KR20050021433A - Microorganisms and processes for enhanced production of pantothenate - Google Patents

Abstract

The present invention features an improved and improved method for producing pantoate and pantothenate using microorganisms with modified pantothenate biosynthetic enzyme activity and modified methylene tetrahydrofolate (MTF) biosynthetic enzyme activity. In particular, the invention features a method of improving the production of the desired product by increasing the amount of ketopantoate, the key intermediate, by increasing the enzyme or substrate that directly or indirectly contributes to the synthesis. Recombinant microorganisms and conditions for culturing them are also characterized. It also features a composition produced by such microorganisms.

Description

MICROORGANISMS AND PROCESSES FOR ENHANCED PRODUCTION OF PANTOTHENATE}

Pantothenate, also known as pantothenic acid or vitamin B5, is a member of the vitamin B complex and is a necessary nutrient for mammals, including livestock and humans (eg from food sources, as water soluble vitamin supplements, or feed additives). Pantothenate in cells is mainly used for biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). This coenzyme functions in the metabolism of acyl moieties that form thioesters with sulfhydryl groups of the 4'-phosphophanate moiety of the molecule. The coenzyme is essential in all cells and is involved in over 100 different intermediate reactions in cell metabolism.

A common method of synthesizing pantothenates (particularly bioactive D isomers) is chemical synthesis from bulk chemicals, which has the disadvantage of excessive substrate cost as well as the need for optical separation of racemic intermediates. Thus, researchers have recently turned to bacterial or microbial systems that produce enzymes useful for pantothenate biosynthesis processes (because bacteria can synthesize pantothenate on their own). In particular, bioconversion methods are being evaluated as advantageous methods for producing the desired pantothenic acid isomers. In recent years, a direct microbial synthesis method is under investigation as a method for easily producing D-pantothenate.

However, there is still a need for improving the pantothenate production process, particularly for timing microbial processes optimized with higher yields of desired products.

Summary of the Invention

The present invention relates to an improved process for pantothenate production (eg microbial synthesis). The pantothenate production process has been presented in the related application, for example characterized by microorganisms engineered to overexpress key enzymes of the pantothenate biosynthetic pathway and the isoleucine-valine biosynthetic pathway (see, eg, FIG. 1). . Strains have been processed to produce> 50 g / l pantothenate in standard fermentation processes (see, eg, International Patent Publication WO01 / 21772 and US Patent Application 60 / 262,995). In particular, an increase in panB, panC, panD and panE1 gene expression and an increase in ilvBNC and ilvD gene expression result in strains that convert glucose (pyruvate) to commercially attractive amounts of pantothenate.

In order to improve the production level of pantothenate, for example, various improvements to the process described above are currently being developed. For example, US patent application Ser. No. 09 / 667,569 describes production strains with modified (eg, deleted or reduced activity) pantothenate kinase enzymes. In such strains, pantothenate levels are effectively increased by reducing the use of pantothenate for coenzyme A ("CoA") synthesis. In addition, US Patent Application 60 / 262,995 discloses the use of various pantothenate biosynthetic enzymes and / or isoleucine-valine biosynthetic enzymes, and / or their respective substrates for [R] -3- (2-hydroxy An improved pantothenate-producing strain has been described that has been engineered to minimize its use in the production of replacement products identified as 3-methylbutyrylamino) -propionic acid ("HMBPA").

The invention features a method of further enhancing pantothenate production by regulating the biosynthetic pathway, ie methylenetetrahydrofolate (“MTF”) biosynthetic pathway, which provides a substrate for the pantothenate biosynthetic pathway. In particular, altering the MTF biosynthetic pathway to increase the level of MTF has been found to improve the levels of the key pantothenate biosynthetic pathway intermediate, ketopantoate. Increasing ketopantoate levels significantly improves pantothenate production levels in suitably engineered strains. Indeed, the inventors have identified a restriction step in the production of panto-compounds (eg pantothenate) by strains engineered to overexpress, for example, the panB, panC, panD, panEI, ilvBNC and ilvD genes. , We describe a method for overcoming this limitation by modifying the MTF biosynthetic pathway.

At least three effective methods of modifying the MTF biosynthetic pathway are described herein. In one aspect, increasing serine levels in the culture medium of pantothenate-producing microorganisms has been demonstrated to improve panto-compound production. In addition, by increasing the synthesis or activity of 3-phosphoglycerate dehydrogenase (serA gene product), or the synthesis or activity of serine hydroxymethyl transferase (glyA gene product), serine in suitably engineered microorganisms And improving the biosynthesis of methylenetetrahydrofolate has been demonstrated to increase panto-compound production. An increase in 3-phosphoglycerate dehydrogenase (serA gene product) synthesis is achieved, for example, by overexpressing serA from a properly-engineered expression cassette. An increase in serine hydroxymethyl transferase (glyA gene product) synthesis is achieved, for example, by overexpressing glyA from a properly-engineered expression cassette. Alternatively, the level of serine hydroxymethyl transferase (glyA gene product) is increased by modifying the regulation of the glyA gene. For example, mutations or deletions in genes encoding glyA, a negative regulator of purR gene expression (ie, a repressor), effectively increase glyA expression. Further suitable methods of increasing MTF levels of panto-compound producing microorganisms include deregulating enzymes (eg, glycine cleavage enzymes) that convert glycine to MTF.

Thus, in one aspect, the present invention includes culturing a microorganism having modified pantothenate biosynthetic enzyme activity and modified methylenetetrahydrofolate (MTF) biosynthetic enzyme activity under conditions in which pantothenate production is improved. A process for improving the production of ates and pantothenates. In another aspect, the present invention provides a modified pantothenate biosynthetic enzyme activity, a modified isoleucine-valine (ilv) biosynthetic enzyme, and a modified methylenetetrahydrofolate (MTF) biosynthetic enzyme activity under conditions that improve pantothenate production. Characterized by a process for improving the production of pantoate and pantothenate, comprising culturing the microorganisms having. In particular, the present invention provides a method for improving the production of the desired product (eg pantoate and / or pantothenate) by increasing the levels of key intermediates, ketopantoate, to enzymes that contribute to their synthesis. It features. Preferred methods are at least 50, 60, 70 g / L after 36 hours of microbial culture, or at least 60, 70, 80, 90, 100, 110, 120 g / L of pantothenate after 36 hours of microbial culture. Improves pantothenate production. Recombinant microorganisms and their culture conditions are also characterized. Also characterized are compositions produced by such microorganisms.

Other features and advantages of the invention are apparent from the following detailed description and claims.

Brief description of the drawings

1 is a schematic of pantothenate and isoleucine-valine (ilv) biosynthetic pathways. Pantothenate biosynthetic enzymes are shown in bold and the corresponding genes are shown in italics. Isoleucine-valine (ilv) biosynthetic enzymes are shown in bold italics and the corresponding genes are shown in italics.

2 is a schematic of the methylenetetrahydrofolate (“MTF”) biosynthetic pathway in E. coli (and possibly in B. subtilis ).

3 is a schematic of the plasmid pAN665 structure.

4 is a schematic of the plasmid pAN670 structure.

5 is a schematic of the plasmid pAN004 structure.

6 is a schematic of the plasmid pAN396 structure.

7 is a schematic of the plasmid pAN393 structure.

8 is a schematic of the pAN835F structure, which is a clone of the B. subtilis purR gene.

9 is a schematic of the plasmid pAN838F structure designed to disrupt the B. subtilis purR gene.

10 is a schematic of the plasmid pAN821 structure designed to select kanamycin resistance by deleting a portion of the serA gene.

FIG. 11 is a schematic of plasmid pAN824 structure designed to select Ser ⁺ by incorporating an unamplified P ₂₆ serA cassette at the serA site.

12 is a schematic of the medium copy plasmid pAN395 structure designed to integrate and amplify the P ₂₆ serA expression cassette at the serA site.

Detailed description of the invention

The present invention relates to an improved method for producing panto-compounds (eg ketopantoate, pantoate and / or pantothenate), and strains engineered for use in the improved method. Strains capable of producing> 50 g / l pantothenate can be prepared as taught in International Patent Application WO01 / 21772 and US Patent Application 60 / 262,995. By increasing the expression of the panB, panC, panD and panE1 genes and increasing the expression of the ilvBNC and ilvD genes, strains that convert glucose (pyruvate) to commercially attractive amounts of pantothenate (e.g., Bacillus ) Strains) can be designed.

Currently, however, high levels of panB gene product, ketopantoate hydroxymethyltransferase (see, for example, PA 824 described in US patent application Ser. No. 09 / 667,569, PA668- described in US patent application Ser. No. 60 / 262,995). In strains engineered to express 24, it has been found that the limiting step to further increasing the production of pantothenate is still the conversion of α-ketoisovalerate (α-KIV) to ketopantoate. Methods of increasing the synthesis of α-KIV have already been described in international patent application WO01 / 21772 and US patent application 60 / 262,995. Disclosed herein is the achievement of further increases in pantothenate production by manipulating panto-compound producing microorganisms to improve or increase the level of MTF, or the rate of MTF synthesis.

Accordingly, the present invention features methods for improving panto compound production, including the regulation of methylenetetrahydrofolate (“MTF”) biosynthetic pathways. In particular, increasing MTF levels in panto-compound producing microorganisms is an effective means of improving ketopantoate production, which enhances pantoate and / or pantothenate production in properly engineered recombinant microorganisms.

Ketopantoate hydroxymethylenetransferase catalyzes the production of ketopantoate from α-ketoisovalerate (“α-KIV”) and MTF (see, eg, FIG. 1). In particular, this enzyme catalyzes the transition of hydroxymethyl groups from MTF to α-KIV to produce ketopantoate. Both α-KIV and MTF are substrates of this reaction and can increase their synthesis to improve the production of ketopantoate. The MTF biosynthetic pathway in E. coli (and also Bacillus subtilis ) is shown in FIG. 2. MTF is synthesized from tetrahydrofolate and serine in a reaction catalyzed by the glyA gene encoding serine hydroxymethyl transferase. In improved MTF synthesis, cells require increased amounts of both substrate and glyA gene product.

In one embodiment, the present invention has (i) a deregulated pantothenate biosynthetic pathway (eg, 1, 2, 3 or 4 deregulated pantothetes) under conditions where pantothenate production is improved. Nate biosynthetic enzymes), (ii) culturing a microorganism having a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway (eg, having one or more deregulated MTF biosynthetic enzymes) Is characterized by a process for improved production of pantothenate. Exemplary pantothenate biosynthetic enzymes include ketopantoate hydroxymethyltransferase, ketopantoate reductase, pantothenate synthetase, and aspartate-α-decarboxylase. Exemplary MTF biosynthetic enzymes include the serA gene product and the glyA gene product.

In another embodiment, the present invention has (i) a deregulated pantothenate biosynthetic pathway (eg, 1, 2, 3 or 4 deregulated pantothenates) under conditions where pantothenate production is improved. Biosynthetic enzymes), (ii) has a deregulated isoleucine-valine (ilv) biosynthetic pathway (eg, has one, two or three deregulated ilv biosynthetic enzymes), (iii) deregulated A process for improved production of pantothenate, comprising culturing a microorganism having an MTF biosynthetic pathway (eg, having one or two or more deregulated MTF biosynthetic enzymes). Exemplary ilv biosynthetic enzymes include acetohydroxy acid synthetase, acetohydroxy acid isomerdeductase, and dihydroxyacid dehydratase.

In another embodiment, the present invention is directed to produce at least 50 g / L of pantothenate after 36 hours of microbial culture, preferably to at least 60 g / L of pantothenate after 36 hours of microbial culture, more preferably Most preferably, at least 70 g / L pantothenate is produced after 36 hours of culture, most preferably at least 80 g / L pantothenate, at least 90 g / L pantothenate, at least 100 g / L pantothene after 36 hours of microbial culture. , Pantothenate biosynthetic pathway deregulated to produce at least 110 g / L pantothenate, or at least 120 g / L pantothenate (or more) after 36 hours of microbial incubation, deregulated ilv biosynthetic pathway, and Characterized by a process for producing pantothenate, comprising culturing a microorganism with a regulated MTF biosynthetic pathway.

In another embodiment, the present invention is directed to produce at least 70 g / L of pantothenate after 48 hours of microbial culture, preferably at least 80 g / L of pantothenate after 48 hours of microbial culture, more preferably Culturing a microorganism having a deregulated pantothenate biosynthetic pathway, a deregulated ilv biosynthetic pathway, and a deregulated MTF biosynthetic pathway so that at least 90 g / L of pantothenate is produced after 48 hours of culture It is characterized by the production process of pantothenate.

In one exemplary embodiment, deregulation of the MTF biosynthetic pathway can be achieved by, for example, constitutive expression of the serA gene or by introducing a feedback resistant allele of serA to the serA gene in a panto-compound producing strain. This is accomplished by deregulating the product. In another exemplary embodiment, deregulation of the MTF biosynthetic pathway modulates glyA gene product in a panto-compound producing strain by, for example, overexpressing the glyA gene or modulating glyA gene inhibition by mutation or destruction of the purR gene product. By deregulation. In other exemplary embodiments, MTF biosynthesis is modulated by increasing serine or deregulating glycine cleavage enzymes in the culture medium.

The present invention also features the above-described methods, wherein pantothenate production is further improved by modulating pantothenate kinase activity (eg, reduced pantothenate kinase activity). In one embodiment, CoaA is deleted and CoaX is downregulated. In other embodiments, CoaX is deleted and CoaA is downregulated. In another embodiment, CoaX and CoaA are down regulated. The invention also features the above-described methods, wherein the microorganisms are cultured under excess serine conditions. The invention also features the above-described methods, wherein the microorganism has a pantothenate biosynthetic pathway that is deregulated such that pantothenate production is independent of β-alanine supply.

The product synthesized according to the process of the invention is also characterized in that it is a composition comprising pantothenate produced according to the above process. Recombinant microorganisms for use in the process of the invention are also featured. In one embodiment, the invention features recombinant microorganisms for enhancing pantothenate production, having a deregulated pantothenate biosynthetic pathway and a deregulated MTF biosynthetic pathway. In another embodiment, the invention features recombinant microorganisms for enhancing pantothenate production, having a deregulated pantothenate biosynthetic pathway, a deregulated MTF biosynthetic pathway, and a deregulated ilv pathway. The microorganism may also have reduced pantothenate kinase activity. Preferred microorganisms belong to the genus Bacillus, for example Bacillus subtilis.

As described above, certain aspects of the invention include the production of panto-compounds (eg, pantoate and / or pantothenate) comprising culturing microorganisms having at least a deregulated pantothenate biosynthetic pathway. Characterized in a process for improving the. The term “pantothenate biosynthetic pathway” refers to a pantothenate biosynthetic enzyme (eg, a polypeptide encoded by a biosynthetic enzyme-coding gene) used in the formation or synthesis of pantothenate, a compound (eg, a substrate , Intermediates or products), cofactors, and the like. The term “pantothenate biosynthetic pathway” includes biosynthetic pathways leading to pantothenate synthesis in microorganisms (eg, in vivo), and biosynthetic pathways leading to in vitro pantothenate synthesis.

As used herein, microorganisms having “deregulated pantothenate biosynthetic pathways” may improve pantothenate production (eg, as compared to pantothenate production in the microorganisms prior to deregulation of the biosynthetic enzymes). Or microorganisms having one or more deregulated pantothenate biosynthetic enzymes (eg, overexpressed) (as both terms are defined herein), relative to wild-type microorganisms. The term “pantothenate” refers to pantothenate in the free acid form, also referred to as “pantothenic acid,” as well as any salts thereof also referred to as “pantothenate salt” (eg, pantothenate or acidic hydrogen of pantothenic acid). In the presence of a cation such as calcium, sodium, potassium, ammonium, magnesium). The term "pantothenate" also includes alcohol derivatives of pantothenate. Preferred pantothenate salts are calcium pantothenate or sodium pantothenate. Preferred alcohol derivatives are pantothenol. Pantothenate salts and / or alcohols of the present invention include salts and / or alcohols prepared by conventional methods from the free acids described herein. In another embodiment, the pantothenate salt is synthesized directly by the microorganism of the present invention. The pantothenate salts of the present invention can likewise be converted to the free acid form of pantothenate or pantothenic acid by conventional means. The term "pantothenate" is also abbreviated herein as "pan".

Preferably, the microorganism "having a deregulated pantothenate biosynthetic pathway" has one or more deregulated pantothenate biosynthetic enzymes (eg, overexpressed) such that pantothenate production is at least 1 g / L. Contains microorganisms. More preferably, the microorganism “having a deregulated pantothenate biosynthetic pathway” may be characterized by one or more deregulated pantothenate biosynthetic enzymes (eg, overexpressed) such that pantothenate production is at least 2 g / L. It includes microorganisms having. Even more preferably, the microorganism "having a deregulated pantothenate biosynthetic pathway" has a pantothenate production of 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, Microorganisms having one or more deregulated pantothenate biosynthetic enzymes (eg, overexpressed) to be at least 60 g / L, 70 g / L, 80 g / L, 90 g / L.

The term “pantothenate biosynthetic enzyme” includes any enzyme used to synthesize a compound (eg, an intermediate or a product) of the pantothenate biosynthetic pathway. For example, the synthesis of pantoate from α-ketoisovalerate (α-KIV) proceeds with the intermediate, ketopantoate. The formation of ketopantoate is catalyzed by pantothenate biosynthetic enzyme PanB or ketopantoate hydroxymethyltransferase (panB gene product). The formation of pantoate is catalyzed by pantothenate biosynthetic enzyme PanE1 or ketopantoate reductase (panE1 gene product). The synthesis of β-alanine from aspartate is catalyzed by pantothenate biosynthetic enzyme PanD or aspartate-α-decarboxylase (panD gene product). Formation (eg, condensation) of pantothenate from pantoate and β-alanine is catalyzed by pantothenate biosynthetic enzyme PanC or pantothenate synthetase (panC gene product). Pantothenate biosynthetic enzymes may also perform an alternative function as enzymes in the HMBPA biosynthetic pathways described herein.

Thus, in one embodiment the present invention relates to pantothenate production comprising culturing a microorganism having one or more deregulated pantothenate biosynthetic enzymes (eg, deregulated to enhance pantothenate production). Characterized by methods for improving, said enzymes are for example PanB (or ketopantoate hydroxymethyltransferase), PanC (or pantothenate synthetase), PanD (or aspartate-α-decarboxyl Laze), PanE1 (or ketopantoate reductase). In another embodiment, the invention features a method for enhancing pantothenate production comprising culturing a microorganism having two or more deregulated pantothenate biosynthetic enzymes, wherein the enzyme is for example PanB (or Ketopantoate hydroxymethyltransferase), PanC (or pantothenate synthetase), PanD (or aspartate-α-decarboxylase) and PanE1 (or ketopantoate reductase) . In another embodiment, the invention features a method for enhancing pantothenate production comprising culturing a microorganism having three or more deregulated pantothenate biosynthetic enzymes, wherein the enzyme is for example PanB (or Ketopantoate hydroxymethyltransferase), PanC (or pantothenate synthetase), PanD (or aspartate-α-decarboxylase) and PanE1 (or ketopantoate reductase) . In another embodiment, the invention provides a microorganism having four or more deregulated pantothenate biosynthetic enzymes, such as deregulated PanB (or ketopantoate hydroxymethyltransferase), PanC (or pantothenate synthetase). ), PanD (or aspartate-α-decarboxylase) and PanE1 (or ketopantoate reductase) are characterized by a method for improving pantothenate production comprising culturing microorganisms.

In another aspect, the invention features a process for improving pantothenate production, comprising culturing a microorganism having a deregulated isoleucine-valine biosynthetic pathway. The term “isoleucine-valine biosynthetic pathway” refers to an isoleucine-valine biosynthetic enzyme (eg, a polypeptide encoded by a biosynthetic enzyme-coding gene), a compound (used to form or synthesize pyruvate conversion to valine or isoleucine). Biosynthetic pathways including, for example, substrates, intermediates or products), cofactors, and the like. The term “isoleucine-valine biosynthetic pathway” includes biosynthetic pathways that synthesize valine or isoleucine in microorganisms (eg, in vivo), as well as biosynthetic pathways that synthesize valine or isoleucine in vitro.

