ttaacttaaa tatcaataat aatagtaatt accttctacc cattattaca gcaggaaaat   2280 

tcattaataa aggtaattca atatatttac cgctatcttt acaggtacat cattctgttt   2340 

gtgatggtta tcatgcagga ttgtttatga actctattca ggaattgtca gataggccta   2400 

atgactggct tttataatat gagataatgc cgactgtact ttttacagtc ggttttctaa   2460 

tgtcactaac ctgccccgtt agttgaagaa ggtttttata ttacagctcc cgggagatct   2520 

gggatcacta gtccaaacga cagaaggcga ccacctgcat ggatttttga ttgaaaaagc   2580 

aaaacgttta tctctcgctg caccagtatt agaaaccgtt tatgcgaatc tgcaaatgta   2640 

tgaagcagaa aaataaaaaa aggaggcgga aaagcctcct tttatttact taaaaagccc   2700 

aatttccgtt tctgaagata ggctctcttt tcccgtctgc cgtaattccg tcaatattca   2760 

tatccttaga accgatcata aagtccacgt gtgtaatgct ttcatttagg ccttctttga   2820 

caagctcttc acgagacatc tgctttccgc cttcaatatt aaaggcatag gcgcttccga   2880 

tcgccaaatg atttgacgcg ttttcatcaa acagcgtgtt atagaaaaga atgtttgatt   2940 

gtgatatagg cgaatcgtaa ggaacaagtg ccacttcacc taaatagtga gaaccttcat   3000 

ctgtttccac cagttctttt aaaatatcct cacctttttc agctttaatg tcgactatac   3060 

ggccattttc aaacgtcagg gtgaaatttt caataatatt tccgccgtag cttaatggtt   3120 

ttgtgcttga taccactccg tcaaccccgt ctttttgcgg cagcgtgaac acttcttctg   3180 

tcggcatatt ggccataaac tcatggccac tttcattcac gcttcccgca cctgcccaaa   3240 

catgcttcct aggcagctta attgttagat cagttccttc tgcttgataa tgtaaggcag   3300 

cgtaatgtct ctcgttcaaa tggtcaactt tttcatgaag attttggtca tgattgatcc   3360 

acgcctgaac cgggttgtct tcatttacgc gcgtcgcttt aaaaatttct tcccacagaa   3420 

ggtggatcgc ttcctcctct gatttgccag gaaacacctt gtgagcccag cctgctgatg   3480 

ccgcacctac gacagtccag ctgactttgt ctgattgaat atattgtctg tatgtatgta   3540 

atgctttgcc tgctgctttt tggaatgccg caatccgttt ggaatctata ccttttagca   3600 

agtctgggtt cgacgacaca acagaaatga aagcagctcc atttttggca agctcttctc   3660 

tgccttttgc ttcccattca ggatattctt caaatgcttc aaacggcgca agttcgtatt   3720 

ttaatttggc gacttcgtca tcctgccaat tcacggtgac gttttttgcg cccttttcat   3780 

atgcgtgttt tacaattaaa cggacaaaat cccgaacgtc tgttgaagca tttacgacta   3840 

catactggcc tttttggaca ttaacgc                                       3867 

 
           
             25  
             8704  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence vector  
             
