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WO2004113510A2 - Production de pantothenate au moyen de micro-organismes inaptes a la sporulation - Google Patents

Production de pantothenate au moyen de micro-organismes inaptes a la sporulation Download PDF

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Publication number
WO2004113510A2
WO2004113510A2 PCT/EP2004/006619 EP2004006619W WO2004113510A2 WO 2004113510 A2 WO2004113510 A2 WO 2004113510A2 EP 2004006619 W EP2004006619 W EP 2004006619W WO 2004113510 A2 WO2004113510 A2 WO 2004113510A2
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Prior art keywords
microorganism
pantothenate
gene
sporulation
spooa
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WO2004113510A3 (fr
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John Perkins
Zoltan Pragai
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DSM IP Assets BV
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DSM IP Assets BV
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Priority to JP2006515998A priority Critical patent/JP4713469B2/ja
Priority to CN2004800171442A priority patent/CN1809631B/zh
Priority to KR1020057024232A priority patent/KR101185656B1/ko
Publication of WO2004113510A2 publication Critical patent/WO2004113510A2/fr
Publication of WO2004113510A3 publication Critical patent/WO2004113510A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N3/00—Spore forming or isolating processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

Definitions

  • Pantothenate is a member of the B complex of vitamins and is a nutritional requirement for mammals including humans, e.g., from food, sources, as a water-soluble vitamin supplement or as a feed additive. In cells, pantothenate is used primarily for the biosynthesis of coenzyme A and acyl carrier protein. These essential coenzymes function in the metabolism of acyl moieties, which form thioesters with the sulfhydryl group of the 4'- phosphopantethein portion of these molecules.
  • Pantothenate has been synthesized conventionally via chemical synthesis from bulk chemicals.
  • the substrates required for chemical synthesis are expensive and the racemic intermediates have to be optically resolved.
  • bacterial or microbial systems have been employed that produce enzymes useful in pantothenate biosynthesis processes.
  • bioconversion processes have been evaluated as a means of favoring production of preferred isomer of pantothenic acid.
  • methods of direct microbial synthesis have recently been examined as a means of facilitating D-pantothenate production.
  • Patent applications WO 01/21772, WO 02/057474, and WO 02/061108 described a method to produce pantothenate using strains of B. subtilis 168 that have higher expression levels of biosynthetic genes involved in pantothenate production. These genes include panB, panC, panD, panE (ylbQ), ilvB, ilvN, ilvC, ilvD, glyA, and serA. To achieve higher expression levels of these genes, standard genetic recombinant methods were used that are known in the art. Pantothenate production of these engineered strains ranged from between 37 g/liter to 85 g/liter in 48 hour fed-batch fermentation.
  • sporulation-deficient strains of B. subtilis are used in fermentative processes (e.g. riboflavin - US5,837,528; biotin - US6,057,136).
  • the spoOA mutation is used to arrest sporulation.
  • the spoOA gene encodes a protein that regulates the initiation of sporulation.
  • WO 97/03185 describes a method to produce commercially important enzymes that uses a mutation in the Bacillus licheniformis spollAC which encodes a sporulation-specific transcription sigma factor, ⁇ F , to obtain bacteria of the genus Bacillus other than B. subtilis that are incapable of sporulating.
  • Spo0A ⁇ P concentration then reaches a higher critical level, it activates genes (sinl, spollG, spollE, spollA, etc.) required for the entry and commitment to sporulation. Since Spo0A ⁇ P represses transcription of abrB and AbrB represses transcription of sigH, therefore Spo0A ⁇ P indirectly activates genes that are in the regulon for ⁇ H , an alternative sigma factor governing the transcription of genes (spoOA, spoOF, spollA, phrC, phrE, etc.) involved in stationary growth phase and the early stages of sporulation (Britton et al., 2002).
  • genes and gene products that increase or decrease the level of Spo0A ⁇ P and AbrB include, but are not limited to, abrB, kapB, kbaA, kinA, kinB, kinC, kinD, kinE, kipA, kipl, obg, phrC, phrE, rapA, rapB, rapE, sigH, spoOA, spoOB, spoOE and spoOF.
  • proteins that influence SpoOA ⁇ P-dependent binding at promoter sites to activate gene expression include, but are not limited to, SpoOJA and SpoOJB.