As used herein, microorganisms "having a deregulated isoleucine-valine (ilv) pathway" enhance the production of isoleucine and / or valine and / or valine precursors, α-kenoisovalerate (α-KIV). One or more deregulated (eg, relative to the production of isoleucine and / or valine and / or α-KIV in the microorganism prior to deregulation of the biosynthetic enzyme, or relative to wild-type microorganisms) And microorganisms with isoleucine-valine (ilv) biosynthetic enzymes (both terms defined herein), overexpressed. 1 includes a schematic of the isoleucine-valine biosynthetic pathway. Isoleucine-valine biosynthetic enzymes are shown in bold italics and the corresponding genes are shown in italics. The term “isoleucine-valine biosynthetic enzyme” includes any enzyme used to form a compound (eg, an intermediate or a product) of the isoleucine-valine biosynthetic pathway. According to FIG. 1, the synthesis of valine from pyruvate is via intermediate, acetolactate, α, β-dihydroxyisovalerate (α, β-DHIV) and α-ketoisovalerate (α-KIV) Proceed. Formation of acetolactate from pyruvate is catalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxy acid synthetase (ilvBN gene product, or alsS gene product instead). The synthesis of α, β-DHIV from acetolactate is catalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxy acid isomer reductase (ilvC gene product). The synthesis of α-KIV from α, β-DHIV is catalyzed by the isoleucine-valine biosynthetic enzyme dihydroxy acid dehydratase (ilvD gene product). In addition, valine and isoleucine can be interconverted to their respective α-keto compounds by branched chain amino acid transaminase. Isoleucine-valine biosynthetic enzymes may also perform an alternative function as enzymes in the HMBPA biosynthetic pathways described herein.

Thus, in one embodiment the present invention relates to one or more deregulated isoleucine-valine (ilv) biosynthetic enzymes (eg, deregulated to enhance valine and / or isoleucine and / or α-KIV production). And a method for improving the production of pantothenate comprising culturing the microorganism having the enzyme, wherein the enzyme is for example IlvBN, AlsS (or acetohydroxy acid synthetase), IlvC (or acetohydroxy acid isomers). Ductase) and IlvD (or dihydroxy acid dehydratase). In another embodiment, the invention features a method for enhancing pantothenate production comprising culturing a microorganism having two or more deregulated isoleucine-valine (ilv) biosynthetic enzymes, wherein the enzyme is, for example, IIvBN, AlsS (or acetohydroxy acid synthetase), IlvC (or acetohydroxy acid isomer reductase) and IlvD (or dihydroxy acid dehydratase). In another embodiment, the invention features a method for enhancing pantothenate production comprising culturing a microorganism having three or more deregulated isoleucine-ilv biosynthetic enzymes, e.g. Deregulated IlvBN or AlsS (or acetohydroxy acid synthetase), IlvC (or acetohydroxy acid isomer reductase) and IlvD (or dihydroxy acid dehydratase).

As mentioned herein, enzymes of the pantothenate biosynthetic pathway and / or the isoleucine-valine (ilv) pathway are [R] -3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid (" HMBPA ") has been found to have altered activity in the synthesis or in the [R] -3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid (" HMBPA ") biosynthetic pathway. The term “[R] -3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid (“ HMBPA ”) biosynthetic pathway” refers to the pantothenate biosynthetic pathway traditionally used for the formation or synthesis of HMBPA. And / or alternative biosynthetic pathways comprising biosynthetic enzymes and compounds (eg, substrates, etc.) associated with the isoleucine-valine biosynthetic pathway. The term “HMBPA biosynthetic pathway” includes biosynthetic pathways that synthesize HMBPA in microorganisms (eg, in vivo), and biosynthetic pathways that synthesize HMBPA in vitro.

The term “HMBPA biosynthetic enzyme” includes any enzyme used to form a compound (eg, an intermediate or a product) of the HMBPA biosynthetic pathway. For example, the synthesis of 2-hydroxyisovaleric acid (α-HIV) from α-ketoisovalerate (α-KIV) is known as panE1 or panE2 gene product (panE1 is otherwise referred to herein as ketopantoate reductase. And / or catalyzed by the ilvC gene product (hereafter referred to as acetohydroxy acid isomer reductase). The formation of HMBPA from β-alanine and α-HIV is catalyzed by the panC gene product (hereafter referred to as pantothenate synthetase).

The term "[R] -3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid (" HMBPA ")" means "[R] -3- (2-hydroxy-3-methyl- HMBPA in free acid form, also referred to as "butyrylamino) -propionate, as well as its" 3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid salt "or" HMBPA salt " Acidic hydrogen of any salt (eg, 3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid or 3- (2-hydroxy-3-methyl-butyrylamino) -propionate In the presence of a cation such as calcium, sodium, potassium, ammonium, magnesium). Preferred HMBPA salts are calcium HMBPA or sodium HMBPA. HMBPA salts of the present invention include salts prepared by conventional methods from the free acid described herein. HMBPA salts of the present invention may also be prepared by conventional methods, such as 3- (2-hydroxy-3-methyl-butyrylamino) -propionic acid or 3- (2-hydroxy-3-methyl-butyrylamino) -prop It can be converted to the free acid form of cypionate.

In a preferred embodiment, the invention relates to the preparation of a panto-compound (eg pantoate and / or pantothenate) comprising culturing a microorganism having a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway. Characterized by a process for improving production. The term “methylenetetrahydrofolate (MTF) biosynthetic pathway” refers to a PanB substrate, an MTF biosynthetic enzyme (eg, a polypeptide encoded by a biosynthetic enzyme-coding gene) used for the formation or synthesis of MTF, a compound (eg For example, substrates, intermediates or products), cofactors, and the like. The term “methylenetetrahydrofolate (MTF) biosynthetic pathway” refers to a biosynthetic pathway (eg, a pathway in E. coli as shown in FIG. 2) that synthesizes MTF in vivo, as well as an MTF in vitro Biosynthetic pathway for synthesizing The term “methylenetetrahydrofolate (MTF) biosynthetic enzyme” includes any enzyme used to form a compound (eg, an intermediate or a product) of the methylenetetrahydrofolate (MTF) biosynthetic pathway.

The present invention is based, at least in part, on the discovery that deregulation of certain MTF biosynthetic enzymes improves MTF production. MTF biosynthetic enzymes are called "MTF biosynthetic-enhancing enzymes" because of their improved MTF production. Exemplary "MTF biosynthesis-enhancing enzymes" are the serA gene product (3-phosphoglycerate dehydrogenase) and the glyA gene product (serine hydroxymethyl transferase). Microorganisms having “deregulated methylenetetrahydrofolate (MTF) biosynthetic pathways” improve MTF production or biosynthesis (eg, compared to MTF production in the microorganisms or in wild-type microorganisms prior to deregulation of the biosynthetic enzymes). Microorganisms with one or more deregulated MTF biosynthetic-enhancing enzymes (eg, overexpressed).

In one embodiment, the present invention comprises culturing a microorganism having a deregulated "methylenetetrahydrofolate (MTF) biosynthetic pathway" as defined herein, such as a panto-compound (eg pantoate and ( Or) pantothenate). In another embodiment, the invention provides a method for enhancing panto-compound (eg, pantoate and / or pantothenate) production comprising culturing a microorganism with deregulated MTF biosynthesis-enhancing enzyme. It is characterized by. In a preferred embodiment, the present invention provides a method for culturing microorganisms with deregulated glyA gene product (serine hydroxymethyl transferase) and / or deregulated serA gene product (3-phosphoglycerate dehydrogenase). A process for enhancing panto-compound (eg pantoate and / or pantothenate) production, including.

Another aspect of the invention is a pantothenate comprising culturing the microorganism under selected culture conditions to favor pantothenate production, for example, culturing the microorganism with an excess of serine (glyA substrate) in the medium. Characterized by a process for improving production. The term "excess serine" includes increased or higher serine levels than are routinely used to culture the microorganisms of interest. For example, the culturing of Bacillus microorganisms described in the Examples of the present invention is typically in the presence of about 0-2.5 g / L serine. Thus, excess serine levels may include levels above 2.5 g / L serine, for example about 2.5 to 10 g / L serine. Excess serine levels may include levels above 5 g / L serine, for example about 5-10 g / L serine.

Another aspect of the present invention is to increase, for example, the pantothenate and / or isoleucine-valine (ilv) biosynthetic pathway precursors and / or intermediates (e.g., excess β-alanine in a microorganism). Cultured in the presence of valine and / or α-KIV, or alternatively by further modifying the microorganism (ie, to produce a significant level of β-alanine in the absence of β-alanine supply) (Beta) -alanine-independent microorganisms such as those described in US patent application Ser. No. 09/09 / 667,569) characterized by culturing the microorganisms described herein under conditions such that pantothenate production is further increased.

Another aspect of the invention is characterized by further regulating the pantothenate kinase activity of the pantothenate-producing strain so that pantothenate production is improved. Pantothenate kinases are key enzymes that catalyze the formation of coenzyme A (CoA) from pantothenate (see, eg, US patent application Ser. No. 09/09 / 667,569). Modulation of pantothenate kinase (eg, reduction in activity or level of pantothenate kinase) reduces the production of CoA, making pantothenate accumulation advantageous. In one embodiment, pantothenate kinase activity is reduced by deleting CoaA and downregulating CoaX activity (both CoaA and CoaX can catalyze the first step of CoA biosynthesis in a particular microorganism). In another embodiment, pantothenate kinase activity is reduced by deleting CoaX and downregulating CoaA. In another embodiment, pantothenate kinase activity is reduced by downregulating CoaA and CoaX activity.

Various aspects of the invention are described in detail below.

I. Targeting genes encoding various pantothenates and / or isoleucine-valine (ilv) and / or methylenetetrahydrofolate (MTF) biosynthetic enzymes

In one embodiment, the invention is characterized by modifying or increasing the levels of various biosynthetic enzymes of pantothenate and / or isoleucine-valine (ilv) and / or methylenetetrahydrofolate (MTF) biosynthetic pathways do. In particular, the invention is characterized by modifying various enzyme activities associated with the pathway by modifying or modifying the gene encoding the biosynthetic enzyme.

As used herein, the term "gene" refers to intergenic DNA (i.e. intervening or spacer DNA that is naturally adjacent to and / or separates a gene from the chromosomal DNA of the organism). Nucleic acid molecules (eg, DNA molecules or portions thereof) that can be separated from other genes or other genes. Alternatively, the gene may slightly overlap with other genes (eg, the first gene at the 3 'end overlaps the 5' end of the second gene) and the overlapping gene may be separated from the other gene by intergenic DNA. Genes may directly synthesize enzymes or other protein molecules (eg may include coding sequences, eg, adjacent open reading frames (ORFs) that encode proteins), or may function on their own within an organism. have. Genes in an organism can be clustered within operons, and as described herein, the operons are separated from other genes and / or operons by intergenic DNA. As used herein, an “isolated gene” includes a gene that is substantially free of sequences naturally adjacent to the gene in the chromosomal DNA of the organism from which the gene is derived (ie, encodes a second or separate protein adjacent to a structural sequence or the like). Without adjacent coding sequences), optionally 5 'and 3' regulatory sequences such as promoter sequences and / or terminator sequences. In one embodiment, the isolated gene mainly comprises a coding sequence for the protein (eg, a sequence encoding a Bacillus protein). In other embodiments, an isolated gene is a coding sequence for a protein (eg, for a Bacillus protein), and adjacent 5 ′ and / or 3 ′ regulatory sequences (eg, from chromosomal DNA of the organism from which the gene is derived). Eg, contiguous 5 ′ and / or 3 ′ Bacillus regulatory sequences). Preferably, the isolated gene is about 10 kb, 5 kb, 2 kb, 1 kb, 0.5 kb, 0.2 kb, 0.1 kb, 50 bp, naturally adjacent to the gene in the chromosomal DNA of the organism from which the gene is derived. Nucleotide sequences of less than 25 bp or 10 bp.

The term "operon" includes one or more genes or two or more contiguous genes or ORFs with optionally overlapping sequences at the 5 'or 3' end of the ORF. The term “operon” refers to a coordinated unit of gene expression comprising a promoter and possibly regulatory elements associated with one or more adjacent genes or ORFs (eg structural genes encoding enzymes, eg biosynthetic enzymes). It includes. Expression of genes (eg, structural genes) can be coordinated by, for example, regulatory proteins that bind to regulatory elements, or by anti-termination of transcription. The gene of the operon (eg, a structural gene) can be transcribed into a single mRNA encoding all proteins.

As used herein, a “mutant gene” or “mutant gene” refers to one or more modifications such that the polypeptide or protein encoded by the mutation exhibits different activity than the polypeptide or protein encoded by the wild-type nucleic acid molecule or gene. Genes with nucleotide sequences, including (eg, substitutions, insertions, deletions). In one embodiment, the gene with the mutation or the mutant gene is increased relative to the polypeptide or protein encoded by the wild type gene, for example when analyzed under similar conditions (eg, in a microorganism cultured at the same temperature). Encodes a polypeptide or protein that has active activity. As used herein, "increased activity" or "increased enzymatic activity" means at least 5% higher activity than the activity of a polypeptide or protein encoded by a wild type nucleic acid molecule or gene, encoded by a wild type nucleic acid molecule or gene. Over at least 5-10% higher activity, more preferably at least 10-25% higher activity, even more preferably at least 25-50%, 50-75% or 75-100 relative to the activity of the polypeptide or protein % High activity. The intermediate ranges of the above mentioned values, for example 75-85%, 85-90%, 90-95%, are also intended to be included in the present invention. As used herein, “increased activity” or “increased enzymatic activity” is also at least 1.25 times higher than the activity of the polypeptide or protein encoded by the wild-type gene, activity of the polypeptide or protein encoded by the wild-type gene. More preferably at least 1.5 times higher activity, more preferably at least 2 times higher activity, even more preferably at least 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times higher activity do.

In other embodiments, the gene with the mutation or the mutant gene is reduced compared to the polypeptide or protein encoded by the wild-type gene, for example when analyzed under similar conditions (eg, in a microorganism cultured at the same temperature). Encoding a polypeptide or protein having activity. Mutant genes may also encode polypeptides or produce reduced levels of wild type polypeptides. As used herein, "reduced activity" or "reduced enzyme activity" means at least 5% less activity than the activity of a polypeptide or protein encoded by a wild type nucleic acid molecule or gene, preferably a wild type nucleic acid molecule or gene. Is an activity that is at least 5-10% less, more preferably at least 10-25%, even more preferably less than 25-50%, 50-75% or 75-100% less than the activity of the polypeptide or protein encoded by . Intermediate ranges of the aforementioned values, for example 75-85%, 85-90%, 90-95%, are also intended to be included in the present invention. As used herein, "reduced activity" or "reduced enzyme activity" is also about 100 greater than the activity of a deleted or "knocked out" activity (eg, a polypeptide or protein encoded by a wild type nucleic acid molecule or gene). Less than% activity).

Activity can be measured according to any well-known assay for measuring the activity of a particular protein of interest. Activity can be measured or assayed directly, for example, by measuring the activity of proteins in crude cell extracts, or proteins isolated or purified from cells or microorganisms. In general, activity may be measured or assayed in cells or microorganisms, or in extracellular medium. For example, assays of mutant genes (ie, said mutations encoding reduced enzymatic activity) express mutated genes in microorganisms, for example mutant microorganisms in which the enzymes are temperature-sensitive, and are temperature sensitive to enzymatic activity. (Ts) can be achieved by analyzing mutant genes for their ability to complement mutations. Mutant genes encoding “increased enzyme activity” may be, for example, to more effectively complement Ts mutations than the corresponding wild type genes. Mutant genes encoding "reduced enzymatic activity" are, for example, less effective complementary to Ts mutations than the corresponding wild type genes.

Those skilled in the art will appreciate that only one substitution in a nucleic acid or gene sequence (eg, a base substitution that encodes an amino acid change in the corresponding amino acid sequence) will significantly affect the activity of the encoded polypeptide or protein relative to the corresponding wild type polypeptide or protein. It will be appreciated. As defined herein, a mutant gene (eg, encoding a mutant polypeptide or protein) expresses the mutant gene or produces the mutant protein or polypeptide as compared to the corresponding microorganism that optionally expresses the wild type gene. Can be readily distinguished from nucleic acids or genes encoding protein homologues in that they encode proteins or polypeptides with modified activity that can be observed in different or completely different phenotypes in a microorganism (ie mutant microorganism). In contrast, protein homologues, when produced in a microorganism, may have the same or substantially similar activity, optionally phenotypicly indistinguishable from corresponding microorganisms expressing wild-type genes. Thus, for example, distinguishing homologues and mutations is not the degree of sequence identity between nucleic acid molecules, genes, proteins or polypeptides, but distinguishing homologues and mutations is the activity of the encoded protein or polypeptide. For example, having low sequence identity (eg, 30-50% sequence identity) but having substantially equivalent functional activity, mutations share, for example, 99% sequence identity but are significantly different or distinct functional activity ).

Those skilled in the art will also appreciate that the nucleic acid molecules, genes, proteins or polypeptides for use in the present invention may be derived from any microorganism having an MTF biosynthetic pathway, an ilv biosynthetic pathway or a pantothenate biosynthetic pathway. Such nucleic acid molecules, genes, proteins or polypeptides can be identified by those skilled in the art using known techniques such as homology screening, sequence comparisons, etc., wherein the expression or production of the nucleic acid molecule, gene, protein or polypeptide is recombinant. Modification of the gene sequence in a manner that occurs within the microorganism (e.g., by using appropriate promoters, ribosomal binding sites, expression or integration vectors, according to the techniques described herein and those known in the art). Modifications (possibly by taking into account preferred codon usage), etc., by one skilled in the art.

In one embodiment, the genes of the present invention are derived from Gram positive microbial organisms (eg, microorganisms having basic dyes, eg crystal violet due to the presence of Gram-positive walls surrounding the microorganisms). The term “derived from” (eg, “derived from a gram positive microorganism”) refers to a gene that is found naturally in a microorganism (eg, naturally found in a gram positive microorganism). In a preferred embodiment, the genes of the invention are Bacillus, Cornyebacterium (e.g., Cornyebacterium glutamicum ), Lactobacillus , Lactococci and It is derived from a microorganism belonging to the genus selected from the group consisting of Streptomyces . In a more preferred embodiment, the gene of the present invention is derived from a microorganism of the genus Bacillus. In another preferred embodiment, the genes of the invention are Bacillus subtilis, Bacillus lentimorbus , Bacillus lentus , Bacillus pertus , Bacillus firmus , Bacillus panthothenticus , Bacillus amyloliquefaciens , Bacillus cereus , Bacillus circulans , Bacillus coagulans , Bacillus licheniformis , Bacillus licheniformis (Bacillus megaterium), Bacillus pumil Ruth (Bacillus pumilus), Bacillus-to Lindsay N-Sys (Bacillus thuringiensis), Bacillus halo two lance (Bacillus halodurans), and for example, from the group consisting of other group 1 Bacillus species, which is characterized by 16SrRNA form Derived from the microorganism selected. In another preferred embodiment, the gene is derived from Bacillus brevis or Bacillus stearothermophilus . In another preferred embodiment, the genes of the present invention consists of a Bacillus piece you formate miss (Bacillus licheniformis), Bacillus amyl Lowry quinolyl Pacific Enschede (Bacillus amyloliquefaciens), Bacillus subtilis (Bacillus subtilis) and Bacillus pumil Ruth (Bacillus pumilus) Derived from a microorganism selected from the group consisting of: In a particularly preferred embodiment, the gene is derived from Bacillus subtilis (eg, Bacillus subtilis-derived). The terms “derived from Bacillus subtilis” or “Bacillus subtilis-derived” include genes found naturally in microbial Bacillus subtilis. Bacillus-derived genes (eg, B. subtilis -derived genes) included in the scope of the present invention are, for example, Bacillus or B. subtilis purR gene, serA gene, glyA gene, coaX gene, coaA gene, pan gene. And / or the ilv gene.