           
            25 

gcggccgctt cgtcgaccga aacagcagtt ataaggcatg aagctgtccg gtttttgcaa     60 

aagtggctgt gactgtaaaa agaaatcgaa aaagaccgtt ttgtgtgaaa acggtctttt    120 

tgtttccttt taaccaactg ccataactcg aggcctacct agcttccaag aaagatatcc    180 

taacagcaca agagcggaaa gatgttttgt tctacatcca gaacaacctc tgctaaaatt    240 

cctgaaaaat tttgcaaaaa gttgttgact ttatctacaa ggtgtggtat aataatctta    300 

acaacagcag gacgctctag aggaggagac taacatgaaa tttttggttg tcggagcagg    360 

tggagtaggc gggtatattg gcggacggct ttcggagaaa ggaaatgatg tgacatttct    420 

cgtgcgccaa aaacgagctg agcagcttaa aaaaaccggg cttgtcatcc atagtgaaaa    480 

agggaatgta tcatttcagc ccgaactaat cagtgccgga gaaacagggc aatttgatgt    540 

cgttatcatt gcttctaaag catactcgct tggtcaagtg atagaggatg tcaaaccatt    600 

tatccatcaa gaatctgtca ttatcccttt tttaaatggg taccgccact atgagcagct    660 

atttgcggca ttttcaaaag aacaggtgct gggcggcctg tgttttatag aaagtgcttt    720 

agacaacaaa ggagaaattc atcatacgag cgcatcgcat cgttttgtat ttggagaatg    780 

gaacggcgag cgtacggagc ggataagagc gcttgaagag gcattttcag gtgtgaaggc    840 

tgaagtcatc attagcgggc atatcgagaa ggacatttgg aaaaagtatc tctttattgc    900 

agcgcaagcg gggatcacaa cgttatttca acgaccgctt ggcccaatcc tcgccacaga    960 

agccggacgt cacacggccc aaactcttat tggggaaatt tgcactgttt tacgaaaaga   1020 

aggtgttccg gctgatccgg ctcttgagga agagagcttt cgtacgatga ccagcatgtc   1080 

ttaccatatg aagtcctcca tgcttcggga tatggaaaac ggccaaacga cagaaggcga   1140 

ccacctgcat ggatttttga ttgaaaaagc aaaacgttta tctctcgctg caccagtatt   1200 

agaaaccgtt tatgcgaatc tgcaaatgta tgaagcagaa aaataaaaaa aggaggcgga   1260 

aaagcctcct tttatttact taaaaagccc aatttccgtt tctgaagata ggctctcttt   1320 

tcccgtctgc cgggatcctg ttttggcgga tgagagaaga ttttcagcct gatacagatt   1380 

aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg   1440 

gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg   1500 

gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc   1560 

gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac   1620 

aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg   1680 

acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct   1740 

ttttgcgttt ctacaaactc ttggtaccca gaaaaagcgg caaaagcggc tgttaaaaaa   1800 

gcgaaatcga agaagctgtc tgccgctaag acggaatatc aaaagcgttc tgctgttgtg   1860 

tcatctttaa aagtcacagc cgatgaatcc cagcaagatg tcctaaaata cttgaacacc   1920 

cagaaagata aaggaaatgc agaccaaatt cattcttatt atgtggtgaa cgggattgct   1980 

gttcatgcct caaaagaggt tatggaaaaa gtggtgcagt ttcccgaagt ggaaaaggtg   2040 

cttcctaatg agaaacggca gctttttaag tcatcctccc catttaatat gaaaaaagca   2100 

cagaaagcta ttaaagcaac tgacggtgtg gaatggaatg tagaccaaat cgatgcccca   2160 

aaagcttggg cacttggata tgatggaact ggcacggttg ttgcgtccat tgataccggg   2220 

gtggaatgga atcatccggc attaaaagag aaatatcgcg gatataatcc ggaaaatcct   2280 

aatgagcctg aaaatgaaat gaactggtat gatgccgtag caggcgaggc aagcccttat   2340 

gatgatttgg ctcatggaac ccacgtgaca ggcacgatgg tgggctctga acctgatgga   2400 

acaaatcaaa tcggtgtagc acctggcgca aaatggattg ctgttaaagc gttctctgaa   2460 

gatggcggca ctgatgctga cattttggaa gctggtgaat gggttttagc accaaaggac   2520 

gcggaaggaa atccccaccc ggaaatggct cctgatgttg tcaataactc atggggaggg   2580 

ggctctggac ttgatgaatg gtacagagac atggtcaatg cctggcgttc ggccgatatt   2640 

ttccctgagt tttcagcggg gaatacggat ctctttattc ccggcgggcc tggttctatc   2700 

gcaaatccgg caaactatcc agaatcgttt gcaactggag cgactgagaa ttccaattcc   2760 

ccatggagag aaaagaaaat cgctaatgtt gattactttg aacttctgca tattcttgaa   2820 

tttaaaaagg ctgaaagagt aaaagattgt gctgaaatat tagagtataa acaaaatcgt   2880 

gaaacaggcg aaagaaagtt gtatcgagtg tggttttgta aatccaggct ttgtccaatg   2940 

tgcaactgga ggagagcaat gaaacatggc attcagtcac aaaaggttgt tgctgaagtt   3000 

attaaacaaa agccaacagt tcgttggttg tttctcacat taacagttaa aaatgtttat   3060 

gatggcgaag aattaaataa gagtttgtca gatatggctc aaggatttcg ccgaatgatg   3120 

caatataaaa aaattaataa aaatcttgtt ggttttatgc gtgcaacgga agtgacaata   3180 

aataataaag ataattctta taatcagcac atgcatgtat tggtatgtgt ggaaccaact   3240 

tattttaaga atacagaaaa ctacgtgaat caaaaacaat ggattcaatt ttggaaaaag   3300 

gcaatgaaat tagactatga tccaaatgta aaagttcaaa tgattcgacc gaaaaataaa   3360 

tataaatcgg atatacaatc ggcaattgac gaaactgcaa aatatcctgt aaaggatacg   3420 

gattttatga ccgatgatga agaaaagaat ttgaaacgtt tgtctgattt ggaggaaggt   3480 

ttacaccgta aaaggttaat ctcctatggt ggtttgttaa aagaaataca taaaaaatta   3540 

aaccttgatg acacagaaga aggcgatttg attcatacag atgatgacga aaaagccgat   3600 

gaagatggat tttctattat tgcaatgtgg aattgggaac ggaaaaatta ttttattaaa   3660 

gagtagttca acaaacgggc catattgttg tataagtgat gaaatactga atttaaaact   3720 

tagtttatat gtggtaaaat gttttaatca agtttaggag gaattaatta tgaagtgtaa   3780 

tgaatgtaac agggttcaat taaaagaggg aagcgtatca ttaaccctat aaactacgtc   3840 

tgccctcatt attggagggt gaaatgtgaa tacatcctat tcacaatcga atttacgaca   3900 

caaccaaatt ttaatttggc tttgcatttt atcttttttt agcgtattaa atgaaatggt   3960 

tttgaacgtc tcattacctg atattgcaaa tgattttaat aaaccacctg cgagtacaaa   4020 

ctgggtgaac acagccttta tgttaacctt ttccattgga acagctgtat atggaaagct   4080 

atctgatcaa ttaggcatca aaaggttact cctatttgga attataataa attgtttcgg   4140 

gtcggtaatt gggtttgttg gccattcttt cttttcctta cttattatgg ctcgttttat   4200 