  • signals that increase or decrease the level of Spo0A ⁇ P and AbrB include, but are not limited to, nutritional, metabolic, DNA status, cell density (pheromones and quorum-sensing molecules) and cell cycle signals.
  • the cell divides asymmetrically to generate two compartments of unequal size and dissimilar developmental fates (Piggot and Losick, 2002).
  • the smaller compartment, the forespore develops into the spore, whereas the larger compartment, the mother cell, nurtures the developing spore.
  • the mother cell lyses to liberate the mature spore. Differentiation involves the action of four cell-specific sigma factors: ⁇ F , ⁇ E , ⁇ G and ⁇ ⁇ .
  • ⁇ F and ⁇ E factors are activated shortly after asymmetric division (polar septation, stage II.) when ⁇ F directs gene expression in the forespore (Margoiis et al., 1991) and ⁇ E directs gene expression in the mother cell (Dirks and Losick, 1991 ). Later in sporulation ⁇ F is replaced in the forespore by ⁇ G and ⁇ E is replaced by ⁇ ⁇ in the mother cell (Losick and Stragier, 1992; Li and Piggot, 2001).
  • ⁇ E is derived from an inactive proprotein called pro- ⁇ E (LaBell et al., 1987). Synthesis of pro- ⁇ E commences prior to septation (Satola et al., 1992), but the proteolytic processing of pro- ⁇ E to mature ⁇ E occurs after asymmetric division in the mother cell. This reaction is mediated by a sporulation-specific protease, SpolIGA, that cleaves 27 amino acids from the pro- ⁇ E amino terminus (Stragier et al., 1988; Jonas et al., 1988; Peters and Haldenwang, 1994).
  • SpolIGA sporulation-specific protease
  • pantothenate-producing B. subtilis 168 strains that are described in WO 01/21772, WO 02/057474, and WO 02/061108 are all wild-type for sporulation. No example is provided that shows the production of pantothenate in a sporulation-deficient strain.
  • the present invention therefore relates to a sporulation-deficient microorganism being capable of overproducing a pantothenate compound.
  • the microorganism may be modified such that the activity of SigE is reduced compared to the non-modified form of the microorganism.
  • SigE is the gene product encoded by the gene spollGB.
  • Reduced SigE activity may be caused by lack of spollGB expression, a decrease or lack of expression of the protease encoded by spollGA, a decrease or lack of expression of other upstream regulating genes or proteins, and the like.
  • the modification of the microorganism comprises a mutation that causes reduced SigE activity. More preferably, the mutation affects one or more genes selected from the group consisting of the genes spollGA, spollGB, spollR and spoil AC.
  • the microorganism may be modified such that the activity of both SpoOA and AbrB is reduced compared to the non-modified form of the microorganism.
  • SpoOA and AbrB are gene products encoded by the spoOA and abrB genes, respectively.
  • Reduced SpoOA and AbrB activity may be caused by lack of gene expression, a decrease or lack of signals (nutritional, metabolic, DNA status, cell density [pheromones and quorum-sensing molecules] and cell cycle signals), a decrease or lack of expression of other upstream regulating genes or proteins, and the like.
  • the modification of the microorganism comprises a mutation that causes reduced SpoOA/AbrB activity.
  • the mutation affects one, two or more genes selected from the group consisting of the genes abrB, kapB, kbaA, kinA, kinB, kinC, kinD, kinE, kipA, kipl, obg, phrC, phrE, rapA, rapB, rapE, scoC, sigH, sinR, sinl, spoOA, spoOB, spoOE, spoOF, spoOJA and spoOJB.
  • the microorganism may be eukaryotic or prokaryotic.
  • the microorganism is prokaryotic.
  • the prokaryotic microorganism may be Gram positive or Gram negative.
  • Gram positive microorganisms include but are not limited to microorganisms belonging to one of the genera Bacillus, Corynebacterium, Lactobacillus, Lactococci and Streptomyces.
  • the microorganism belongs to the genus Bacillus. Examples are Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus puntis, Bacillus halodurans, etc.
  • the microorganism is Bacillus subtilis.
  • the corresponding wild type forms of the microorganisms of the invention are spore-forming microorganisms.
  • pantothenate compound preferably is pantothenate.
  • Pantothenate compounds further include intermediate compounds in the biosynthetic pathway of pantothenate biosynthesis such as pantoate, ⁇ -ketopantoate, ⁇ -ketoisovalerate and the like.