In another embodiment, the gene of the present invention is derived from a gram negative (retroduced basic dye) microorganism. In a preferred embodiment, the gene of the present invention, Salmonella (Salmonella) (e.g., S. typhimurium (Salmohella typhimurium)), Escherichia (Escherichia), Klebsiella (Klebsiella), Serratia marcescens (Serratia) and It is derived from a microorganism belonging to the genus selected from the group consisting of Proteus . In a more preferred embodiment, the gene of the present invention is derived from a microorganism of the genus Escherichia. In an even more preferred embodiment, the genes of the invention are derived from Escherichia coli . In another embodiment, the genes of the present invention (e.g., in my process serenity busy as Saccharomyces (Saccharomyces cerevisiae)) My process (Saccharomyces) are derived from a saccharide.

II. Recombinant Nucleic Acid Molecules and Vectors

The invention also relates to the genes described herein (eg, isolated genes), preferably the Bacillus gene, more preferably the Bacillus subtilis gene, even more preferably the Bacillus subtilis pantothenate biosynthesis gene. And / or a recombinant nucleic acid molecule (eg, a recombinant DNA molecule) comprising an isoleucine-valine (ilv) biosynthesis gene and / or a methylenetetrahydrofolate (MTF) biosynthesis gene. The term “recombinant nucleic acid molecule” is modified, modified, or engineered so that the nucleotide sequence differs (eg, by the addition, deletion or substitution of one or more nucleotides) from a native or natural nucleic acid molecule from which the recombinant nucleic acid molecule is derived. Nucleic acid molecules (eg, DNA molecules). Preferably, the recombinant nucleic acid molecule (eg, recombinant DNA molecule) comprises an isolated gene of the present invention operably linked to a regulatory sequence. The phrase “operably linked to regulatory sequence (s)” allows a gene to be expressed (eg, enhanced, increased, always expressive, basal, attenuated, reduced or suppressed expression). Nucleotides of the gene of interest, preferably in a manner such that the gene product encoded by the gene is expressed (e.g., when a recombinant nucleic acid molecule is included in a recombinant vector and introduced into a microorganism as described herein) By sequences linked to regulatory sequence (s).

The term "regulatory sequence" includes nucleic acid sequences that affect (eg, modulate or regulate) the expression of other nucleic acid sequences (ie, genes). In one embodiment the regulatory sequence is similar or identical to a particular gene of interest when viewed with respect to the regulatory sequence and to a gene of interest that appears in nature, for example, a natural position and / or orientation. (Or) is incorporated into the recombinant nucleic acid molecule in an orientation. For example, a gene of interest may be operably linked to a regulatory sequence that accompanies or is adjacent to a gene of interest in a natural organism (eg, to a "native" regulatory sequence (eg, to a "native" promoter)). Operably linked) into a recombinant nucleic acid molecule. Alternatively, the gene of interest can be included in a recombinant nucleic acid molecule that is operably linked to regulatory sequences that carry or are adjacent to other (eg, different) genes in a natural organism. Alternatively, the gene of interest can be included in a recombinant nucleic acid molecule operably linked to regulatory sequences from other organisms. For example, regulatory sequences from other microorganisms (eg, other bacterial regulatory sequences, bacteriophage regulatory sequences, etc.) may be operably linked to a particular gene of interest.

In one embodiment, the regulatory sequence is a non-native or non-native sequence (eg, a modified, mutated, substituted, derived, deleted sequence comprising a chemically synthesized sequence). Preferred regulatory sequences include promoters, enhancers, termination signals, anti-termination signals, and other expression control elements (e.g., sequences to which an inhibitor or inducer binds, and / or for example, transcription and transcription in mRNA). (Or) binding sites for translational regulatory proteins). Such regulatory sequences are described, for example, in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning : A Laboratory Manual. 2nd, ed., Cold Sprig Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Regulatory sequences include the constant expression of nucleotide sequences in a microorganism (e.g., a constitutive promoter and a strong constitutive promoter), or the inducible expression of a nucleotide sequence in a microorganism (e.g., For example, inducible promoters such as xylose inducible promoters, and attenuating or inhibiting the expression of nucleotide sequences in a microorganism (eg, attenuating signal or inhibitor binding sequences such as PurR binding sites) ). It is also within the scope of the present invention to regulate expression of the gene of interest by removing or deleting regulatory sequences. For example, sequences related to negative regulation of transcription can be removed to enhance expression of the gene of interest.

In one embodiment, a recombinant nucleic acid molecule of the invention comprises one or more bacterial gene products (eg, pantothenate biosynthetic enzyme, isoleucine-valine biosynthetic enzyme, and / or methylene, operably linked to a promoter or promoter sequence. Nucleic acid sequences or genes encoding tetrahydrofolate (MTF) biosynthetic enzymes). Preferred promoters of the invention include Bacillus promoters and / or bacteriophage promoters (eg, bacteriophage infecting Bacillus). In one embodiment, the promoter is a Bacillus promoter, preferably a Bacillus strong promoter (e.g., a promoter associated with the biochemical housekeeping gene of Bacillus, or a promoter associated with the corresponding pathway gene of Bacillus). In other embodiments, the promoter is a bacteriophage promoter. In a preferred embodiment, the promoter is a promoter of bacteriophage SP01. In a particularly preferred embodiment, the promoter is for example selected from the group consisting of P ₁₅ , P ₂₆ or P _veg having the respective sequence:

Further preferred promoters include tef (translation elongation factor (TEF) promoter) and pyc (pyruvate carboxylase (PYC) promoter) that promote high levels of expression in Bacillus (eg, Bacillus subtilis). do. For example, further preferred promoters for use in Gram positive microorganisms include, but are not limited to, amy and SPO2 promoters. For example, further preferred promoters for use in Gram-negative microorganisms are cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIQ, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL, including but not limited to.

In other embodiments, recombinant nucleic acid molecules of the invention comprise terminator sequences or terminator sequences (eg, transcription terminator sequences). The term "terminator sequence" includes regulatory sequences that act to terminate transcription of mRNA. Terminator sequences (or tandem transcription terminators) may also function to stabilize mRNA (eg, by adding structure to mRNA), for example, against nucleases.

In another embodiment, a recombinant nucleic acid molecule of the invention encodes a sequence (i.e., a detectable and / or selectable marker) such as an antibiotic resistant sequence or encodes a nutritive mutation, allowing detection of a vector comprising said sequence. For example genes that overcome trpC, drug markers, fluorescent markers, and / or colorimetric markers (eg lacZ / β-galactosidase). In another embodiment, a recombinant nucleic acid molecule of the invention comprises an artificial ribosomal binding site (RBS), or a sequence that is transcribed into artificial RBS. The term “artificial ribosomal binding site (RBS)” refers to a ribosome that binds (eg, initiates translation) that differs from innate RBS (eg, RBS found in native genes) and one or more nucleotides. sites within an mRNA molecule (eg, encoded in DNA). Preferred artificial RBSs are 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 (of which about 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-15 or more are born RBS (eg, interest Congenital RBS of the gene of interest, e.g., congenital panB RBS Or born panD RBS And nucleotides). Preferably, the different nucleotides are identical to one or more nucleotides of the ideal RBS when optimally arranged for comparison. Ideal RBS It includes, but is not limited to. Artificial RBS can be used to replace natural or innate RBS associated with a particular gene. Artificial RBS preferably increases the translation of certain genes. Preferred artificial RBS (e.g., RBS to increase translation of panB, e.g. Bacillus subtilis panB)

And

It includes. Preferred artificial RBS (eg, RBS to increase translation of panD, eg, Bacillus subtilis panD)

It includes.

The invention also features a vector (eg, a recombinant vector) comprising a nucleic acid molecule (eg, a gene or recombinant nucleic acid molecule comprising the gene) as described herein. The term “recombinant vector” refers to a vector (eg, modified, modified, or engineered to include more, less, or different nucleic acid sequences than those contained in the native or natural nucleic acid molecule from which the recombinant vector is derived (eg, Plasmids, phages, phasmids, viruses, cosmids or other purified nucleic acid vectors). Preferably, the recombinant vector comprises a biosynthetic enzyme comprising said gene operably linked to regulatory sequences, eg, promoter sequences, terminator sequences, and / or artificial ribosomal binding sites (RBS) as described herein. Coding genes or recombinant nucleic acid molecules. In other embodiments, recombinant vectors of the invention comprise sequences that enhance replication in bacteria (eg, replication-enhancing sequences). In one embodiment, the replication-enhancing sequence functions in E. coli . In other embodiments, the replication-enhancing sequence is derived from pBR322.

In another embodiment, the recombinant vector of the invention comprises an antibiotic resistance sequence. The term “antibiotic resistance sequence” includes sequences that promote or confer resistance to antibiotics in a host organism (eg, Bacillus). In one embodiment, the antibiotic resistance sequence comprises a cat (chloramphenicol resistant) sequence, a tet (tetracycline resistant) sequence, an erm (erythromycin resistant) sequence, a neo (neomycin resistant) sequence, a kan (kanamycin resistant) sequence and a spec ( Spectinomycin resistance). Recombinant vectors of the invention may also include homologous recombination sequences (eg, sequences designed to recombine a gene of interest into the chromosome of a host organism). For example, a bpr, vpr or amyE sequence can be used as a homology target for recombination into a host chromosome. Those skilled in the art will also understand that vectors may be custom designed depending on factors such as the choice of genetically engineered microorganisms, the level of expression of the desired gene product, and the like.

III. Recombinant microorganism

The invention also features microorganisms, ie recombinant microorganisms, comprising vectors or genes (eg, wild type and / or mutated genes) as described herein. As used herein, the term "recombinant microorganism" is intended to have a modification, modification, or different genotype and / or phenotype as compared to the native microorganism from which it is derived (e.g., genetic modification is applied to the coding nucleic acid sequence of the microorganism). Affecting) genetically modified, modified or engineered (eg, genetically engineered) microorganisms (eg, bacteria, yeast cells, fungal cells, etc.).

In one embodiment, the recombinant microorganism of the present invention is a gram positive organism (eg, a microorganism having a basic dye, eg, 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 the genus selected from the group consisting of Bacillus, Corynebacterium (eg, Corynebacterium glutamicum), Lactobacillus, Lactokcockki and Streptomyces. In a more preferred embodiment, the recombinant microorganism is of the genus Bacillus. In another preferred embodiment, the recombinant microorganism is Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus permus, Bacillus pantotenticus, Bacillus amyloriquifaciens, Bacillus cereus, Bacillus circus, Bacillus coagulum Lances, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurance, and other Group 1 Bacillus species, for example characterized by 16SrRNA form. In another preferred embodiment, the recombinant microorganism is Bacillus brevis or Bacillus stearothermophilus. In another preferred embodiment, the recombinant microorganism is selected from the group consisting of Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus subtilis and Bacillus pumilus.

In other embodiments, the recombinant microorganism is a gram negative (retroducing base dye) organism. In a preferred embodiment, the recombinant microorganism is a microorganism belonging to the genus selected from the group consisting of Salmonella (eg Salmonella typhimurium), 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 other embodiments, the recombinant microorganism is Saccharomyces (eg, Saccharomyces cerevisiae).

Preferred "recombinant" microorganisms of the present invention include deregulated pantothenate biosynthetic pathways or enzymes, deregulated isoleucine-valine (ilv) biosynthetic pathways or enzymes, and / or modified or deregulated methylenetetrahydrofolate ( MTF) is a microorganism with a biosynthetic pathway or enzyme. The term “deregulated” or “deregulated” includes modifying or modifying one or more genes in a microorganism that encodes an enzyme in the biosynthetic pathway such that the level or activity of the biosynthetic enzyme in the microorganism is modified or modified. . Preferably, one or more genes encoding enzymes in the biosynthetic pathway are modified or modified to enhance or increase the gene product. The phrase “deregulated pathway” includes a biosynthetic pathway in which one or more genes encoding enzymes in the biosynthetic pathway have been modified or modified such that the level or activity of the one or more biosynthetic enzymes is modified or modified. In some cases, the ability to “deregulate” a pathway in a microorganism (eg, to simultaneously deregulate one or more genes in a given biosynthetic pathway) may include one or more enzymes (eg, two or three biosynthetic enzymes). ) Originates from the unique phenomenon of microorganisms, which are encoded by adjacent genes on adjacent pieces of a genetic material called "operon" (defined herein). Because of the coordinated regulation included in the operon, the modification or modification of a single promoter and / or regulatory element may modify or modify the expression of each gene product encoded by the operon. Alteration or modification of regulatory elements may include removal of endogenous and / or regulatory factors, addition of strong promoters, inducible promoters or multiple promoters, removal of regulatory sequences such that expression of the gene product is modified, modification of the chromosome position of the gene or operon Alteration of a nucleic acid sequence adjacent to (or within an operon) a gene or operon, such as a ribosome binding site, increased copy number of the gene or operon, transcription of the gene or operon and / or translation of the gene product or operon or gene product thereof, respectively. Modifying the protein involved (e.g., regulatory protein, inhibitor, enhancer, transcriptional activator, etc.), or any conventional means in the art to deregulate expression of the gene pathway (e.g. block the expression of the suppressor protein) Including but not limited to the use of antisense nucleic acid molecules for Not limited. Deregulation may also be involved in altering the coding region of one or more genes to provide enzymes that are, for example, feedback resistant or have greater or smaller specific activity.

In another preferred aspect, the recombinant microorganism is designed or engineered to overexpress one or more pantothenate biosynthetic enzymes, one or more isoleucine-valine biosynthetic enzymes, and / or one or more MTF biosynthetic enzymes. The term “overexpressed” or “overexpressed” includes expressing a gene product (eg a biosynthetic enzyme) in an amount greater than that expressed prior to the manipulation or expressed in a significant microorganism not manipulated. In another embodiment, the microorganism is processed or engineered to overexpress an amount of more gene product than is expressed in a significant microorganism that is not genetically engineered.

Genetic engineering involves the regulation or alteration of 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 so that expression is expressed at all times), Altering the chromosomal position of a protein, altering a nucleic acid sequence adjacent to a particular gene, such as a ribosome binding site, translating a particular gene product and / or proteins involved in transcription of the particular gene (eg, regulatory proteins, inhibitors) , Modification of enhancers, transcriptional activators, etc.), or the use of any other conventional means (eg, antisense nucleic acid molecules to block expression of suppressor proteins) in the industry to deregulate expression of specific gene pathways. But not limited to). Genetic engineering can include deletion of a gene, for example, to block pathways or remove inhibitors.

In another aspect, the microorganism may be physically or environmentally engineered to overexpress levels of gene products more than in a microorganism that is more or not manipulated than is expressed prior to manipulation of the microorganism. For example, by processing or culturing in the presence of a reagent known or suspected to increase transcription of a particular gene and / or translation of a particular gene product, transcription and / or translation may be augmented or enhanced. Alternatively, the microorganism may be cultured at a temperature selected to increase transcription of a particular gene and / or translation of a particular gene product so that transcription and / or replication may be enhanced or enhanced.

IV. Cultivation and Fermentation of Recombinant Microorganisms

The term "culture" includes growing and / or maintaining (eg, maintaining and / or growing a culture or strain) of the living microorganisms of the present invention. In one embodiment, the microorganisms of the invention are cultured in liquid medium. In another embodiment, the organisms of the invention are cultured in solid or semi-solid media. In a preferred embodiment, the microorganisms of the present invention may contain nutrients essential or beneficial for the maintenance and / or growth of microorganisms (for example carbon sources or carbon substrates such as carbohydrates, hydrocarbons, oils, fats, fatty acids, organic acids and alcohols; nitrogen sources such as Peptone, yeast extract, meat extract, malt extract, soybean meal, soybean powder, soybean flour, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate, phosphorus sources such as phosphoric acid, potassium thereof and Sodium salts; medium containing trace elements such as 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) For example in sterile, liquid medium).

Preferably, the microorganism of the present invention is incubated at a controlled pH. The term "regulated pH" includes any pH that results in the desired product (eg pantoate and / or pantothenate). In one embodiment, the microorganism is incubated at pH about 7. In another embodiment, the microorganism is incubated at a pH of 6.0-8.5. Preferred pH can be maintained by a variety of methods known in the art.

Also, preferably, the microorganism of the present invention is cultured under controlled aeration. The term "regulated aeration" includes sufficient aeration (eg oxygen) to produce the desired product (eg pantoate and / or pantothenate). In one embodiment, the aeration is controlled by controlling the amount of oxygen in the culture. Preferably, the aeration of the culture is controlled by stirring the culture. It can be agitated by rotating or vibrating a culture vessel (eg a tube or flask) by a propeller or similar mechanical stirring device, or by various pumping devices. Aeration may be further controlled by the passage of sterile air or oxygen through the medium (eg, through the fermentation mixture). Also preferably, the microorganisms of the invention are cultured without excess foaming (eg by the addition of antifoaming agents).

Moreover, the microorganisms of the present invention can be cultured at a controlled temperature. The term "controlled temperature" includes any temperature that allows the production of the desired product (eg pantoate and / or pantothenate). In one embodiment, the controlled temperature comprises a temperature of 15 ° C. to 95 ° C. In another embodiment, the temperature controlled includes 15 ° C. to 70 ° C. Preferable temperature is 20 degreeC-55 degreeC, More preferably, it is 30 degreeC-50 degreeC.

The microorganisms can be cultured (maintained and / or grown) in liquid medium, preferably continuously or intermittently in conventional culture methods such as standing culture, test tube culture, shaking culture (e.g., rotary shaking culture, shake flask culture And the like, and cultured by 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 (eg fermentation process). Fermentation processes of the present invention include batches, feed batches and continuous processes or methods of fermentation. "Batch process" or "batch fermentation" means a system in which the composition, nutrients, supplemental additives, etc. of the medium are determined at the beginning of the fermentation and are not altered during the fermentation, however, excess media oxidation and / or microbial Attempts may be made to adjust factors such as pH and oxygen concentration to prevent death. The term “feed batch process” or “feed batch” fermentation refers to the same as batch fermentation, except that one or more substrates or supplements are added (eg, increased or continuously) as the fermentation proceeds. The term "continuous process" or "continuous fermentation" means that the same amount or "adjusted" medium in which the designated fermentation medium is fed continuously into the fermentor and used is preferably a desired product (eg pantoate and / or pantothenate). ) Means a system that is removed simultaneously to recover. These various processes are developed and are well known in the art.

The term "culturing under conditions that allow a desired compound to be produced" refers to appropriate or sufficient conditions (eg, temperature, pressure, pH, sustainability) to obtain the production of the desired compound or to obtain the desired harvest of the particular compound produced. Etc.) to maintain and / or grow the microorganisms. For example, the culturing is continued for a time sufficient to produce the desired amount of compound (eg pantoate and / or pantothenate). Preferably, substantially the production of the compound (e.g., a time sufficient to reach a suitable ratio of pantoate and / or pantothenate: HMBPA or a suitable concentration of pantoate and / or pantothenate) Continue incubation for a time sufficient to be different. In one embodiment, the incubation is continued for about 12 to 24 hours. In another embodiment, the culture 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 more than 144 hours. In another embodiment, the microorganism is about 5-10 g / L or more of the compound about 36 hours old, about 10 to 20 g / L compound about 48 hours old, or about 20 to 30 g / L of the compound about 72 hours old Incubated under conditions such that they are produced at the time point. In another embodiment, the microorganism is about 5-20 g / L or more of the compound about 36 hours old, about 20-30 g / L of the compound about 48 hours old, or about 30-50 or 60 g / L of the compound about 72 hours old Incubate under conditions such that they are produced at a time elapsed time. In another embodiment, the microorganism is cultured under conditions such that at least about 40 to 60 g / L of the compound is produced at about 36 hours later and at about 60 to 90 g / L compound at about 48 hours later. It will be appreciated by those skilled in the art that values above the upper limit of the stated ranges may also be obtainable, for example, by performing special fermentation or by the methods described herein as specially engineered strains.