tcaaggggct ggtgcagctg catttccagc actcgtaatg gttgtagttg cgcgctatat   4260 

tccaaaggaa aataggggta aagcatttgg tcttattgga tcgatagtag ccatgggaga   4320 

aggagtcggt ccagcgattg gtggaatgat agcccattat attcattggt cctatcttct   4380 

actcattcct atgataacaa ttatcactgt tccgtttctt atgaaattat taaagaaaga   4440 

agtaaggata aaaggtcatt ttgatatcaa aggaattata ctaatgtctg taggcattgt   4500 

attttttatg ttgtttacaa catcatatag catttctttt cttatcgtta gcgtgctgtc   4560 

attcctgata tttgtaaaac atatcaggaa agtaacagat ccttttgttg atcccggatt   4620 

agggaaaaat atacctttta tgattggagt tctttgtggg ggaattatat ttggaacagt   4680 

agcagggttt gtctctatgg ttccttatat gatgaaagat gttcaccagc taagtactgc   4740 

cgaaatcgga agtgtaatta ttttccctgg aacaatgagt gtcattattt tcggctacat   4800 

tggtgggata cttgttgata gaagaggtcc tttatacgtg ttaaacatcg gagttacatt   4860 

tctttctgtt agctttttaa ctgcttcctt tcttttagaa acaacatcat ggttcatgac   4920 

aattataatc gtatttgttt taggtgggct ttcgttcacc aaaacagtta tatcaacaat   4980 

tgtttcaagt agcttgaaac agcaggaagc tggtgctgga atgagtttgc ttaactttac   5040 

cagcttttta tcagagggaa caggtattgc aattgtaggt ggtttattat ccataccctt   5100 

acttgatcaa aggttgttac ctatggaagt tgatcagtca acttatctgt atagtaattt   5160 

gttattactt ttttcaggaa tcattgtcat tagttggctg gttaccttga atgtatataa   5220 

acattctcaa agggatttct aaatcgttaa gggatcaact ttgggagaga gttcaaaatt   5280 

gatccttttt ttataacagt tcgaagcggc cgcaattctt gaagacgaaa gggcctcgtg   5340 

atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc   5400 

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat   5460 

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag   5520 

agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt   5580 

cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt   5640 

gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc   5700 

cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta   5760 

tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac   5820 

ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa   5880 

ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg   5940 

atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc   6000 

cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg   6060 

atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta   6120 

gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg   6180 

cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg   6240 

tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc   6300 

tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt   6360 

gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt   6420 

gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc   6480 

atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag   6540 

atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa   6600 

aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg   6660 

aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag   6720 

ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg   6780 

ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga   6840 

tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc   6900 

ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc   6960 

acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga   7020 

gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt   7080 

cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg   7140 

aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac   7200 

atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga   7260 

gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg   7320 

gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata   7380 

tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc   7440 

tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc   7500 

cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga   7560 

gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa   7620 

gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct   7680 

cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg   7740 

cggttttttc ctgtttggtc acttgatgcc tccgtgtaag ggggaatttc tgttcatggg   7800 

ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca   7860 

tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca   7920 

gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca   7980 

gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt   8040 

ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt   8100 

cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg   8160 

ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat   8220 

gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat   8280 

ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa   8340 

gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc   8400 

attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg   8460 

tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa   8520 

atcgccgtga cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cgcgagcgat   8580 

ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg   8640 

ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc   8700 

gtcg                                                                8704