  • the invention encompasses any mutation to a gene of the microorganism resulting in a reduced function of the sigE gene product (SigE protein). SigE function may be assayed in a sporulation assay known to those skilled in the art.
  • the microorganism of the invention may have a mutation in its spollGB (sigE) gene resulting in the absence or decreased expression of functional sigE gene product.
  • the invention further encompasses any mutation in genes known to activate spollGB (sigE). These include, but are not limited to, spollGA, spollR, and spollAC (sigF) (Helmann and Moran, 2002).
  • the microorganism of the invention may have a mutation in its spollGA gene resulting in the absence or decreased expression of functional spollGA gene product.
  • the microorganism of the invention may have a mutation in its spollR gene resulting in the absence or decreased expression of functional spollR gene product.
  • the microorganism of the invention may have a mutation in its spollAC gene resulting in the absence or decreased expression of functional spollAC gene product.
  • expression denotes the transcription of a nucleic acid sequence and/or the subsequent translation of the transcribed sequence into an amino acid sequence.
  • a decreased expression of a nucleic acid may be achieved by introducing a mutation to a gene, e.g. by deleting a gene; nucleotide additions or subtractions that inactivate or decrease the activity of the encoded protein through formation of a frame shift of the reading frame or premature termination of translation through introduction of a stop codon; modifying regulatory sequences such as promoters, ribosome binding sites, and other techniques described herein.
  • a “mutation” may be a deletion, substitution or addition in the sequence of a gene.
  • the mutation may be a disruption, frame shift mutation or nonsense mutation.
  • the mutation may be a disruption affecting the entire sequence of the gene or a portion thereof.
  • the mutation may affect the coding or the non-coding sequence, e.g., the promoter, of the gene.
  • the mutation may result in complete lack of transcription of the gene or in premature termination of translation.
  • the mutation may be located in a preceding (or "upstream") gene, which disrupts transcription of the adjacent (or "downstream”) gene(s) by a process referred to as polarity.
  • the mutation may have the effect that the translated protein carries a mutation in comparison with the wild type protein that renders the protein nonfunctional.
  • Methods of introducing suitable mutations into a gene are known to those skilled in the art. These methods include, but are not limited to, introduction of point mutations into the bacteria chromosome (Cutting and Vander Horn, 1990), removal of a chromosomal DNA segment and replacing the segment with an antibiotic resistance gene (Perego, 1993), direct insertion of a transposable element, such a transposon or mini (Youngman, 1990; Petit et al., 1990), or plasmids such as pMUTIN (Vagner et al., 1998).
  • the sporulation-deficient microorganism having a reduced function of its sigE gene product is capable of overexpressing spoOA, more preferably it overexpresses spoOA.
  • Overexpression of spoOA may be achieved as described herein.
  • Increased expression or overexpression of a gene may be achieved in a variety of ways.
  • One approach to obtain microorganisms overexpressing one or more genes is to alter or modify regulatory sequences or sites associated with the expression of a particular gene, e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive. Further techniques are described infra.
  • a “promoter” is a DNA sequence upstream from the start of transcription of a gene and involved in recognition and binding of RNA polymerase and/or other proteins to initiate transcription of the gene. Usually, the promoter determines under what conditions the gene is expressed.
  • an "inducible promoter” is one which causes mRNA synthesis of a gene to be initiated temporally under specific conditions, (i) Promoters may be regulated primarily by an ancillary factor such as a repressor or an activator.
  • the repressors are sequence-specific DNA binding proteins that repress promoter activity. The transcription can be initiated from this promoter in the presence of an inducer that prevents binding of the repressor to the operator of the promoter.
  • promoters from Gram-positive microorganisms include, but are not limited to, gnt (gluconate operon promoter); penP from Bacillus licheniformis; glnA (glutamine synthetase); xylAB (xylose operon); araABD (L-arabinose operon) and P spac promoter, a hybrid SPO1//ac promoter that can be controlled by inducers such as isopropyl- ⁇ -D-thiogalactopyranoside [IPTG] (Yansura and Henner, 1984).
  • inducers such as isopropyl- ⁇ -D-thiogalactopyranoside [IPTG] (Yansura and Henner, 1984).