Preferably, the production process of the present invention results in producing levels of pantothenate "improved compared to a suitable control". As used herein, the term “suitable control” includes any control that will be recognized by those skilled in the art as being suitable for determining an improved, increased, elevated amount of the desired product. For example, if the method is characterized by culturing a microorganism having a deregulated pantothenate biosynthetic pathway and the microorganism further has a deregulated MTF biosynthetic pathway (eg, one or more MTF biosynthetic enzymes are deregulated). Suitable controls include, for example, cultures of pre- or non-engineered microorganisms of MTF enzymes or pathways (ie, having only a deregulated pantothenate biosynthetic pathway). Likewise, the method is characterized by a microorganism having a deregulated pantothenate biosynthetic pathway and a deregulated ilv biosynthetic pathway, wherein the microorganism is further deregulated by an MTF biosynthetic pathway (ie, one or more MTF biosynthetic enzymes are deregulated, For example, with overexpressed) suitable controls include cultures of non-engineered microorganisms prior to or prior to manipulation of MTF enzymes or pathways (ie, only with deregulated pantothenate biosynthetic pathways and ilv biosynthetic pathways). It is not necessary to make a comparison in each method carried out in accordance with the present invention. For example, one skilled in the art can empirically determine the appropriate control by, for example, performing a series of reactions (eg, test tube culture, shake flask culture, fermentation) under the same or similar conditions. For example, by recognizing routine amounts of production by special strains, one skilled in the art can recognize amounts that have been improved, increased or elevated above this level. In other words, comparison with a suitable control includes a comparison with a predetermined value (eg, a predetermined control).

As such, suitably engineered strains produce 50, 60, 70 g / L and more pantothenates in an aspect that produces 40 g / L within 36 hours (prior to manipulation to increase pantothenate production). E.g. after treatment that causes one or more MTF biosynthetic enzymes to be overexpressed) is an improved production. Likewise, in an embodiment in which a suitably engineered strain produces 50 g / L pantothenate after 48 hours (prior to manipulation to improve pantothenate production), 60, 70, 80, 90 g / L or more The production of pantothenate of (after manipulation, for example after treatment which causes one or more MTF biosynthetic enzymes to overexpress) is an improved production example.

The method of the present invention further comprises recovering the desired compound (eg pantoate and / or pantothenate). The term "recovery" of a desired compound includes the extraction, harvesting, isolation, or purification of the compound from the culture medium. Recovery of the compound is carried out according to conventional isolation, purification methods known in the art and treatment of conventional resins (eg anionic or cation exchange resins, nonionic adsorptive resins, etc.), conventional absorbents (eg activated carbon, Treatment with silicic acid, silica gel, cellulose, alumina, etc., changing pH, solvent extraction (e.g. alcohol, ethyl acetate, hexane, etc.), dialysis, filtration, concentration, crystallization, recrystallization, pH control, lyophilization, etc. Including but not limited to. For example, the compound can be recovered from the culture medium by first removing the microorganisms from the culture. The medium is then passed through a cation exchange resin to remove inorganic anions and organic acids having a stronger acidity than the desired compound. The compound obtained can then be converted to the salts described herein (eg calcium salts).

Preferably, the formulation obtained by "extracting", "isolating", or "purifying" the desired compound of the present invention is substantially free of other media components (eg, media components and / or fermentation by-products). The term "substantially free of other media" includes the preparation of the desired compound from which the compound is isolated from the fermentation by-products or media of the culture to be produced. In one embodiment, the formulation comprises more than about 80% (by dry weight) of the desired compound, more preferably more than about 90% (eg less than about 10% of the fermentation by-products or other media components), more preferably Greater than about 95% of the compound (e.g., less than about 5% of the fermentation by-products or other media components), and even more preferably about 98-99% of the scavenging compound (e.g., about 1- of the other media components or fermentation by-products) Less than 2%). When the scavenging compound is led to a salt, the compound is preferably free of chemical contaminants associated with salt formation. If the scavenging compound is led to an alcohol, then the compound is preferably free of chemical contaminants associated with the formation of the alcohol.

In an alternative embodiment, the desired compound is not purified from the microorganism, for example if the microorganism is biologically harmless (ie safe). For example, the entire culture (or culture supernatant) can be used as a source of product (eg as a crude product). In another aspect, the culture (or culture supernatant) is used without deformation. In another embodiment, the culture (or culture supernatant) is concentrated. In another embodiment, the culture (or culture supernatant) is dried or lyophilized.

In another embodiment, the desired compound is partially purified. The term “partially purified” includes media formulations that have at least some processing, such as, for example, treatment with a commercial resin (eg, batch treatment). In a preferred embodiment, the "partially purified" formulation has about 30% (dry weight) of desired compounds, preferably greater than about 40%, more preferably 50%, even more preferably greater than about 70%. A "partially purified" formulation preferably has less than 80% (dry weight) of the desired compound (ie, less pure than an "extracted", "isolated" or "purified" formulation as specified herein).

Depending on the combination of biosynthetic enzymes or biosynthetic enzymes being engineered, it may be desirable or necessary to provide one or more biosynthetic precursors to the microorganisms of the present invention to produce the desired compound or compounds. The term “biosynthetic precursor” or “precursor” includes reagents or compounds that, when provided, brought into contact with, or included in the culture medium of a microorganism, serve to enhance or increase the 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 such that it is concentrated in a culture medium sufficient to improve the productivity of the microorganisms (e.g., sufficient to improve the production of pantoate and / or pantothenate). density). The biosynthetic precursors of the invention can be added in the form of concentrated solutions or suspensions (eg in a suitable solvent such as water or buffer). Moreover, the biosynthetic precursors of the present invention can be added continuously or intermittently for a given time as a single aliquot. The term “excess β-alanine” includes β-alanine that is increased or higher than those routinely used to culture microorganisms. For example, culturing the Bacillus microorganisms described in this example is done in the presence of about 0 to 0.01 g / L β-alanine. Thus, the excess amount of β-alanine may comprise an amount of about 0.01 to 1, preferably about 1 to 20 g / L.

In another embodiment, the biosynthetic precursor is valine. In another embodiment, the biosynthetic precursor is α-ketoisovalerate. Preferably, valine or α-ketoisovalerate is added in an amount that results in a concentrate in the medium sufficient to produce the desired product (eg pantoate and / or pantothenate). The term “excess α-KIV” includes increased or higher α-KIV than those routinely used to culture the desired microorganisms. For example, culturing the Bacillus microorganisms described in this example is routinely performed at about 0 to 0.01 g / L α-KIV. Thus, the amount of excess α-KIV may comprise about 0.01 to 1, preferably about 1 to 20 g / L α-KIV. The term “excess valine” includes increased or higher valine amounts than those routinely used to culture the desired microorganisms. For example, it is used at about 0 to 0.5 g / L for culturing the Bacillus microorganism described in this example. Thus, the excess amount of valine may comprise about 0.5 to 5 g / L, preferably about 5 to 20 g / L valine.

In another embodiment, the biosynthetic precursor is serine. Preferably, serine is added in an amount that concentrates in a medium sufficient to produce the desired product (eg pantoate and / or pantothenate). Excess serine (as mentioned above) can be added for example for improved production of pantothenate according to the production process described herein. Those skilled in the art will appreciate that very excess biosynthetic precursors can result in microbial toxicity. Biosynthetic precursors are also referred to herein as "supplementary biosynthetic substrates."

Another aspect of the invention is a bioconversion method having the characteristics of the recombinant microorganisms described herein. The term "bioconversion method", referred to as the term "bioconversion method", includes biological methods that result in the production of suitable substrates and / or intermediate compounds as desired products.

Microorganisms and / or enzymes used in biotransformation reactions are in a form such that they perform their intended function (eg to produce the desired compound). The microorganism may be an entire cell or only a portion of the cell necessary to obtain the desired desired value. The microorganisms are suspended (eg in a buffer solution or a suitable solution such as a medium), washed (washed so that there is no culture medium for culturing the microorganisms), dried with acetone, immobilized (eg, with polyacrylamide gel or k-carrageenan). Or in a synthetic support such as beads, matrices, etc.), and can adhere, crosslink, and permeate (eg, having a permeable membrane and / or wall so that compounds such as substrates, intermediates or products can easily pass through the membrane or wall). can do).

This invention will be further illustrated by the following examples which are not limiting further. The contents of all references, patents and published patent applications mentioned throughout this application are incorporated herein by reference.

Example 1: Panto-Compound Production Strains

In the development of Bacillus strains for the production of pantothenate, it is associated with the pantothenate biosynthetic pathway and the isoleucine-valine (ilv) pathway as described in US Patent Application No. 09 / 400,494 and US Patent Application No. 09 / 667,569. Various genetic manipulations have been performed on genes and enzymes. For example, strains with deregulated panBCD operon and / or strains with deregulated panE1 have shown improved pantothenate production (α-ketoisovalerate (α-KIV) and β-alanine) Incubated in presence). The more deregulated strains for ilvBNC and ilvD only showed enhanced pantothenate production in the presence of β-alanine. Moreover, it is possible to obtain β-alanine independence by further deregulating panD.

Exemplary pantothenate producing strains are tryptophan prototroph, Spec and Tet resistant, deregulated for panBCD at panBCD site and deregulated for panE1 at panE1 site (two genes of B. subtilis genome) Is homologous to E. coli panE, panE1 and panE2, panE1 encodes the major ketopantoate reductase involved in pantothenate production, whereas panE2 does not contribute to pantothenate synthesis (US Patent Application No. 09 / 400,494), PA824, which is deregulated for ilvD at the ilvD site, overexpresses the ilvBNC cassette at the amyE site, and overexpresses the panD at the bpr site, PA824, when incubated for 48 hours in a 14 L fermenter according to standard fermentation procedures. Routinely yields about 40-50 g / L pantothenate (see, eg, Provisional Patent Application No. 60 / 263,053 or Provisional Patent Application No. 60 / 262,995, incorporated herein by reference). The elemental batch medium (4.5 L) is inoculated with a shake flask culture of PA824. The fermentation is controlled for temperature (eg 43 ° C.), dissolved O ₂ , and pH, and is carried out in a glucose-limited feed batch process. After the glucose in the first batch has been consumed, the glucose concentration is maintained between about 0 and 1 g / L by continuously feeding fresh FEED medium The pH is set at 7.2, monitored and NH ₃ -or H ₃ PO ₄ Maintain solution by feeding in. Dissolved oxygen concentration [pO ₂ ] is maintained by controlling the agitation and aeration rate at about 10-30% Foaming is controlled by adding a suitable antifoaming agent. After removal pantothenate titers in the fermentation broth were determined (by HPLC analysis).

The second exemplary strain is PA668. PA668 is a derivative of PA824 containing an extra copy of P ₂₆ panB amplified at vpr and / or panB sites. PA668 was constructed using a panB expression vector (pAN636) that allows for the selection of multiple copies using chloramphenicol. In summary, the pAN636 NotI restriction fragment (excluding the vector sequence) is ligated and then a plate containing 5 μg / ml chloramphenicol is used to convert PA824. A transformant resistant to 30 μg / ml chloramphenicol is isolated and screened for pantothenate production in a 48 hour test tube culture. Isolates produce about 10 percent more than PA824. In 10 L fermentation, the first strain, PA668-2A, produces pantothenate in an amount corresponding to PA824 cultured under similar conditions (eg, ˜45-50 g / L at 36 hours). After 36 hours when pantothenate production starts routinely slow against PA824, PA668-2A continues to produce significant amounts of pantothenate (e.g. ˜60-65 g / L pantothenate at 48 hours). The second strain, PA668-24, produces pantothenate at a much faster rate, reaching 60-70 g / L after 48 hours.

A third production strain, PA721B-39, is engineered to further introduce an amplifying P ₂₆ panBpanD cassette as follows. First, construct a single expression cassette capable of integrating both panB and panD into the bpr site. By removing antibiotic resistance markers, the strain obtained by combining both genes into one expression cassette is simplified. The P ₂₆ panBpanD expression cassette is constructed to include two different panD ribosomal binding sites (RBS previously synthesized and tested in International Application Document WO01 / 21772 and US Patent Application No. 60/262995). The cassette further comprises a synthetic panB gene ribosomal binding site (RBS1), but this design allows for subsequent alteration of panB RBS by simple oligonucleotide cassette substitutions. In the first step of construction, the panB gene is combined with two panD gene cassettes for the construction of pAN665 as shown in FIG. Next, the panBpanD cassette obtained as described in FIG. 4 is transferred to the B. subtilis expression vector pOTP61. A summary of the main features of each plasmid (pAN670 and pAN674) is provided as in Table 1.

TABLE 1

Plasmids containing various B. subtilis panBpanD gene expression cassettes

These new plasmids are expected to combine the production of extra PanB and PanD from a single vector and produce an elevated amount of PanB compared to the panB expression vector (pAN636) present in PA668. The strategy for installing the P26panBpanD vector in pantothenate producing strains uses the genetic link between bpr and panE1. Derivatives of PA824 are initially constructed by converting strains to chromosomal DNA isolated from PA930 (panE1 :: cat) and selecting for resistance to chloramphenicol. The resulting transducer is screened for tetracycline sensitivity. Then, two Tet-sensitive isolates named PA715 were stored. This strain is a host strain for testing the P26 panBpanD vector (see below). In order to preserve the P26 panE1 cassette in PA715, each vector was first converted to a strain (PA328) containing P26 panE1 but not containing the cassette integrated at the bpr site. PA328 has a P26 panBCD site but has not been engineered for overproduction of α-KIV. A convertor of PA328 resistant to tetracycline was obtained from two vectors using suitable NotI restriction fragments and the resulting strains were named PA710 and PA714.

The next step was to transfer this cassette into the PA715 so that they could be evaluated in the PA824 background. This was accomplished by isolating chromosomal DNA from strains PA710 and PA714 and selecting for resistance to tetracycline by converting PA715 using each of the two DNA separately. Tetracycline resistance convertors were screened for sensitivity to chloramphenicol. It was identified at the bpr site by linking with P26 panBpanD to obtain a desired transformant obtained the P26 panE1 gene from the donor DNA. Chloramphenicol-sensitive isolates were obtained from conversions in which PA710 or PA714 chromosomal DNA was used as donor. The isolate that produced the largest pantothenate titer in the test tube culture assay was stored. These strains were named PA717 and PA721, respectively. Duplicate test tube cultures of new strains as well as PA824 and PA721 were incubated in SVY + 10 g / L aspartate for 48 hours at 43 ° C. and then assayed for pantothenate, HMBPA, and β-alanine. In addition, extracts from each strain were performed on SDS-PAGE gels. The results of this test tube culture assay are provided in Table 2.

TABLE 2

Production of Pantothenate by Strains PA717 and PA721 Grown in SVY Plus 10g / l Aspartate

As expected, each of the new strains produced more pantothenate and β-alanine than PA715. Two of the strains (PA717-24 and PA721-39) produced approximately the same amount of pantothenate as PA824, while PA721-35 produced more pantothenate than PA824. All three new strains produced less HMBPA than PA824. Protein gel analysis showed that it produced more PanB than any of the control strains.

Strains PA717-24, PA721-35 and PA721-39 were also evaluated in shake flask culture in a medium based on soy flour. In, P ₂₆ strains having panBpanD cassette is the same level as distinct as possible amplification P ₂₆ P ₂₆ panB and panD level shown by a PA668-2 and PA668-24 each containing cassette as shown in Figure 3, the plate Tottenate and HMBPA were produced.

TABLE 3

48 hour shake flask experiment

Conditions: Flask with 40 ml medium / 200 ml bulkhead, 4X Bioshield cover, 300 rpm, 2.5% inoculum (1.0 ml).

Soy medium: 20 g / l Cargill 200/20 soy flour, 8 g / l (NH ₄ ) ₂ SO ₄ , 5 g / l glutamate, 1 × PSTE, 0.1M phosphate pH 7.2 and 0.3M MOPS pH 7.2. 60 g / l glucose or maltose with 10 mM Mg and 1.4 mM Ca.

Average of two flasks with duplicate test.

In addition to the production of pantothenate (other panto-compounds depicted in and described herein in Figure 1), certain strains engineered to produce the desired panto-compounds in commercial quantities are also referred to as 3- (2-hydroxy-3 -Methyl-butyrylamino) -propionic acid (HMBPA) (herein "β-alanine 2- (R) -hydroxyisovalerate", "β-alanine 2-hydroxyisovalerate", "β-alanyl It is described to produce by-products identified as " -α-hydroxyisovalerate " and / or " ringtothenate ". (The term "hwantothenate" is abbreviated herein as "fan").

HMBPA is a condensation product of [R] -α-hydroxyisovaleric acid (α-HIV) and β-alanine catalyzed by the PanC enzyme. α-HIV is produced by reduction of α-KIV, a reaction catalyzed by α-keto reductase PanE (eg PanE1 and / or PanE2) and / or IlvC. Thus, it has been suggested that there are two or more pathways within the microorganism that compete for α-KIV, the substrate for the biosynthetic enzyme PanB, namely the pantothenate biosynthetic pathway and the HMBPA biosynthetic pathway (third and third competing for α-KIV). Fourth pathways are those due to the production of valine or leucine from α-KIV, see eg, FIG. 1). At least, the pantothenate biosynthetic pathway and the HMBPA biosynthetic pathway further produce a competitive substrate for the enzymes PanC ie α-HIV and pantothenate. The production of HMBPA can have a significant impact on pantothenate production. For example, the HMBPA pathway can compete with the pantothenate pathway for precursors (α-KIV and β-alanine) and for some of the enzymes (PanC, PanD, PanE1 and / or IlvC). In addition, since the structure of HMBPA is similar to that of pantothenate, it may have undesirable properties of negatively controlling one or more steps in the pantothenate pathway. Based on the identification of HMBPA, U.S. Provisional Application No. 60/262995 shows that production of pantothenate is performed by the substrate (α-KIV and β-alanine) and / or enzyme (PanC) methods in the pantothenate biosynthesis method compared to the HMBPA biosynthesis method. , PanD, PanE1, and / or IlvC) can be improved or optimized by any means that benefit from the use.

Example II Increasing Pantonenate Production by Increasing Serine Availability

One or more methods of optimizing pantothenate production include modulating the serine availability of the microbial culture. In particular, it has been demonstrated that increasing the usefulness of serine leads to an increase in pantothenate production (for example as compared to HMBPA production), while a decrease in the usefulness of serine leads to a decrease in pantothenate production compared to HMBPA production. Can be. This method is understood that methylenetetrahydrofolate (MTF) derived from the compound, serine, provides the hydroxymethyl group to α-KIV during the pantothenate biosynthesis reaction, resulting in ketopantoate (see FIGS. 1 and 2). Is based on Thus, controlling the amount of serine is one means of effectively controlling the amount of ketopantoate and in turn controlling pantoate and / or pantothenate production in suitably engineered microorganisms. To demonstrate this control, PA824 is grown for 48 hours at 43 ° C. in SVY glucose test tube cultures with 5 g / L β-alanine and ± 5 g / L serine.

TABLE 4

Production of Pantothenate and HMBPA by PA824 with and without Serine

As demonstrated by the data presented in Table 4, the addition of serine increases the amount of pantothenate production (as opposed to reducing HMBPA production).

Example 3 Manipulation of Microbial Cells with Increased Amount of Serine Hydroxymethyl Transferase, a glyA Gene Product

As an alternative method of supplying serine, 3-phosphoglycerate dehydrogenase or serine hydroxymethyl transferase (an alternative method of increasing serine levels and / or serine utilization to control the amount of pantothenate production) By increasing the synthesis or activity of serA and glyA gene products, respectively, to increase serine and methylenetetrahydrofolate biosynthesis of suitably engineered microorganisms.

The expression of the glyA gene can be increased by transcribing B. subtilis cells with an expression cassette containing the cloned B. subtilis glyA gene downstream of the always-expressing strong promoter. The glyA gene was amplified by PCR from chromosomal DNA isolated from B. subtilis PY79 using RY417 and RY418 described in Table 5 to construct the expression cassette.

TABLE 5

B. Primers Used for Amplification of Subtilis glyA and serA

RY417 contains an RBS2 synthetic ribosomal binding site immediately downstream from the XbaI site. The amplified DNA was then cut into XbaI and BamHI and cloned between the XbaI and BamHI sites in vector pAN004 (FIG. 5) to yield plasmid pAN396 (FIG. 6: SEQ ID NO: 24). The pAN004 vector has a phage SP01 P26 promoter immediately upstream of the XbaI cloning site to drive expression of the cloned glyA gene. Immediately downstream of the expression cassette, pAN396 contains a cat gene that functions in B. subtilis. To convert B. subtilis, NotI DNA fragments containing the P ₂₆ glyA cassette and cat gene were isolated from pAN396, self-ligated and converted into B. subtilis PY79 antibody-competent cells. Switch. Several chloramphenicol resistant convertors were selected and named PA1007 and PA1008. Chromosomal DNA was isolated from each of these strains and used to convert the antibody-capable cells of PA721B-39 and PA824 to yield PA 1011 and PA1014 strains, respectively. Cell extracts of selected isolates of PA1011 and PA1014 were subjected to SDS polyacrylamide gel electrophoresis so that these strains were glyA compared to their parent strains PA721B-39 (described in Example I) and PA824 (International Application Publication WO01 / 21772). It was found to contain increased amounts of gene products. To test the effect of increasing glyA expression on pantothenate production, PA1011 and PA1014 were grown for 48 hours at 43 ° C. in test tubes of SVY glucose plus 5 g / L β-alanine. As shown in the data provided in Table 6, PA1014 produced more pantothenate (4.5 g / L) than the parent strain PA824 (3.2 g / L). Likewise, PA1011 produced more pantothenate (4.35 g / L) than the parental strain PA721B-39 (4.05 g / L).

TABLE 6

Production of Pantothenate and HMBPA by PA1011 and PA1014 Compared to PA721B-39 and PA824

Example IV. Manipulation of microbial cells with increased amounts of 3-phosphoglycerate dehydrogenase, the serA gene product

3-phosphoglycerate dehydrogenase, the product of the serA gene, is the first enzyme introduced in the pathway of serine biosynthesis (see FIG. 2). Since serine is one of the substrates for the synthesis of MTF, overexpression of the serA gene was engineered to increase the amount of serine in the cells. In a manner similar to that described above for the glyA gene in Example III, expression of the serA gene was induced in the expression cassette containing the B. subtilis serA gene cloned from the B. subtilis cells at the bottom of the constitutively strong promoter. Increased by switching to have To construct the expression cassette, the serA gene was amplified by PCR from chromosomal DNA isolated from B. subtilis PY79 using primers RY405 and RY 406 depicted in Table 5. The amplified DNA was then cleaved with XbaI and BamHI and cloned between the XbaI and BamHI sites in vector pAN004 (FIG. 5) to obtain plasmid pAN393 (FIG. 7; SEQ ID NO: 25). To convert B. subtilis, NotI DNA fragments containing P ₂₆ serA cassette and cat gene were isolated from pAN393, self-ligated and converted to B. subtilis PY9 cells capable of antibody reactivity. Several chromosomal resistant transducers were selected and named PA1004 and PA1005. Chromosomal DNA was isolated from each of these strains and the cells capable of antibody response of PA721B-39 and PA824 were converted to yield PA1010 and PA1013 strains, respectively. Cell extracts of selected isolates of PA1010 and PA1013 were subjected to SDS polyacrylamide gel electrophoresis to confirm that these strains contained increased amounts of serA gene product, as compared to their parent strains PA721B-39 and PA824. .

To test the effect of increasing serA expression of pantothenate production, PA1010 and PA1013 were grown for 48 hours at 43 ° C. in test tube cultures of SVY glucose plus 5 g / L β-alanine. As indicated by the data provided in Table 7, PA1010 produced more pantothenate than the parent strain PA721B-39 (4.1 g / L) (4.7 g / L). Likewise, PA1013 produced, on average, higher amounts of pantothenate (4.1 g / L) than PA 824 (3.1 g / L).

TABLE 7

Production of Pantothenate and HMBPA by PA1010 and PA1013 as Compared to PA721B-39 and PA824

Example V. Shake Flask and Fermenter Experiments on Strains with Increased Expression of serA and glyA

Based on performance in test tubes, two strains with an amplifiable serA cassette and two strains with an amplifiable glyA cassette were selected, respectively, from two parents PA824 and PA721B-39. These 4 strains were grown by the parent in the shake flask (FIG. 8). In soy flour MOPS glucose (SMG) medium, all four strains produced more pantothenate than these parent strains. In soy flour MOPS Maltose (SMM) medium, one from these four strains appeared to be superior to the parent strain.

SerA overexpressing strains and glyA overexpressing strains from each parent were run simultaneously in a 10 liter Chemap bench fermenter. The glyA overexpressing strain derived from PA824, PA1014-3, which gave the highest pantothenate titer in SMM, was also performed with the best results in the fermentor (Table 9). Compared to parent PA824, which produces 41 g / L and 46 g / L pantothenate, respectively, strain PA1014-3 produces 71 g / L pantothenate at 36 hours in culture supernatant and 86 g / L pantothenate at 48 hours. Each produced. The serA strain, PA1012-4, also produced more pantothenate at 52 g / L at 36 hours and 60 g / L at 48 hours, respectively, than in the PA824 control supernatant. This result clearly demonstrates the increased efficacy of glyA and serA.

The serA overexpression and glyA overexpression derivatives of PA721B-39 were further improved than their parent strains. Both produced approximately 80 g / L pantothenate (82 g / L and 79 g / L, respectively) in the culture supernatant at 48 hours. The effect of increased PanB levels on PA721B-39 derivatives versus PA824 derivatives is evident in itself in the reduction of HMBPA. PA721B-39 and its derivatives produced less HMBPA after 48 hours than PA824 or PA668-24. Increasing GlyA seems to lower the carbon flow to HMBPA.

TABLE 8

Shake Flask Evaluation of Pantothenate Producing Strains Overexpressing serA or glyA

All data are the average of duplicate shake flasks after 48 hours.

Conditions: 40ml medium / 200ml bulkhead shake flask, 4X Bioshield Cover, 300rpm, 2.5% inoculum and 43 ° C

Medium: 20 g / L Cargill 200/20 soy flour, 1 × PSTE, 8 g / l (NH ₄ ) ₂ SO ₄ and 5 g / L glutamate.

Buffer: 0.1 M phosphate pH 7.2 and 0.3 M MOPS pH 7.2

Carbon source (sterilized separately with 20 × stocks): 60 g / L glucose or maltose with 10 mM Mg and 1.4 mM Ca.

TABLE 9

Evaluation of 10 L Fermenter of Pantothenate Producing Strains for Overexpressing serA or glyA

The medium used in the table above is PFM-222. This is the same with the PFM-155 described in US Application Serial No. 60 / 262,995, filed Jan. 19, 2001, except for the following differences: (1) In batch material, there is no Amberberx 1003. 40 g / L of Cargill 2002/20 (bean flour) was changed to 50 g / L of Cargill 20-80 (grit), MgSO ₄ · 7H ₂ O was replaced with MgCl ₂ · 7H ₂ O 1 g / L, SM-1000X is PSTE-1000X (PSTE-1000X = MnCl ₂ · 4H2O, 2.0 g / L; ZnSO ₄ · 7H ₂ O 1.5 g / L; CoCl ₂ · 6H ₂ O, 2.0 g / L; CuSO ₄ · 5H ₂ 0, 0.25 g / L; Na ₂ MoO ₄ 2H ₂ O, 0.75 g / L). In the feed material, SM-1000X has been replaced by PSTE-1000X.

Pantothenate production can be increased by combining overexpression of serA and glyA in a single strain and / or by introducing mutations that lead to feedback resistant to serA or glyA or both.

Example VI. Increased glyA gene expression by mutating the purR gene

As described in Examples III and V, the expression of the gly gene can be increased by adding a copy of one or more cassettes in which the glyA gene is driven by the constitutively potent promoter. An alternative way to increase glyA expression is to alter regulation. In the literature describing glyA :: lacZ fusion, the gly promoter has mild intensity under normal conditions (about 400 Miller Units), but if its negative regulator, purR gene, is deleted (Saxild et al (2001) J. Bacteriol. 183: 6175-6183) suggest that this promoter can be induced up to relatively high amounts (1800 Miller units). Therefore, experiments were conducted to determine whether glyA expression and consequently pantothenate production increased by deleting purR from pantothenate producing strains.

The B. subtilis purR gene was amplified from PY79 chromosomal DNA by PCR, and the obtained fragment was cloned into PvuII cleaved pGEM5-Zf (+) vector DNA to obtain plasmid pAN835F (SEQ ID NO: 26, FIG. 8). This step removes the PvuII sites at both ends of the insert, leaving a specific PvuII site in the middle of the purR open reading frame. Next, a blunt PCR DNA fragment (SEQ ID NO: 27) containing a Gram-positive kanamycin resistance gene from pAN363F was ligated to a specific PvuII site of pAN838F to obtain pAN838F (SEQ ID NO: 28, Figure 9).

PAN838F was then converted to PY79, PA66824, and PA824 and selected for kanamycin resistance at 10 mg / L to obtain a new set of strains named PA1059, PA1060 and PA1061, respectively. PCR showed that all new isolates contained the disrupted purR :: kan allele expected from the double crossover. Several isolates of PA1060 and PA1061 were tested for pantothenate production in test tube cultures grown in SVY glucose plus β-alanine (Table 10). The best isolates derived from PA668-24, PA1060-2 and PA1060-4 were improved with pantothenate from 3.0 g / L to 5.3 to 5.1 g / L, respectively, which is a 75% improvement. Likewise, the best isolates derived from PA824, PA1061-1 and PA1061-2 showed an increase of 5.4 g / L at 3.1 g / L, which is a 75% improvement. These results suggest that the glyA gene is substantially induced in new strains by the destruction of the purR gene. Alternatively, improvements in pantothenate production in PA1060 and PA1061 may be due to more complex pleiotropic effects. In either case, deregulation of purR regulatoryon has a positive effect on pantothenate production.

In another embodiment, purR disruption can be mounted in other pantothenate producing strains, such as those having more than one copy or integrated P26serA allele of P ₂₆ panBCD operon. The purR gene can be used as a site for the addition of a desired expression cassette such as P26panB. In addition, guanine resistant derivatives such as 8-azaguanine can be used as the selection of purR mutations.

TABLE 10

Production of Pantothenates and Ringtothenates by Derivatives of PA824 and PA668-24 Containing Broken RPR in Test Tube Cultures Grown in SVY Glucose Plus 5g / L β-Alanine

b.d = less than detection

* Concentration of antibiotics in Petri plates from which inoculum colonies are taken

Example VII. Overexpression of the serA Gene from an Unamplified Cassette

In this example, another method for inserting the always-expressing strong promoter (P ₂₆ ) in front of the chromosome serA gene in two steps to increase serine production will be described. Make up two plasmids, each containing about 700 base pairs of DNA sequence from the region immediately upstream of the native serA gene. The first plasmid, pAN821, also contains 3 ′ half of the serA coding region, and a kanamycin resistance gene (SEQ ID NO: 30, FIG. 10) between the two sequences. When switching to B. subtilis, to select kanamycin resistance, pAN821 destroys the serA gene, making it a serine requirement. This produces a gene sequence called the ΔserA :: kan allele.

A second plasmid designed to introduce a P ₂₆ serA structure is constructed by inserting a serA upstream sequence at the 5 'end of the P ₂₆ promoter at pAN395. The obtained plasmid, pAN824, is shown in Figure 11 (SEQ ID NO: 31). Plasmid pAN395 is similar to pAN393 described in Example IV. An open reading frame of the serA gene was synthesized by PCR using B. subtilis PY79 DNA as a template. The upstream primer contains an XbaI site and a moderately strong synthetic ribosomal binding site, RBS2. The downstream primer contains a BamHI site. Using this serA open reading frame, panBCD was substituted in the intermediate copy plasmid pAN006 to obtain pAN395 (SEQ ID NO: 29, FIG. 12). This plasmid contained the serA gene expressed from the P ₂₆ promoter and RBS2 ribosomal binding site.

ΔserA :: kan allele from pAN821 was introduced into strain PA824 to obtain PA1026. As expected, PA1026 was not grown in minimal medium. In the second step, a P ₂₆ serA cassette from plasmid pAN824 was introduced into PA1026 to select for serine prototrophy to obtain strain PA1028. Diagnostic PCR confirmed that several PA1028 isolates had the expected chromosome structure (P26 serA). These isolates were then tested for pantothenate production in test tube cultures grown for 48 hours in SVY plus 5 g / L β-alanine (Table 11). PA1028 isolate (derived from PA824) showed an increase of 10% to 20% in pantothenate production. As shown in Table 12, in shake flask experiments, PA824 produced about 7 g / L pantothenate, while PA1028 produced 11 g / L.

Example VIII. Integrated non-amplified P ₂₆  serA cassette and amplifiable P ₂₆  Construction of Pantothenates Producing Strains Containing All glyA Cassettes

Because the unamplified P26serA cassette integrated at serA leads to higher pantothenate synthesis (see Table 12, for example), and because the chloramphenicol amplifying P ₂₆ glyA cassette at glyA leads to higher pantothenate synthesis (eg For example, see Table 8 PA1014-3). It is suggested that these two combinations will be synergistic. The strain PA1028-4, a derivative of PA824 containing a non-amplified P ₂₆ serA cassette in serA, was converted from PA1014-3 to chlorophenicol resistance at 5 mg / L using chromosomal DNA to convert the chloramphenicol amplifying P ₂₆ glyA cassette. A strain set named PA1038 was obtained. PA1038 isolates were tested for pantothenate production using standard test tube cultures grown in SVY plus β-alanine (Table 13). As expected, a dramatic increase in pantothenate production was seen in PA1038 from about 4.2 g / L in PA824 to 6.6 to 7.5 g / L by the PA1038 set. Isolates PA1038-3 and PA1038-12 were further tested in shake flasks as shown in Table 12. Both produced an average of 13.6 g / L pantothenate, compared to 7.4 g / L pantothenate produced by PA824.

TABLE 11

Production of Pantothenates and Ringtothenates by Derivatives of PA824 Containing a Single Copy of P ₂₆ serA at SerA Sites in 48 Hours in Test Tube Cultures Grown in SVY plus β-alanine

TABLE 12

Shake Flask Evaluation of Pantothenate Producing Strains Overexpressing serA and / or glyA

All data are the average of two identical reaction shake flasks after 48 hours.

Conditions: 40ml medium / 200ml bulkhead shake flask, 4X Bioshield Cover, 300rpm, 2.5% inoculum and 43 ° C

Inoculator: SVY base with maltose 24 hours 43 ℃

Medium: 20 g / L Cargill 200/20 soy flour, 8 g / L (NH ₄ ) ₂ SO ₄ , 5 g / L glutamate and 1 × PSTE.

Buffer: 0.1 M phosphate pH 7.2 and 0.3 M MOPS pH 7.2

Carbon source (sterilized separately with 20X stock): 30 g / L maltose, 5 mM MgCl ₂ and 0.7 mM CaCl ₂

TABLE 13

Pantothenate production by PA1038, a PA824 derivative containing non-amplified P26 serA cassette in serA and amplifiable P26 glyA in glyA

Test tube cultures were incubated with SVY glucose plus 5 g / L β-alanine at 43 ° C. for 48 hours.

Example IX. Increasing the Production of MTF by Altering the Glycine Cleavage Pathway

As described in the examples above, increasing the MTF production of bacteria increases the production of pantothenate in strains engineered to produce more pantothenate by manipulation of the panBCD and / or panE genes. It has been demonstrated that production of pantothenate can be increased by increasing the expression of the glyA or serA genes. A strong promoter or ribosomal binding site can be used to increase glyA or serA expression as described in Examples III-V and VII-VIII. Alternatively, expression of the glyA gene can be deregulated in Bacillus by disrupting the purR inhibitor gene as described in Example VI.

Another way to increase MTF production is to enhance enzyme expression of the glycine cleavage pathway. For example, enzymes encoded by the gcvT, gcvPA, gcvPB, gcvH and pdhD genes catalyze the breakdown of glycine to MTF, CO ₂ and NH ₃ . The co-expressing strong promoters previously described, such as the SP01 phage P ₂₆ promoter, can be cloned in front of the gcvT-gcvPA-gcvPB operon or before the gcvH or pdhD genes to enhance their expression. In addition to the aforementioned approach, inexpensive additional glycine can be added to the medium to further enhance MTF production by all strains engineered as described herein.

Equivalent

Those skilled in the art will be able to recognize or assure that there are many equivalents of the particular aspects of the invention described herein, even with routine experimentation. Such equivalents are intended to be encompassed by the claims.

Related Applications

The present application is filed on January 18, 2002, filed with international patent applications PCT / US02 / 00925 (pending) and entitled September 21, 2000, entitled "Microorganisms and Methods for Enhancing the Production of Pantothenate". International patent application PCT / US00 / 25993 entitled “Methods and Microorganisms for the Production of Panto-Compounds”. The entire contents of each of the aforementioned applications are incorporated herein by reference.