  • Activators are also sequence-specific DNA binding proteins that induce promoter activity.
  • promoters from Gram-positive microorganisms include, but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS, SpoOA-Phosphorelay), LevR, Mry and GltC.
  • Production of secondary sigma factors can be primarily responsible for the transcription from specific promoters.
  • Examples from Gram-positive microorganisms include, but are not limited to, the promoters activated by sporulation specific sigma factors: ⁇ F , ⁇ E , ⁇ G and ⁇ ⁇ and general stress sigma factor, ⁇ B .
  • the ⁇ B -mediated response is induced by energy limitation and environmental stresses (Hecker and V ⁇ lker, 1998).
  • Attenuation and antitermination also regulates transcription.
  • Gram-positive microorganisms include, but are not limited to, trp operon and sacB gene,
  • Other regulated promoters in expression vectors are based on the temperature-sensitive immunity repressor from phage ⁇ 105 (Osbourne et al., 1985) and the sacR regulatory system conferring sucrose inducibility (Klier and Rapoport, 1988).
  • a “constitutive” promoter is one that permits the gene to be expressed under virtually all environmental conditions, i.e. a promoter that directs constant, non-specific gene expression.
  • a “strong constitutive promoter” is one which causes mRNAs to be initiated at high frequency compared to a native host cell. Strong constitutive promoters are well known and an appropriate one may be selected according to the specific sequence to be controlled in the host cell. Examples of such strong constitutive promoters from Gram-positive microorganisms include, but are not limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and amyE.
  • promoters from Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, 73, gal, trc, are, SP6, ⁇ -P R , and ⁇ -P L .
  • the invention further encompasses any mutation to one or more genes resulting in a reduced function of both SpoOA and AbrB.
  • SpoOA and AbrB function may be assayed in an enzyme assay for ⁇ -glutamyltranspeptidase (Xu and Strauch, 1996) and with microarray technology known to those skilled in the art.
  • SpoOA function may be assayed in a sporulation assay; and AbrB function may be assayed with lacZ reporter gene fused to AbrB-regulated promoters (spoOVG, tycA, abrB, etc.).
  • the microorganism may have at least one mutation in its spoOA gene and at least one mutation in its abrB gene. The mutations result in the absence or decreased expression of functional spoOA gene product and in the absence or decreased expression of functional abrB gene product.
  • Mutations to one or more genes resulting in a reduced function of SpoOA include mutations to genes known to activate spoOA. These include, but are not limited to, spoOB, spoOF, kinA, kinB, kinC, kinD, kinE and sigH.
  • the microorganism of the invention may have a mutation in its spoOB resulting in the absence or decreased expression of functional spoOB gene product.
  • the microorganism of the invention may have a mutation in its spoOF resulting in the absence or decreased expression of functional spoOF gene product.
  • the mutation resulting in a reduced SigE function and the mutations resulting in a reduced function of both SpoOA and AbrB may be combined. Accordingly, the preferred embodiments of the various mutations and modifications described herein may be combined. This applies to the method described infra mutatis mutandis.
  • the microorganism of the invention is capable of overproducing a pantothenate compound under suitable conditions.
  • the term "overproducing a pantothenate compound” refers to significantly increased production of the pantothenate compound by the microorganism of the invention in comparison to the wild type form of said microorganism.
  • overproduction of pantothenate means production of at least 50 mg, 100 mg, 200 mg, 500 mg, 1 g, 3 g, 5 g or 10 g pantothenate per liter culture medium.
  • a population of the microorganisms of the invention produces at least 100 mg, preferably at least 125 mg, more preferably at least 140 mg, more preferably at least 200 mg, still more preferably at least 300 mg, even more preferably at least 400 mg, most preferably at least 500 mg pantothenate per liter culture medium when cultured under the conditions as described in Example II. In this example culturing in minimal medium is described.
  • a population of the microorganisms of the invention may produce at least 100 mg, preferably at least 125 mg, more preferably at least 140 mg, more preferably at least 200 mg, still more preferably at least 300 mg, even more preferably at least 400 mg, most preferably at least 500 mg pantothenate per liter culture medium when cultured under the conditions as described in Example IV or V.
  • a population of the microorganisms of the invention produces at least 5 g, preferably at least 7.5 g, most preferably at least 10 g pantothenate per liter culture medium when cultured under conditions as described in Example III.