SEQUENCE LISTING <110> OmniGene Bioproducts, Inc., et al. <120> MICROORGANISMS AND PROCESSES FOR ENHANCED PRODUCTION OF PANTOTHENATE <130> BGI-154PC2 <150> 60/393826 <151> 2002-07-03 <160> 31 <170> Patent In Ver. 2.0 <210> 1 <211> 194 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Promoter sequence <220> <221> -35_signal <222> (136) .. (141) <220> <221> -10_signal <159> (159) .. (164) <400> 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 <210> 2 <211> 163 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Promoter sequence <220> <221> -35_signal (222) (113) .. (118) <220> <221> -10_signal <222> (136) .. (141) <400> 2 gcctacctag cttccaagaa agatatccta acagcacaag agcggaaaga tgttttgttc 60 tacatccaga acaacctctg ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt 120 atctacaagg tgtggtataa taatcttaac aacagcagga cgc 163 <210> 3 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Promoter sequence <220> <221> -35_signal <222> (34) .. (39) <220> <221> -10_signal (222) (58) .. (63) <220> <221> -35_signal (222) (75) .. (80) <220> <221> -10_signal (222) (98) .. (103) <400> 3 gaggaatcat agaattttgt caaaataatt ttattgacaa cgtcttatta acgttgatat 60 aatttaaatt ttatttgaca aaaatgggct cgtgttgtac aataaatgta gtgaggtgga 120 tgcaatg 127 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 4 taaacatgag gaggagaaaa catg 24 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 5 attcgagaaa tggagagaat ataatatg 28 <210> 6 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 6 agaaaggagg tga 13 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <220> <221> misc_feature <222> 17, 18, 19, 20 N = a, t, c, or g <400> 7 ttaagaaagg aggtgannnn atg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <220> <221> misc_feature <222> 16, 17, 18, 19, 20 N = a, c, t, or g <400> 8 ttagaaagga ggtgannnnn atg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <220> <221> misc_feature <222> 14, 15, 16, 17, 18, 19, 20 N = a, c, t, or g <400> 9 agaaaggagg tgannnnnnn atg 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <220> <221> misc_feature <222> 14, 15, 16, 17, 18, 19 N = a, c, t, or g <400> 10 agaaaggagg tgannnnnna tg 22 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 11 ccctctagaa ggaggagaaa acatg 25 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 12 ccctctagag gaggagaaaa catg 24 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 13 ttagaaagga ggatttaaat atg 23 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 14 ttagaaagga ggtttaatta atg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 15 ttagaaagga ggtgatttaa atg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 16 ttagaaagga ggtgtttaaa atg 23 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 17 attcgagaaa ggaggtgaat ataatatg 28 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 18 attcgagaaa ggaggtgaat aataatg 27 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: ribosome binding site <400> 19 attcgtagaa aggaggtgaa ttaatatg 28 <210> 20 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5 'PCR primer <223> for serA gene <400> 20 ccctctagag gaggagaaaa catgtttcga gtattggtct cagacaaaat g 51 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3 'PCR primer <223> for serA gene <400> 21 cccggatcca attatggcag atcaatgagc ttcacagaca caa 43 <210> 22 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5 'PCR primer <223> for gly A gene <400> 22 ggatctagag gaggtgtaaa catgaaacat ttacctgcgc aagacgaa 48 <210> 23 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3 'PCR primer <223> for gly A gene <400> 23 cggggatccc ccatcaacaa ttacacactt ctattgattc tac 43 <210> 24 <211> 7926 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: serA overexpression <223> plasmid <400> 24 gaattttgcg gccgcttcga aagctgtaat ataaaaacct tcttcaacta acggggcagg 60 ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120 gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180 aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240 tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300 ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360 gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420 ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480 tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540 cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600 aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660 tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720 cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780 tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840 ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900 ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa 960 aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct 1020 tatcttgata ataagggtaa ctattgaatt cggtaccaag agtttgtaga aacgcaaaaa 1080 ggccatccgt caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc 1140 ctgcccgcca ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg 1200 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg 1260 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc 1320 acactaccat cggcgctacg gcgtttcact tctgagttcg gcatggggtc aggtgggacc 1380 accgcgctac tgccgccagg caaattctgt tttatcagac cgcttctgcg ttctgattta 1440 atctgtatca ggctgaaaat cttctctcat ccgccaaaac aggatccaat tatggcagat 1500 caatgagctt cacagacaca atatcaggga catttgttag ttctttcaca attttatctt 1560 ccagatgtct gtcaaaggaa agcatcatga tggcttctcc gcctttttcc ttacggccaa 1620 cctgcatagt tgcaatgtta atatcattat ctccgagaat acgtcctact cggccgatga 1680 cacctgttgt atcttgatgc tggatataca ccaagtgacc agtcggataa aaatcaatat 1740 taaatccatt gatctcgaca attcgttctc cgaaatgagg aatatacgta gccgttacag 1800 taaaggtgct gcggtctcct gtcactttta cgctgatgca gttatcgtat ccagattcag 1860 aagaggaaat tttttcactg aagctaatgc cgcgttcttt tgcgacaccc ccggcattga 1920 cctcattaac agtagagtct acgcgcggtt ttaaaaagcc tgacagaagg gcttttgtaa 1980 tgaacgatgt ttcaagttta gcaattgtgc cttcatattg aatggcaaca tcctgtactg 2040 gttctttcat gcactgtgat acaaggctgc caatttttcc tgcaatttga tggtaaggct 2100 taattttagc aaattcatct tttgtcatgg caggcaggtt gatagctgac atgacaggca 2160 ggccttttgc gaactgcaga acttcttctg acacttgggc ggcgacattg agctgtgctt 2220 ctttcgttga tgctcccaag tgaggagtgg caatgactaa tggatgatca acaagtttgt 2280 tgtcaactgg cggttcgact tcgaaaacgt caagcgctgc tcccgcaaca tgcccgtttt 2340 ccaaagcttc gagaagtgct gcttcatcga taattccgcc tcgcgcacag ttaattaagc 2400 gaacgccttt tttcgttttt gcaatcgttt ctttattcaa taagcctttt gtttcttttg 2460 ttaaaggcgt gtgaacggta atgatatccg cactttcaag cacttcttca aatgtacggc 2520 tgtttacgcc gatttttttc gctctttctt ccgttaagaa aggatcaaaa acgtgcacag 2580 tcataccgaa cgctcctcga cgctgtgcaa tttcacttcc gattcggcct aatcctacaa 2640 taccaagcgt ttttccataa agctctgaac cgacataagc tgtgcggttc cactctctgg 2700 atttcactga gatattagcc tgcggaatgt gtctcattaa agaagagatc attgcaaatg 2760 tatgctcagc tgtcgaaatg gtgttgccgt tcggagcatt gatcacgatt accccgtgtt 2820 tcgtagcctc atcaatatcg atattatcga caccgacacc ggctcttccg acaattttta 2880 aagaagtcat tttgttgaaa aggtcttctg ttacttttgt cgcgcttcgc accaaaagag 2940 catcaaaagt atgtaattca tcttctgcat ctgctacgtt tttttgaacg atttcaataa 3000 agtctgattc aataagtggc tgtaaaccgt cgttgctcat tttgtctgag accaatactc 3060 gaaacatgtt ttctcctcct ctagagcgtc ctgctgttgt taagattatt ataccacacc 3120 ttgtagataa agtcaacaac tttttgcaaa atttttcagg aattttagca gaggttgttc 3180 tggatgtaga acaaaacatc tttccgctct tgtgctgtta ggatatcttt cttggaagct 3240 aggtaggcct cgagttatgg cagttggtta aaaggaaaca aaaagaccgt tttcacacaa 3300 aacggtcttt ttcgatttct ttttacagtc acagccactt ttgcaaaaac cggacagctt 3360 catgccttat aactgctgtt tcggtcgaca agcttcgcga agcggccgca aaattcactg 3420 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 3480 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 3540 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 3600 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 3660 gcatagttaa gccagccccg acacccgcca acacccgctg actatgcttg taaaccgttt 3720 tgtgaaaaaa tttttaaaat aaaaaagggg acctctaggg tccccaatta attagtaata 3780 taatctatta aaggtcattc aaaaggtcat ccaccggatc agcttagtaa agccctcgct 3840 agattttaat gcggatgttg cgattacttc gccaactatt gcgataacaa gaaaaagcca 3900 gcctttcatg atatatctcc caatttgtgt agggcttatt atgcacgctt aaaaataata 3960 aaagcagact tgacctgata gtttggctgt gagcaattat gtgcttagtg catctaacgc 4020 ttgagttaag ccgcgccgcg aagcggcgtc ggcttgaacg aattgttaga cattatttgc 4080 cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc 4140 gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg 4200 gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga 4260 ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat 4320 cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata 4380 gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc 4440 tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga 4500 agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg 4560 gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa 4620 tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg 4680 ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca 4740 ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc 4800 gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc 4860 tcatgatgtt taactttgtt ttagggcgac tgccctgctg cgtaacatcg ttgctgctcc 4920 ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc cgaggcatag 4980 actgtacccc aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac 5040 cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca 5100 gcttacgaac cgaacaggct tatgtccact gggttcgtgc cttcatccgt ttccacggtg 5160 tgcgtcaccc ggcaaccttg ggcagcagcg aagtcgaggc atttctgtcc tggctggcga 5220 acgagcgcaa ggtttcggtc tccacgcatc gtcaggcatt ggcggccttg ctgttcttct 5280 acggcaaggt gctgtgcacg gatctgccct ggcttcagga gatcggaaga cctcggccgt 5340 cgcggcgctt gccggtggtg ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg 5400 aaggcgagca tcgtttgttc gcccagcttc tgtatggaac gggcatgcgg atcagtgagg 5460 gtttgcaact gcgggtcaag gatctggatt tcgatcacgg cacgatcatc gtgcgggagg 5520 gcaagggctc caaggatcgg gccttgatgt tacccgagag cttggcaccc agcctgcgcg 5580 agcaggggaa ttgatccggt ggatgacctt ttgaatgacc tttaatagat tatattacta 5640 attaattggg gaccctagag gtcccctttt ttattttaaa aattttttca caaaacggtt 5700 tacaagcata acgggttttg ctgcccgcaa acgggctgtt ctggtgttgc tagtttgtta 5760 tcagaatcgc agatccggct tcaggtttgc cggctgaaag cgctatttct tccagaattg 5820 ccatgatttt ttccccacgg gaggcgtcac tggctcccgt gttgtcggca gctttgattc 5880 gataagcagc atcgcctgtt tcaggctgtc tatgtgtgac tgttgagctg taacaagttg 5940 tctcaggtgt tcaatttcat gttctagttg ctttgtttta ctggtttcac ctgttctatt 6000 aggtgttaca tgctgttcat ctgttacatt gtcgatctgt tcatggtgaa cagctttaaa 6060 tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt tttacaccgt tttcatctgt 6120 gcatatggac agttttccct ttgatatcta acggtgaaca gttgttctac ttttgtttgt 6180 tagtcttgat gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt 6240 tagccagtat gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat 6300 tgagatcatg cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa 6360 tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc gttacgtagg taggaatctg 6420 atgtaatggt tgttggtatt ttgtcaccat tcatttttat ctggttgttc tcaagttcgg 6480 ttacgagatc catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc 6540 ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt 6600 tcaaaaccca ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga 6660 acttaaattc atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt 6720 cttttaataa ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt 6780 ccagattata ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa 6840 aaactaattc taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa 6900 agcctttaac caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt 6960 tagctaatac accataagca ttttccctac tgatgttcat catctgagcg tattggttat 7020 aagtgaacga taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg 7080 ccacacagca taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta 7140 gttcatttgc tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt 7200 aatcactata ccaattgaga tgggctagtc aatgataatt actagtcctt ttcctttgag 7260 ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac 7320 cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa 7380 tttatagaat aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata 7440 actcactact ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc 7500 ctctacaaaa cagaccttaa aaccctaaag gcttaagtag caccctcgca agctcgggca 7560 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt 7620 gacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg 7680 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc 7740 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag 7800 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt 7860 cagactggct aatgcaccca gtaaggcagc ggtatcatca acaggcttac ccgtcttact 7920 gtcaac 7926 <210> 25 <211> 7701 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: glyA overexpression <223> plasmid <400> 25 gaattttgcg gccgcttcga aagctgtaat ataaaaacct tcttcaacta acggggcagg 60 ttagtgacat tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120 gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180 aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240 tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300 ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360 gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420 ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480 tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540 cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600 aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660 tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720 cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780 tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840 ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900 ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa 960 aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct 1020 tatcttgata ataagggtaa ctattgaatt cggtaccaag agtttgtaga aacgcaaaaa 1080 ggccatccgt caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc 1140 ctgcccgcca ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg 1200 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg 1260 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc 1320 acactaccat cggcgctacg gcgtttcact tctgagttcg gcatggggtc aggtgggacc 1380 accgcgctac tgccgccagg caaattctgt tttatcagac cgcttctgcg ttctgattta 1440 atctgtatca ggctgaaaat cttctctcat ccgccaaaac aggatccccc atcaacaatt 1500 acacacttct attgattcta caaaaaaaga cattgagttt caagaacatc gtcaaaaaac 1560 ccgccgggca taagcccaag cgggttttag gatcttaata atctaattct ttatataaag 1620 gaaatttatc agtcagagca gctacacgct gtcttgcttc ttcaagtttt ccttcatctt 1680 cgtggttttt caatgcaagc gcaatgatag caccgacttc ttctaatgcg tctccgtcaa 1740 aaccgcggct ggttacagca gctgtaccaa gacggatgcc gcttgttacg aaaggttttt 1800 caggatcata tggaatcgcg tttttgttag acgtaatacc aatttcatca agtacatgct 1860 ccgcaacctt accagtcagt ccgagcgaac gaaggtcaac aaggataagg tggttgtctg 1920 ttccgcctga aacgagctgg atgccctctt tcgttaaggc ttcagccaga cgtttcgcgt 1980 ttgaaatgac gttttgtgca tatgttttga aatcgtcctg caatacttca ccgaatgaaa 2040 cagcttttgc ggcaataacg tgcatcagag ggccgccttg aattccaggg aagatcgatt 2100 tatcaatttt cttgccaaac tcttcacggc aaaggatcat accgccgcga ggaccgcgaa 2160 gtgttttatg tgttgttgtt gtaacgaaat cagcgtaagg aaccgggttt ggatgaaggc 2220 ctgccgcaac aagtcctgcg atatgtgcca tatccaccat gaagtaagcg ccgacttcat 2280 cagcaatttc acggaatttc ttaaagtcga ttgtacgagg atacgcactt gctcctgcta 2340 cgataagctt cggtttatga gcgagggctt tttcacgcac gtcatcgtaa tcaatatatt 2400 gagtttcttt atctacgccg tactcaacaa agttatattg aacaccgctg aagttgactg 2460 ggcttccgtg tgttaaatgg ccgccgtggg agaggttcat cccaagtaca gtatcgcctt 2520 gctccaaaat cgtgaagtac actgccatgt ttgcttgtgc gcctgaatga ggctgaacgt 2580 ttacatgctc cgctccaaag atttccttcg cgcggtcacg ggcgatatct tcaacgacat 2640 cgacgtgctc gcatccgccg tagtagcgtt tgcccggata tccttctgcg tacttatttg 2700 tcaaaacaga tccttgtgct tccataaccg cttcacttac aaagttctca gaagcaatca 2760 attcgatctt agtctgttgg cgttcacgct catttttaat ggcgttaaac acttgttcgt 2820 cttgcgcagg taaatgtttc atgtttacac ctcctctaga gcgtcctgct gttgttaaga 2880 ttattatacc acaccttgta gataaagtca acaacttttt gcaaaatttt tcaggaattt 2940 tagcagaggt tgttctggat gtagaacaaa acatctttcc gctcttgtgc tgttaggata 3000 tctttcttgg aagctaggta ggcctcgagt tatggcagtt ggttaaaagg aaacaaaaag 3060 accgttttca cacaaaacgg tctttttcga tttcttttta cagtcacagc cacttttgca 3120 aaaaccggac agcttcatgc cttataactg ctgtttcggt cgacaagctt cgcgaagcgg 3180 ccgcaaaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 3240 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 3300 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg 3360 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 3420 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgactat 3480 gcttgtaaac cgttttgtga aaaaattttt aaaataaaaa aggggacctc tagggtcccc 3540 aattaattag taatataatc tattaaaggt cattcaaaag gtcatccacc ggatcagctt 3600 agtaaagccc tcgctagatt ttaatgcgga tgttgcgatt acttcgccaa ctattgcgat 3660 aacaagaaaa agccagcctt tcatgatata tctcccaatt tgtgtagggc ttattatgca 3720 cgcttaaaaa taataaaagc agacttgacc tgatagtttg gctgtgagca attatgtgct 3780 tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg gcgtcggctt gaacgaattg 3840 ttagacatta tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt 3900 ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag 3960 cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga 4020 catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca 4080 ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat 4140 ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac 4200 ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga 4260 tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca 4320 gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt 4380 ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga 4440 tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat 4500 cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg 4560 tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg 4620 cgatcaccgc ttccctcatg atgtttaact ttgttttagg gcgactgccc tgctgcgtaa 4680 catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct tgctgcttgg 4740 atgcccgagg catagactgt accccaaaaa aacagtcata acaagccatg aaaaccgcca 4800 ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac 4860 gctacttgca ttacagctta cgaaccgaac aggcttatgt ccactgggtt cgtgccttca 4920 tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc gaggcatttc 4980 tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag gcattggcgg 5040 ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt caggagatcg 5100 gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa gtggttcgca 5160 tcctcggttt tctggaaggc gagcatcgtt tgttcgccca gcttctgtat ggaacgggca 5220 tgcggatcag tgagggtttg caactgcggg tcaaggatct ggatttcgat cacggcacga 5280 tcatcgtgcg ggagggcaag ggctccaagg atcgggcctt gatgttaccc gagagcttgg 5340 cacccagcct gcgcgagcag gggaattgat ccggtggatg accttttgaa tgacctttaa 5400 tagattatat tactaattaa ttggggaccc tagaggtccc cttttttatt ttaaaaattt 5460 tttcacaaaa cggtttacaa gcataacggg ttttgctgcc cgcaaacggg ctgttctggt 5520 gttgctagtt tgttatcaga atcgcagatc cggcttcagg tttgccggct gaaagcgcta 5580 tttcttccag aattgccatg attttttccc cacgggaggc gtcactggct cccgtgttgt 5640 cggcagcttt gattcgataa gcagcatcgc ctgtttcagg ctgtctatgt gtgactgttg 5700 agctgtaaca agttgtctca ggtgttcaat ttcatgttct agttgctttg ttttactggt 5760 ttcacctgtt ctattaggtg ttacatgctg ttcatctgtt acattgtcga tctgttcatg 5820 gtgaacagct ttaaatgcac caaaaactcg taaaagctct gatgtatcta tcttttttac 5880 accgttttca tctgtgcata tggacagttt tccctttgat atctaacggt gaacagttgt 5940 tctacttttg tttgttagtc ttgatgcttc actgatagat acaagagcca taagaacctc 6000 agatccttcc gtatttagcc agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc 6060 atgagaacga accattgaga tcatgcttac tttgcatgtc actcaaaaat tttgcctcaa 6120 aactggtgag ctgaattttt gcagttaaag catcgtgtag tgtttttctt agtccgttac 6180 gtaggtagga atctgatgta atggttgttg gtattttgtc accattcatt tttatctggt 6240 tgttctcaag ttcggttacg agatccattt gtctatctag ttcaacttgg aaaatcaacg 6300 tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat attgctgtaa gtgtttaaat 6360 ctttacttat tggtttcaaa acccattggt taagcctttt aaactcatgg tagttatttt 6420 caagcattaa catgaactta aattcatcaa ggctaatctc tatatttgcc ttgtgagttt 6480 tcttttgtgt tagttctttt aataaccact cataaatcct catagagtat ttgttttcaa 6540 aagacttaac atgttccaga ttatatttta tgaatttttt taactggaaa agataaggca 6600 atatctcttc actaaaaact aattctaatt tttcgcttga gaacttggca tagtttgtcc 6660 actggaaaat ctcaaagcct ttaaccaaag gattcctgat ttccacagtt ctcgtcatca 6720 gctctctggt tgctttagct aatacaccat aagcattttc cctactgatg ttcatcatct 6780 gagcgtattg gttataagtg aacgataccg tccgttcttt ccttgtaggg ttttcaatcg 6840 tggggttgag tagtgccaca cagcataaaa ttagcttggt ttcatgctcc gttaagtcat 6900 agcgactaat cgctagttca tttgctttga aaacaactaa ttcagacata catctcaatt 6960 ggtctaggtg attttaatca ctataccaat tgagatgggc tagtcaatga taattactag 7020 tccttttcct ttgagttgtg ggtatctgta aattctgcta gacctttgct ggaaaacttg 7080 taaattctgc tagaccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata 7140 ttcaagtggt tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc 7200 ccagccctgt gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc 7260 aaacgctgtt tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc 7320 tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc cgaccatcag gcacctgagt 7380 cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg ctctggcagt gaatgggggt 7440 aaatggcact acaggcgcct tttatggatt catgcaagga aactacccat aatacaagaa 7500 aagcccgtca cgggcttctc agggcgtttt atggcgggtc tgctatgtgg tgctatctga 7560 ctttttgctg ttcagcagtt cctgccctct gattttccag tctgaccact tcggattatc 7620 ccgtgacagg tcattcagac tggctaatgc acccagtaag gcagcggtat catcaacagg 7680 cttacccgtc ttactgtcaa c 7701 <210> 26 <211> 3888 <212> DNA <213> Artificial Sequence <220> <223> plasmid <400> 26 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 60 tcggtgcggg cctcttcgct attacgccag tttgggggtg agttcatgaa gtttcgtcgc 120 agcggcagat tggtggactt aacaaattat ttgttaaccc atccgcacga gttaataccg 180 ctaacctttt tctctgagcg gtatgaatct gcaaaatcat cgatcagtga agatttaaca 240 attattaaac aaacctttga acagcagggg attggtactt tgcttactgt tcccggagct 300 gccggaggcg ttaaatatat tccgaaaatg aagcaggctg aagctgaaga gtttgtgcag 360 acacttggac agtcgctggc aaatcctgag cgtatccttc cgggcggtta tgtatattta 420 acggatatct taggaaagcc atctgtactc tccaaggtag ggaagctgtt tgcttccgtg 480 tttgcagagc gcgaaattga tgttgtcatg accgttgcca cgaaaggcat ccctcttgcg 540 tacgcagctg caagctattt gaatgtgcct gttgtgatcg ttcgtaaaga caataaggta 600 acagagggct ccacagtcag cattaattac gtttcaggct cctcaaaccg cattcaaaca 660 atgtcacttg cgaaaagaag catgaaaacg ggttcaaacg tactcattat tgatgacttt 720 atgaaagcag gcggcaccat taatggtatg attaacctgt tggatgagtt taacgcaaat 780 gtggcgggaa tcggcgtctt agttgaagcc gaaggagtag atgaacgtct tgttgacgaa 840 tatatgtcac ttcttactct ttcaaccatc aacatgaaag agaagtccat tgaaattcag 900 aatggcaatt ttctgcgttt ttttaaagac aatcttttaa agaatggaga gacagaatca 960 tgacaaaagc agtccacaca aaacatgccc cagcggcaat cgggccttat tcacaaggga 1020 ttatcgtcaa caatatgttt tacagctcag gccaaatccc tttgactcct tcaggcgaaa 1080 tggtgaatgg cgatattaag gagcagactc atcaagtatt cagcaattta aaggcggttc 1140 tggaagaagc gggtgcttct tttgaaacag ttgtaaaagc aactgtattt atcgcggata 1200 tggaacagtt tgcggaagta aacgaagtgt acggacaata ttttgacact cacaaaccgg 1260 cgagatcttg tgttgaagtc gcgagactcc cgaaggatgc gttagtcgag atcgaagtta 1320 ttgcactggt gaaataataa gaaaagtgat tctgggagag ccgggatcac ttttttattt 1380 accttatgcc cgaaatgaaa gctttatgac cctgcattaa tgaatcggcc aacgcgcggg 1440 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 1500 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 1560 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 1620 ccgtaaaaag gccgcgttgc tggcgttttt cgataggctc cgcccccctg acgagcatca 1680 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 1740 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 1800 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 1860 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 1920 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 1980 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2040 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 2100 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 2160 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2220 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 2280 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 2340 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 2400 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 2460 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 2520 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 2580 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 2640 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 2700 gcgcaacgtt gttggcattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 2760 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 2820 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 2880 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 2940 cttttctgtg actggtgagt actcaaccaa gtcattctga gaataccgcg cccggcgacc 3000 gagttgctct tgcccggcgt caatacggga taatagtgta tgacatagca gaactttaaa 3060 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 3120 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 3180 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 3240 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 3300 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 3360 aggggttccg cgcacatttc cccgaaaagt gccacctgta tgcggtgtga aataccgcac 3420 agatgcgtaa ggagaaaata ccgcatcagg cgaaattgta aacgttaata ttttgttaaa 3480 attcgcgtta aatatttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 3540 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 3600 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 3660 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttgcggt cgaggtgccg 3720 taaagctcta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 3780 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 3840 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaa 3888 <210> 27 <211> 4606 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 27 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 60 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 120 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 180 ttgtaatacg actcactata gggcgaattg ggcccgacgt cgcatgctcc cggccgccat 240 ggccgcggga tgcggccgcg tcgacgtgaa ataccgcaca gatgcgtaag gagaaaatac 300 cgcatcaggc gataaaccca gcgaaccatt tgaggtgata ggtaagatta taccgaggta 360 tgaaaacgag aattggacct ttacagaatt actctatgaa gcgccatatt taaaaagcta 420 ccaagacgaa gaggatgaag aggatgagga ggcagattgc cttgaatata ttgacaatac 480 tgataagata atatatcttt tatatagaag atatcgccgt atgtaaggat ttcagggggc 540 aaggcatagg cagcgcgctt atcaatatat ctatagaatg ggcaaagcat aaaaacttgc 600 atggactaat gcttgaaacc caggacaata accttatagc ttgtaaattc tatcataatt 660 gtggtttcaa aatcggctcc gtcgatacta tgttatacgc caactttcaa aacaactttg 720 aaaaagctgt tttctggtat ttaaggtttt agaatgcaag gaacagtgaa ttggagttcg 780 tcttgttata attagcttct tggggtatct ttaaatactg tagaaaagag gaaggaaata 840 ataaatggct aaaatgagaa tatcaccgga attgaaaaaa ctgatcgaaa aataccgctg 900 cgtaaaagat acggaaggaa tgtctcctgc taaggtatat aagctggtgg gagaaaatga 960 aaacctatat ttaaaaatga cggacagccg gtataaaggg accacctatg atgtggaacg 1020 ggaaaaggac atgatgctat ggctggaagg aaagctgcct gttccaaagg tcctgcactt 1080 tgaacggcat gatggctgga gcaatctgct catgagtgag gccgatggcg tcctttgctc 1140 ggaagagtat gaagatgaac aaagccctga aaagattatc gagctgtatg cggagtgcat 1200 caggctcttt cactccatcg acatatcgga ttgtccctat acgaatagct tagacagccg 1260 cttagccgaa ttggattact tactgaataa cgatctggcc gatgtggatt gcgaaaactg 1320 ggaagaagac actccattta aagatccgcg cgagctgtat gattttttaa agacggaaaa 1380 gcccgaagag gaacttgtct tttcccacgg cgacctggga gacagcaaca tctttgtgaa 1440 agatggcaaa gtaagtggct ttattgatct tgggagaagc ggcagggcgg acaagtggta 1500 tgacattgcc ttctgcgtcc ggtcgatcag ggaggatatc ggggaagaac agtatgtcga 1560 gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat attatatttt 1620 actggatgaa ttgttttagt acctagattt agatgtctaa aaagctttaa ctacaagctt 1680 tttagacatc taatcttttc tgaagtacat ccgcaactgt ccatactctg atgttttata 1740 tcttttctaa aagttcgcta gataggggtc ccgagcgcct acgaggaatt tgtatcgcca 1800 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggtac cgggatcact 1860 agtgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg atgcatagct 1920 tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg 1980 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 2040 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 2100 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 2160 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2220 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 2280 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2340 gtaaaaaggc cgcgttgctg gcgtttttcg ataggctccg cccccctgac gagcatcaca 2400 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 2460 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 2520 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 2580 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2640 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 2700 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 2760 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 2820 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 2880 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 2940 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3000 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3060 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3120 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 3180 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 3240 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3300 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3360 