  • This example describes fed-batch fermentation.
  • the level of pantothenate production by the sporulation-deficient microorganisms of the invention is comparable to the level of pantothenate production by control cells. Therefore, the amount of the pantothenate compound produced by a population of the microorganisms of the invention may be greater than 50%, preferably greater than 75%, more preferably greater than 100% of the amount of the pantothenate compound produced by a population of the corresponding microorganisms not being sporulation-deficient when cultured under identical conditions.
  • the amount of the pantothenate compound produced by a population of the microorganisms of the invention is greater than 50%, preferably greater than 75% of the amount of the pantothenate compound produced by a population of the corresponding microorganisms not being sporulation-deficient.
  • the amount of the pantothenate compound produced by a population of the microorganisms of the invention is greater than 50%, preferably greater than 75%, most preferably greater than 100% of the amount of the pantothenate compound produced by a population of the corresponding microorganisms not being sporulation-deficient.
  • the amount of the pantothenate compound produced by a population of the microorganisms of the invention is greater than 50%, preferably greater than 75%, most preferably greater than 100% of the amount of the pantothenate compound produced by a population of the corresponding microorganisms not being sporulation-deficient.
  • the amount of the pantothenate compound produced by a population of the microorganisms of the invention is greater than 50%, preferably greater than 75%, most preferably greater than 100% of the amount of the pantothenate compound produced by a population of the corresponding microorganisms not being sporulation-deficient.
  • the "corresponding microorganism not being sporulation-deficient" is preferably a microorganism which has not been modified such that it exhibits reduced SigE function or a reduced SpoOA and ArbB function.
  • the corresponding microorganism not being sporulation- deficient is a microorganism that is substantially identical with the exception that is has no mutation in its spollGA gene. The same applies to mutations in other genes.
  • pantothenate compound can be achieved in a variety of ways. Methods of preparing strains of Bacillus subtilis overproducing pantothenate are described in, e.g., WO 01/21772, WO 02/057474, and WO 02/061108.
  • One approach to obtain pantothenate- overproducing microorganisms is to overexpress one or more genes involved in the pantothenate biosynthetic pathway.
  • the term "overexpressed” or "overexpression” includes expression of a gene product at a level higher than that expressed prior to modification of the microorganism or in a comparable microorganism which has not been modified.
  • the microorganism of the invention overexpresses one or more genes selected from the group consisting of panB, panC, panD, panE, ilvB, ilvN, ilvC, ilvD, glyA, and serA, ylmA as well as the gcv genes involved in the glycine cleavage pathway.
  • Overexpression of a gene in a microorganism can be performed according to any methodology described herein including, but not limited to, deregulation of a gene and/or overexpression of at least one gene.
  • the microorganism can be genetically manipulated, e.g., genetically engineered, to overexpress a level of gene product greater than that expressed prior to modification of the microorganism or in a comparable microorganism which has not been modified.
  • Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with the expression of a particular gene, e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive, modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins, e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like, involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including, but not limited to, the use of antisense nucleic acid molecules, e.g., to block expression of repressor proteins).
  • Suitable promoters are, but are not limited to, P veg , P 15 and P 26 (Lee et al., 1980, Mol. Gen. Genet. 180:57-65 and Moran et al., 1982, Mol. Gen. Genet. 186:339-46.
  • the microorganism can be physically or environmentally manipulated to overexpress a level of gene product greater than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated.
  • a microorganism can be treated with or cultured in the presence of an agent known or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
  • a microorganism can be cultured at a temperature selected to increase the transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
  • deregulated includes the alteration of modification of at least one gene in a microorganism such that the level or activity of the gene product in a microorganism is altered or modified.
  • at least one gene is altered or modified such that the gene product is enhanced or increased.
  • the invention further relates to a method for the preparation of a sporulation-deficient microorganism capable of overproducing a pantothenate compound.
  • a microorganism capable of overproducing a pantothenate compound may be modified such that it contains a mutation in a gene regulating SigE function and optionally an increased function of SpoOA.
  • the method comprises
  • step (c) introducing in the microorganism of step (a) or (b) a mutation that causes reduced SigE function such that a sporulation-deficient microorganism is obtained.
  • the order of steps (b) and (c) may be changed.