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3420 gcaacgttgt tggcattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3480 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3540 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 3600 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 3660 tttctgtgac tggtgagtac tcaaccaagt cattctgaga ataccgcgcc cggcgaccga 3720 gttgctcttg cccggcgtca atacgggata atagtgtatg acatagcaga actttaaaag 3780 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 3840 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 3900 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 3960 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4020 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4080 gggttccgcg cacatttccc cgaaaagtgc cacctgtatg cggtgtgaaa taccgcacag 4140 atgcgtaagg agaaaatacc gcatcaggcg aaattgtaaa cgttaatatt ttgttaaaat 4200 tcgcgttaaa tatttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 4260 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 4320 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 4380 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg aggtgccgta 4440 aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 4500 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 4560 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaa 4606 <210> 28 <211> 5399 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 28 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 60 tcggtgcggg cctcttcgct attacgccag tttgggggtg agttcatgaa gtttcgtcgc 120 agcggcagat tggtggactt aacaaattat ttgttaaccc atccgcacga gttaataccg 180 ctaacctttt tctctgagcg gtatgaatct gcaaaatcat cgatcagtga agatttaaca 240 attattaaac aaacctttga acagcagggg attggtactt tgcttactgt tcccggagct 300 gccggaggcg ttaaatatat tccgaaaatg aagcaggctg aagctgaaga gtttgtgcag 360 acacttggac agtcgctggc aaatcctgag cgtatccttc cgggcggtta tgtatattta 420 acggatatct taggaaagcc atctgtactc tccaaggtag ggaagctgtt tgcttccgtg 480 tttgcagagc gcgaaattga tgttgtcatg accgttgcca cgaaaggcat ccctcttgcg 540 tacgcagctg cggccgcgtc gacaaaccca gtgaaccatt tgaggtgata ggtaagatta 600 taccgaggta tgaaaacgag aattggacct ttacagaatt actctatgaa gcgccatatt 660 taaaaagcta ccaagacgaa gaggatgaag aggatgagga ggcagattgc cttgaatata 720 ttgacaatac tgataagata atatatcttt tatatagaag atatcgccgt atgtaaggat 780 ttcagggggc aaggcatagg cagcgcgctt atcaatatat ctatagaatg ggcaaagcat 840 aaaaacttgc atggactaat gcttgaaacc caggacaata accttatagc ttgtaaattc 900 tatcataatt gtggtttcaa aatcggctcc gtcgatacta tgttatacgc caactttcaa 960 aacaactttg aaaaagctgt tttctggtat ttaaggtttt agaatgcaag gaacagtgaa 1020 ttggagttcg tcttgttata attagcttct tggggtatct ttaaatactg tagaaaagag 1080 gaaggaaata ataaatggct aaaatgagaa tatcaccgga attgaaaaaa ctgatcgaaa 1140 aataccgctg cgtaaaagat acggaaggaa tgtctcctgc taaggtatat aagctggtgg 1200 gagaaaatga aaacctatat ttaaaaatga cggacagccg gtataaaggg accacctatg 1260 atgtggaacg ggaaaaggac atgatgctat ggctggaagg aaagctgcct gttccaaagg 1320 tcctgcactt tgaacggcat gatggctgga gcaatctgct catgagtgag gccgatggcg 1380 tcctttgctc ggaagagtat gaagatgaac aaagccctga aaagattatc gagctgtatg 1440 cggagtgcat caggctcttt cactccatcg acatatcgga ttgtccctat acgaatagct 1500 tagacagccg cttagccgaa ttggattact tactgaataa cgatctggcc gatgtggatt 1560 gcgaaaactg ggaagaagac actccattta aagatccgcg cgagctgtat gattttttaa 1620 agacggaaaa gcccgaagag gaacttgtct tttcccacgg cgacctggga gacagcaaca 1680 tctttgtgaa agatggcaaa gtaagtggct ttattgatct tgggagaagc ggcagggcgg 1740 acaagtggta tgacattgcc ttctgcgtcc ggtcgatcag ggaggatatc ggggaagaac 1800 agtatgtcga gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat 1860 attatatttt actggatgaa ttgttttagt acctagattt agatgtctaa aaagctttaa 1920 ctacaagctt tttagacatc taatcttttc tgaagtacat ccgcaactgt ccatactctg 1980 atgttttata tcttttctaa aagttcgcta gataggggtc ccgagcgcct acgaggaatt 2040 tgtatcacca ggtaccagct gcaagctatt tgaatgtgcc tgttgtgatc gttcgtaaag 2100 acaataaggt aacagagggc tccacagtca gcattaatta cgtttcaggc tcctcaaacc 2160 gcattcaaac aatgtcactt gcgaaaagaa gcatgaaaac gggttcaaac gtactcatta 2220 ttgatgactt tatgaaagca ggcggcacca ttaatggtat gattaacctg ttggatgagt 2280 ttaacgcaaa tgtggcggga atcggcgtct tagttgaagc cgaaggagta gatgaacgtc 2340 ttgttgacga atatatgtca cttcttactc tttcaaccat caacatgaaa gagaagtcca 2400 ttgaaattca gaatggcaat tttctgcgtt tttttaaaga caatctttta aagaatggag 2460 agacagaatc atgacaaaag cagtccacac aaaacatgcc ccagcggcaa tcgggcctta 2520 ttcacaaggg attatcgtca acaatatgtt ttacagctca ggccaaatcc ctttgactcc 2580 ttcaggcgaa atggtgaatg gcgatattaa ggagcagact catcaagtat tcagcaattt 2640 aaaggcggtt ctggaagaag cgggtgcttc ttttgaaaca gttgtaaaag caactgtatt 2700 tatcgcggat atggaacagt ttgcggaagt aaacgaagtg tacggacaat attttgacac 2760 tcacaaaccg gcgagatctt gtgttgaagt cgcgagactc ccgaaggatg cgttagtcga 2820 gatcgaagtt attgcactgg tgaaataata agaaaagtga ttctgggaga gccgggatca 2880 cttttttatt taccttatgc ccgaaatgaa agctttatga ccctgcatta atgaatcggc 2940 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 3000 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 3060 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 3120 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tcgataggct ccgcccccct 3180 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 3240 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 3300 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 3360 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 3420 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 3480 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 3540 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 3600 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 3660 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 3720 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 3780 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 3840 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 3900 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 3960 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 4020 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 4080 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 4140 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 4200 gttaatagtt tgcgcaacgt tgttggcatt gctacaggca tcgtggtgtc acgctcgtcg 4260 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 4320 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 4380 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 4440 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaataccgc 4500 gcccggcgac cgagttgctc ttgcccggcg tcaatacggg ataatagtgt atgacatagc 4560 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 4620 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 4680 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 4740 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 4800 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 4860 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgt atgcggtgtg 4920 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgaaattgt aaacgttaat 4980 attttgttaa aattcgcgtt aaatatttgt taaatcagct cattttttaa ccaataggcc 5040 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 5100 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 5160 accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgcgg 5220 tcgaggtgcc gtaaagctct aaatcggaac cctaaaggga gcccccgatt tagagcttga 5280 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 5340 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaa 5399 <210> 29 <211> 6805 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 29 ttgcggccgc ttcgaaagct gtaatataaa aaccttcttc aactaacggg gcaggttagt 60 gacattagaa aaccgactgt aaaaagtaca gtcggcatta tctcatatta taaaagccag 120 tcattaggcc tatctgacaa ttcctgaata gagttcataa acaatcctgc atgataacca 180 tcacaaacag aatgatgtac ctgtaaagat agcggtaaat atattgaatt acctttatta 240 atgaattttc ctgctgtaat aatgggtaga aggtaattac tattattatt gatatttaag 300 ttaaacccag taaatgaagt ccatggaata atagaaagag aaaaagcatt ttcaggtata 360 ggtgttttgg gaaacaattt ccccgaacca ttatatttct ctacatcaga aaggtataaa 420 tcataaaact ctttgaagtc attctttaca ggagtccaaa taccagagaa tgttttagat 480 acaccatcaa aaattgtata aagtggctct aacttatccc aataacctaa ctctccgtcg 540 ctattgtaac cagttctaaa agctgtattt gagtttatca cccttgtcac taagaaaata 600 aatgcagggt aaaatttata tccttcttgt tttatgtttc ggtataaaac actaatatca 660 atttctgtgg ttatactaaa agtcgtttgt tggttcaaat aatgattaaa tatctctttt 720 ctcttccaat tgtctaaatc aattttatta aagttcattt gatatgcctc ctaaattttt 780 atctaaagtg aatttaggag gcttacttgt ctgctttctt cattagaatc aatccttttt 840 taaaagtcaa tattactgta acataaatat atattttaaa aatatcccac tttatccaat 900 tttcgtttgt tgaactaatg ggtgctttag ttgaagaata aagaccacat taaaaaatgt 960 ggtcttttgt gtttttttaa aggatttgag cgtagcgaaa aatccttttc tttcttatct 1020 tgataataag ggtaactatt gaattcggta ccaagagttt gtagaaacgc aaaaaggcca 1080 tccgtcagga tggccttctg cttaatttga tgcctggcag tttatggcgg gcgtcctgcc 1140 cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta 1200 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga 1260 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag accccacact 1320 accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg ggaccaccgc 1380 gctactgccg ccaggcaaat tctgttttat cagaccgctt ctgcgttctg atttaatctg 1440 tatcaggctg aaaatcttct ctcatccgcc aaaacaggat ccaattatgg cagatcaatg 1500 agcttcacag acacaatatc agggacattt gttagttctt tcacaatttt atcttccaga 1560 tgtctgtcaa aggaaagcat catgatggct tctccgcctt tttccttacg gccaacctgc 1620 atagttgcaa tgttaatatc attatctccg agaatacgtc ctactcggcc gatgacacct 1680 gttgtatctt gatgctggat atacaccaag tgaccagtcg gataaaaatc aatattaaat 1740 ccattgatct cgacaattcg ttctccgaaa tgaggaatat acgtagccgt tacagtaaag 1800 gtgctgcggt ctcctgtcac ttttacgctg atgcagttat cgtatccaga ttcagaagag 1860 gaaatttttt cactgaagct aatgccgcgt tcttttgcga cacccccggc attgacctca 1920 ttaacagtag agtctacgcg cggttttaaa aagcctgaca gaagggcttt tgtaatgaac 1980 gatgtttcaa gtttagcaat tgtgccttca tattgaatgg caacatcctg tactggttct 2040 ttcatgcact gtgatacaag gctgccaatt tttcctgcaa tttgatggta aggcttaatt 2100 ttagcaaatt catcttttgt catggcaggc aggttgatag ctgacatgac aggcaggcct 2160 tttgcgaact gcagaacttc ttctgacact tgggcggcga cattgagctg tgcttctttc 2220 gttgatgctc ccaagtgagg agtggcaatg actaatggat gatcaacaag tttgttgtca 2280 actggcggtt cgacttcgaa aacgtcaagc gctgctcccg caacatgccc gttttccaaa 2340 gcttcgagaa gtgctgcttc atcgataatt ccgcctcgcg cacagttaat taagcgaacg 2400 ccttttttcg tttttgcaat cgtttcttta ttcaataagc cttttgtttc ttttgttaaa 2460 ggcgtgtgaa cggtaatgat atccgcactt tcaagcactt cttcaaatgt acggctgttt 2520 acgccgattt ttttcgctct ttcttccgtt aagaaaggat caaaaacgtg cacagtcata 2580 ccgaacgctc ctcgacgctg tgcaatttca cttccgattc ggcctaatcc tacaatacca 2640 agcgtttttc cataaagctc tgaaccgaca taagctgtgc ggttccactc tctggatttc 2700 actgagatat tagcctgcgg aatgtgtctc attaaagaag agatcattgc aaatgtatgc 2760 tcagctgtcg aaatggtgtt gccgttcgga gcattgatca cgattacccc gtgtttcgta 2820 gcctcatcaa tatcgatatt atcgacaccg acaccggctc ttccgacaat ttttaaagaa 2880 gtcattttgt tgaaaaggtc ttctgttact tttgtcgcgc ttcgcaccaa aagagcatca 2940 aaagtatgta attcatcttc tgcatctgct acgttttttt gaacgatttc aataaagtct 3000 gattcaataa gtggctgtaa accgtcgttg ctcattttgt ctgagaccaa tactcgaaac 3060 atgttttctc ctcctctaga gcgtcctgct gttgttaaga ttattatacc acaccttgta 3120 gataaagtca acaacttttt gcaaaatttt tcaggaattt tagcagaggt tgttctggat 3180 gtagaacaaa acatctttcc gctcttgtgc tgttaggata tctttcttgg aagctaggta 3240 ggcctcgagt tatggcagtt ggttaaaagg aaacaaaaag accgttttca cacaaaacgg 3300 tctttttcga tttcttttta cagtcacagc cacttttgca aaaaccggac agcttcatgc 3360 cttataactg ctgtttcggt cgacctgcag gcatgcaagc ttcgcgaagc ggccgccgac 3420 gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca tcgggatgcc 3480 cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac agcttcaagg 3540 atcgctcgcg gctcttacca gcctaacttc gatcactgga ccgctgatcg tcacggcgat 3600 ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg ccgccctata 3660 ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat 3720 ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc caatcaattc 3780 ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat cgcgtccgcc 3840 atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc 3900 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag 3960 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg 4020 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg 4080 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc 4140 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc 4200 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca 4260 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc 4320 cccatgaaca gaaattcccc cttacacgga ggcatcaagt gaccaaacag gaaaaaaccg 4380 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg 4440 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc 4500 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 4560 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 4620 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata 4680 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca 4740 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 4800 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 4860 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4920 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4980 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 5040 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 5100 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 5160 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5220 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5280 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5340 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5400 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5460 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5520 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5580 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5640 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5700 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5760 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5820 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5880 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 5940 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 6000 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat 6060 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 6120 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 6180 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 6240 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 6300 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6360 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6420 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6480 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6540 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6600 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 6660 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 6720 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 6780 cacgaggccc tttcgtcttc aagaa 6805 <210> 30 <211> 5983 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 30 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 60 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 120 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 180 ttgtaatacg actcactata gggcgaattg ggcccgacgt cgcatgctcc cggccgccat 240 ggccgcggga tatcactagt gcggccgcct gcaggtcgac catatgggag agcccggatc 300 caattatggc agatcaatga gcttcacaga cacaatatca gggacatttg ttagttcttt 360 cacaatttta tcttccagat gtctgtcaaa ggaaagcatc atgatggctt ctccgccttt 420 ttccttacgg ccaacctgca tagttgcaat gttaatatca ttatctccga gaatacgtcc 480 tactcggccg atgacacctg ttgtatcttg atgctggata tacaccaagt gaccagtcgg 540 ataaaaatca atattaaatc cattgatctc gacaattcgt tctccgaaat gaggaatata 600 cgtagccgtt acagtaaagg tgctgcggtc tcctgtcact tttacgctga tgcagttatc 660 gtatccagat tcagaagagg aaattttttc actgaagcta atgccgcgtt cttttgcgac 720 acccccggca ttgacctcat taacagtaga gtctacgcgc ggttttaaaa agcctgacag 780 aagggctttt gtaatgaacg atgtttcaag tttagcaatt gtgccttcat attgaatggc 840 aacatcctgt actggttctt tcatgcactg tgatacaagg ctgccaattt ttcctgcaat 900 ttgatggtaa ggcttaattt tagcaaattc atcttttgtc atggcaggca ggttgatagc 960 tgacatgaca ggcaggcctt ttgcgaactg cagaacttct tctgacactt gggcggcgac 1020 attgagctgt gcttctttcg ttgatgctcc caagtgagga gtggcaatga ctaatggatg 1080 atcaacaagt ttgttgtcaa ctggcggttc gacttcgaaa acgtcaagcg ctgctcccgc 1140 aacatgcccg ttttccaaag ctttttagac atctaaatct aggtactaaa acaattcatc 1200 cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa 1260 tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat 1320 gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc 1380 atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc 1440 gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc 1500 ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc 1560 taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag 1620 cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc 1680 ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg 1740 ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt 1800 ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag 1860 gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt 1920 tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca ttttagccat 1980 ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac 2040 aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct 2100 ttttcaaagt tgttttgaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa 2160 ccacaattat gatagaattt acaagctata aggttattgt cctgggtttc aagcattagt 2220 ccatgcaagt ttttatgctt tgcccattct atagatatat tgataagcgc gctgcctatg 2280 ccttgccccc tgaaatcctt acatacggcg atatcttcta tataaaagat atattatctt 2340 atcagtattg tcaatatatt caaggcaatc tgcctcctca tcctcttcat cctcttcgtc 2400 ttggtagctt tttaaatatg gcgcttcata gagtaattct gtaaaggtcc aattctcgtt 2460 ttcatacctc ggtataatct tacctatcac ctcaaatggt tcgctgggtt tatcgcctga 2520 tgcggtattt tctccttacg catctgtgcg gtatttcacg tcgacgcggc cgccatggcc 2580 gcgggatccc ggtaccgaaa catcgttaga tttcctccta aattgacaaa ctaaatatct 2640 gataatttaa catattctca aaagagtgtc aacgtgtatt gacgcagtaa aggataaaag 2700 taaagcctaa taaatcaatg atctgacagc ttgcaggtaa tatatttaat ttgaagcaat 2760 tctctataca gccaaccagt tatcgtttat aatgtaatta aatttcatat gatcaatctt 2820 cggggcaggg tgaaattccc taccggcggt gatgagccaa tggctctaag cccgcgagct 2880 gtctttacag caggattcgg tgagattccg gagccgacag tacagtctgg atgggagaag 2940 atggaggttc ataagcgttt tgaaattgaa tttttcaaac gtttctttgc ctagcctaat 3000 tttcgaaacc ccgcttttat atatgaagcg gtttttttat tggctggaaa agaacctttc 3060 cgttttcgag taagatgtga tcgaaaagga gagaatgaag tgaaagtaaa aaaattagtt 3120 gtggtcagca tgctgagcag cattgcattt gttttgatgc tgttaaattt cccgtttccg 3180 ggtcttccgg attatttaaa aatcgatttt agcgacgttc ccgcaattat tgccattctg 3240 atttacggac ctttggcggg atcactagag ggctcccaac gcgttggatg catagcttga 3300 gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt 3360 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 3420 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 3480 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3540 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 3600 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 3660 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 3720 aaaaggccgc gttgctggcg tttttcgata ggctccgccc ccctgacgag catcacaaaa 3780 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 3840 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 3900 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 3960 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4020 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4080 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4140 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 4200 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4260 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 4320 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 4380 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 4440 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 4500 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 4560 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 4620 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 4680 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 4740 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 4800 acgttgttgg cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 4860 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 4920 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 4980 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5040 ctgtgactgg tgagtactca accaagtcat tctgagaata ccgcgcccgg cgaccgagtt 5100 gctcttgccc ggcgtcaata cgggataata gtgtatgaca tagcagaact ttaaaagtgc 5160 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 5220 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 5280 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 5340 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 5400 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 5460 ttccgcgcac atttccccga aaagtgccac ctgtatgcgg tgtgaaatac cgcacagatg 5520 cgtaaggaga aaataccgca tcaggcgaaa ttgtaaacgt taatattttg ttaaaattcg 5580 cgttaaatat ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 5640 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 5700 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 5760 atggcccact acgtgaacca tcacccaaat caagtttttt gcggtcgagg tgccgtaaag 5820 ctctaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 5880 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 5940 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taa 5983 <210> 31 <211> 7330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: plasmid <400> 31 ttgcggccgc ttcgaaagct gtaatataaa aaccttcttc aactaacggg gcaggttagt 60 gacattagaa aaccgactgt aaaaagtaca gtcggcatta tctcatatta taaaagccag 120 tcattaggcc tatctgacaa ttcctgaata gagttcataa acaatcctgc atgataacca 180 tcacaaacag aatgatgtac ctgtaaagat agcggtaaat atattgaatt acctttatta 240 atgaattttc ctgctgtaat aatgggtaga aggtaattac tattattatt gatatttaag 300 ttaaacccag taaatgaagt ccatggaata atagaaagag aaaaagcatt ttcaggtata 360 ggtgttttgg gaaacaattt ccccgaacca ttatatttct ctacatcaga aaggtataaa 420 tcataaaact ctttgaagtc attctttaca ggagtccaaa taccagagaa tgttttagat 480 acaccatcaa aaattgtata aagtggctct aacttatccc aataacctaa ctctccgtcg 540 ctattgtaac cagttctaaa agctgtattt gagtttatca cccttgtcac taagaaaata 600 aatgcagggt aaaatttata tccttcttgt tttatgtttc ggtataaaac actaatatca 660 atttctgtgg ttatactaaa agtcgtttgt tggttcaaat aatgattaaa tatctctttt 720 ctcttccaat tgtctaaatc aattttatta aagttcattt gatatgcctc ctaaattttt 780 atctaaagtg aatttaggag gcttacttgt ctgctttctt cattagaatc aatccttttt 840 taaaagtcaa tattactgta acataaatat atattttaaa aatatcccac tttatccaat 900 tttcgtttgt tgaactaatg ggtgctttag ttgaagaata aagaccacat taaaaaatgt 960 ggtcttttgt gtttttttaa aggatttgag cgtagcgaaa aatccttttc tttcttatct 1020 tgataataag ggtaactatt gaattcggta ccaagagttt gtagaaacgc aaaaaggcca 1080 tccgtcagga tggccttctg cttaatttga tgcctggcag tttatggcgg gcgtcctgcc 1140 cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta 1200 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga 1260 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag accccacact 1320 accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg ggaccaccgc 1380 gctactgccg ccaggcaaat tctgttttat cagaccgctt ctgcgttctg atttaatctg 1440 tatcaggctg aaaatcttct ctcatccgcc aaaacaggat ccaattatgg cagatcaatg 1500 agcttcacag acacaatatc agggacattt gttagttctt tcacaatttt atcttccaga 1560 tgtctgtcaa aggaaagcat catgatggct tctccgcctt tttccttacg gccaacctgc 1620 atagttgcaa tgttaatatc attatctccg agaatacgtc ctactcggcc gatgacacct 1680 gttgtatctt gatgctggat atacaccaag tgaccagtcg gataaaaatc aatattaaat 1740 ccattgatct cgacaattcg ttctccgaaa tgaggaatat acgtagccgt tacagtaaag 1800 gtgctgcggt ctcctgtcac ttttacgctg atgcagttat cgtatccaga ttcagaagag 1860 gaaatttttt cactgaagct aatgccgcgt tcttttgcga cacccccggc attgacctca 1920 ttaacagtag agtctacgcg cggttttaaa aagcctgaca gaagggcttt tgtaatgaac 1980 gatgtttcaa gtttagcaat tgtgccttca tattgaatgg caacatcctg tactggttct 2040 ttcatgcact gtgatacaag gctgccaatt tttcctgcaa tttgatggta aggcttaatt 2100 ttagcaaatt catcttttgt catggcaggc aggttgatag ctgacatgac aggcaggcct 2160 tttgcgaact gcagaacttc ttctgacact tgggcggcga cattgagctg tgcttctttc 2220 gttgatgctc ccaagtgagg agtggcaatg actaatggat gatcaacaag tttgttgtca 2280 actggcggtt cgacttcgaa aacgtcaagc gctgctcccg caacatgccc gttttccaaa 2340 gcttcgagaa gtgctgcttc atcgataatt ccgcctcgcg cacagttaat taagcgaacg 2400 ccttttttcg tttttgcaat cgtttcttta ttcaataagc cttttgtttc ttttgttaaa 2460 ggcgtgtgaa cggtaatgat atccgcactt tcaagcactt cttcaaatgt acggctgttt 2520 acgccgattt ttttcgctct ttcttccgtt aagaaaggat caaaaacgtg cacagtcata 2580 ccgaacgctc ctcgacgctg tgcaatttca cttccgattc ggcctaatcc tacaatacca 2640 agcgtttttc cataaagctc tgaaccgaca taagctgtgc ggttccactc tctggatttc 2700 actgagatat tagcctgcgg aatgtgtctc attaaagaag agatcattgc aaatgtatgc 2760 tcagctgtcg aaatggtgtt gccgttcgga gcattgatca cgattacccc gtgtttcgta 2820 gcctcatcaa tatcgatatt atcgacaccg acaccggctc ttccgacaat ttttaaagaa 2880 gtcattttgt tgaaaaggtc ttctgttact tttgtcgcgc ttcgcaccaa aagagcatca 2940 aaagtatgta attcatcttc tgcatctgct acgttttttt gaacgatttc aataaagtct 3000 gattcaataa gtggctgtaa accgtcgttg ctcattttgt ctgagaccaa tactcgaaac 3060 atgttttctc ctcctctaga gcgtcctgct gttgttaaga ttattatacc acaccttgta 3120 gataaagtca acaacttttt gcaaaatttt tcaggaattt tagcagaggt tgttctggat 3180 gtagaacaaa acatctttcc gctcttgtgc tgttaggata tctttcttgg aagctaggta 3240 ggcctcgagt tatggcagtt ggttaaaagg aaacaaaaag accgttttca cacaaaacgg 3300 tctttttcga tttcttttta cagtcacagc cacttttgca aaaaccggac agcttcatgc 3360 cttataactg ctgtttcggt cgacgaaaca tcgttagatt tcctcctaaa ttgacaaact 3420 aaatatctga taatttaaca tattctcaaa agagtgtcaa cgtgtattga cgcagtaaag 3480 gataaaagta aagcctaata aatcaatgat ctgacagctt gcaggtaata tatttaattt 3540 gaagcaattc tctatacagc caaccagtta tcgtttataa tgtaattaaa tttcatatga 3600 tcaatcttcg gggcagggtg aaattcccta ccggcggtga tgagccaatg gctctaagcc 3660 cgcgagctgt ctttacagca ggattcggtg agattccgga gccgacagta cagtctggat 3720 gggagaagat ggaggttcat aagcgttttg aaattgaatt tttcaaacgt ttctttgcct 3780 agcctaattt tcgaaacccc gcttttatat atgaagcggt ttttttattg gctggaaaag 3840 aacctttccg ttttcgagta agatgtgatc gaaaaggaga gaatgaagtg aaagtaaaaa 3900 aattagttgt ggtcagcatg caagcttcgc gaagcggccg ccgacgcgag gctggatggc 3960 cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat 4020 gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct 4080 taccagccta acttcgatca ctggaccgct gatcgtcacg gcgatttatg ccgcctcggc 4140 gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc 4200 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac 4260 ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg 4320 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc 4380 acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg 4440 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg 4500 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca 4560 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc 4620 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca 4680 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc 4740 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta 4800 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat 4860 tcccccttac acggaggcat caagtgacca aacaggaaaa aaccgccctt aacatggccc 4920 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg 4980 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc 5040 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 5100 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 5160 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 5220 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 5280 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac 5340 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 5400 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 5460 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 5520 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 5580 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 5640 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 5700 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 5760 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 5820 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 5880 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 5940 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 6000 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 6060 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 6120 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 6180 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 6240 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 6300 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 6360 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 6420 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 6480 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 6540 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc 6600 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 6660 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 6720 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 6780 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 6840 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 6900 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 6960 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 7020 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 7080 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 7140 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 7200 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 7260 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 7320 tcttcaagaa 7330 - One -

Claims (49)

An improved method of producing pantothenate comprising culturing a microorganism having a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway under conditions in which pantothenate production is improved. (i) deregulated pantothenate biosynthetic pathway and (ii) having a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway An improved method of producing pantothenate comprising culturing the microorganism under conditions such that pantothenate production is improved. The method of claim 2, wherein the microorganism has two or more deregulated pantothenate biosynthetic enzymes. The method of claim 2, wherein the microorganism has three or more deregulated pantothenate biosynthetic enzymes. The method of claim 2, wherein the microorganism has four or more deregulated pantothenate biosynthetic enzymes. 6. The method of claim 5, wherein the microorganism is deregulated ketopantoate hydroxymethyltransferase, deregulated ketopantoate reductase, deregulated pantothenate synthetase, and deregulated aspartate-α-dekar Method of having a cyclosase. The method of claim 1, wherein the microorganism has a further deregulated isoleucine-valine ( ilv ) biosynthetic pathway. 8. The method of claim 7, wherein the microorganism has two or more deregulated isoleucine-valine ( ilv ) biosynthetic enzymes. 8. The method of claim 7, wherein the microorganism has three or more deregulated isoleucine-valine ( ilv ) biosynthetic enzymes. 10. The method of claim 9, wherein the microorganism has a deregulated acetohydroxy acid synthetase, a deregulated acetohydroxy acid isomerdeductase, and a deregulated dihydroxy acid dehydratase. The method of claim 1, wherein the microorganism has one or more deregulated MTF biosynthetic enzymes. The method of claim 11, wherein the microorganism has a deregulated glyA gene. The method of claim 11, wherein the microorganism has a deregulated serA gene. The method of claim 11, wherein the microorganism has a deregulated glyA gene and a deregulated serA gene. The method of claim 12 or 14, wherein the microorganism has a mutated, deleted or destroyed purR gene. Microorganisms with a deregulated pantothenate biosynthetic pathway, a deregulated isoleucine-valine ( ilv ) biosynthetic pathway, and a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway that has been deregulated to improve production of pantothenate Improved production method of pantothenate comprising culturing. Deregulated pantothenate biosynthetic pathway, deregulated isoleucine-valine ( ilv ) biosynthetic pathway, and deregulated methylenetetrahydrofolate (MTF) such that at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms A method of producing pantothenate comprising culturing a microorganism having a biosynthetic pathway. 18. The method for producing pantothenate according to claim 17, comprising culturing the microorganism so that at least 60 g / L of pantothenate is produced after 36 hours of culturing the microorganism. 18. The method for producing pantothenate according to claim 17, comprising culturing the microorganism so that at least 70 g / L of pantothenate is produced after 36 hours of culturing the microorganism. Deregulated pantothenate biosynthetic pathway, deregulated isoleucine-valine ( ilv ) biosynthetic pathway, and deregulated methylenetetrahydrofolate, deregulated to produce at least 60 g / L pantothenate after 48 hours of culture of microorganisms (MTF) A method for producing pantothenate comprising culturing a microorganism having a biosynthetic pathway. The method of claim 20, comprising culturing the microorganism such that at least 70 g / L of pantothenate is produced after 48 hours of culturing the microorganism. The method of claim 20, comprising culturing the microorganism such that at least 80 g / L of pantothenate is produced after 48 hours of culturing the microorganism. 23. The method of any one of claims 1 to 22, wherein pantothenate production is further enhanced by modulating pantothenate kinase activity. The method of claim 23, wherein pantothenate kinase activity is reduced. The method of claim 24, wherein CoaA is deleted and CoaX is downregulated. The method of claim 24, wherein CoaX is deleted and CoaA is downregulated. The method of claim 24, wherein CoaX and CoaA are downregulated. 28. The method of any one of claims 1 to 27, wherein the microorganism is cultured under excessive serine conditions. A method for producing pantothenate comprising culturing a microorganism having a deregulated pantothenate biosynthetic pathway under excess serine conditions to produce pantothenate. The method of claim 1, wherein the microorganism has a pantothenate biosynthetic pathway that is deregulated such that pantothenate production is independent of β-alanine feed. 31. The method of any one of claims 1-30, wherein the microorganism is a gram positive microorganism. 32. The method of any one of the preceding claims, wherein the microorganism belongs to the genus Bacillus. 33. The method of any one of the preceding claims, wherein the microorganism is Bacillus subtilis. A product synthesized according to the method according to any one of claims 1 to 33. 35. A composition comprising pantothenate produced according to any one of claims 1 to 34. A recombinant microorganism for improved production of pentothenate, having a deregulated pantothenate biosynthetic pathway and a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway. A recombinant microorganism for improved production of pentothenate, having a deregulated pantothenate biosynthetic pathway, a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway, and a deregulated isoleucine-valine ( ilv ) pathway. 38. The microorganism of claim 36 or 37, further having reduced pantothenate kinase activity. 39. The microorganism according to any one of claims 36 to 38, which is a gram positive microorganism. 39. The microorganism according to any one of claims 36 to 38, belonging to the genus Bacillus. 39. The microorganism according to any one of claims 36 to 38, which is Bacillus subtilis. Under 36 hours of culture of microorganisms, at least 30 g / L of pantothenate is produced. (a) deregulated panB gene, (b) deregulated panD gene and (c) a method of producing pantothenate comprising culturing a recombinant microorganism having one or more deregulated isoleucine-valine ( ilv ) biosynthetic enzyme-encoding genes. The method of claim 42, wherein the microorganism further has a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway, and the microorganism is cultured under conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of the microorganism. Way. Under excessive serine conditions where at least 50 g / L of pantothenate is produced after 36 hours of culture of the microorganism, (a) deregulated panB gene, and (b) a method of producing pantothenate comprising culturing a recombinant microorganism having a deregulated panD gene. Under excessive valine conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms, (a) deregulated panB gene, (b) deregulated panD gene and (c) a method of producing pantothenate comprising culturing a recombinant microorganism having a deregulated methylenetetrahydrofolate (MTF) biosynthetic pathway. Under excessive valine conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms, (a) deregulated panB gene, (b) deregulated panD gene and (c) a method of producing pantothenate comprising culturing a recombinant microorganism having a deregulated glyA gene. Under excessive valine conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms, (a) deregulated panB gene, (b) deregulated panD gene and (c) a method of producing pantothenate comprising culturing a recombinant microorganism having a mutated, deleted or destroyed purR glyA gene. Under excessive valine conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms, (a) deregulated panB gene, (b) deregulated panD gene and (c) a method of producing pantothenate comprising culturing a recombinant microorganism having a deregulated serA gene. Under excessive valine conditions in which at least 50 g / L of pantothenate is produced after 36 hours of culture of microorganisms, (a) deregulated panB gene, (b) deregulated panD gene, (c) deregulated serA gene, and (d) A method of producing pantothenate comprising culturing a recombinant microorganism having a deregulated glyA gene.