  • the method may comprise
  • step (b) introducing in the microorganism of step (a) mutations that cause reduced SpoOA function and reduced ArbB function such that a sporulation-deficient microorganism is obtained.
  • the mutation of the sigE gene or the mutations of the spoOA gene and of the arbB gene may be introduced prior to carrying out the mutations leading to overproduction of the pantothenate compound.
  • the method comprises
  • step (b) introducing in the microorganism of step (a) a mutation that causes reduced SigE activity or introducing in the microorganism of step (a) mutations that cause reduced SpoOA activity and reduced AbrB activity such that a sporulation-deficient microorganism is obtained, and
  • step (c) modifying the sporulation-deficient microorganism obtained in step (b) such that it is capable of overproducing the pantothenate compound.
  • step (b) of this method may comprise the optional step of introducing in the microorganism of step (a) or (b) or (c) a DNA sequence (e.g. promoter) or a mutation that causes increased SpoOA function.
  • a DNA sequence e.g. promoter
  • Another aspect of the invention is a method for producing a pantothenate compound comprising (a) culturing a microorganism according to the invention under conditions such that the pantothenate compound is produced; and (b) optionally recovering the pantothenate compound from the cell culture medium.
  • the method of the invention comprises the step of culturing the modified microorganisms under conditions such that a pantothenate compound is produced.
  • the term "culturing” includes maintaining and/or growing a living microorganism of the present invention.
  • a microorganism of the invention is cultured in liquid media.
  • a microorganism is cultured in solid media or semi-solid media.
  • the microorganism of the invention is cultured in liquid media comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism.
  • nutrients include, but are not limited to, carbon sources or carbon substrates, e.g. complex carbohydrates such as bean or grain meal, starches, sugars, sugar alcohols, hydrocarbons, oils, fats, fatty acids, organic acids and alcohols; nitrogen sources, e.g.
  • animal sources such as meat, milk and animal byproducts such as peptones, meat extracts and casein hydrolysates
  • inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium nit
  • microorganisms are preferably cultured under controlled pH.
  • microorganisms are cultured at a pH of between 6.0 and 8.5, more preferably at a pH of about 7.
  • the desired pH may be maintained by any method known to those skilled in the art.
  • the microorganisms are further cultured under controlled aeration and under controlled temperatures.
  • the controlled temperatures include temperatures between 15 and 70°C, preferably the temperatures are between 20 and 55°C, more preferably between 30 and 45°C or between 30 and 50°C.
  • the microorganisms may be cultured in liquid media either continuously or intermittently, by conventional culturing methods such as standing culture, test tube culture, shaking culture, aeration spinner culture or fermentation.
  • the microorganisms are cultured in a fermentor.
  • Fermentation processes of the invention include batch, fed-batch and continuous methods of fermentation. A variety of such processes have been developed and are well known in the art. The culturing is usually continued for a time sufficient to produce the desired amount of the pantothenate compound.
  • the method further comprises the step of recovering the pantothenate compound.
  • the term "recovering” includes isolating, extracting, harvesting, separating or purifying the compound from culture media. Isolating the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin, treatment with a conventional adsorbent, alteration of pH, solvent extraction, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilisation and the like.
  • the pantothenate compound can be recovered from culture medium by first removing the microorganisms from the culture.
  • Media is then passed through or over a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids having stronger acidities than the compound of interest, e.g. pantothenate.
  • a cation exchange resin to remove unwanted cations
  • an anion exchange resin to remove unwanted inorganic anions and organic acids having stronger acidities than the compound of interest, e.g. pantothenate.
  • the resulting pantothenate can subsequently be converted to salt as described herein.
  • the compound is "isolated" when the resulting preparation is substantially free of other components.
  • the preparation has a purity of greater than about 80% (by dry weight) of the desired compound (e.g. less than about 20% of all the media, components or fermentation byproducts) more preferably greater than about 90% of the desired compound, even more preferably greater than about 95% of the desired compound and most preferably greater than about 98 to 99% of the desired compound.
  • the desired compound is not purified from the microorganism or the culture.
  • the entire culture or the culture supernatant may be used as a source of the product.
  • the culture or the culture supernatant is used without modification.
  • the culture or the culture supernatant is concentrated, dried and/or lyophilized.
  • Bacillus subtilis strains of the present invention are derived from strain 1A747 (Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio 43210 USA), which is a prototrophic derivative of B. subtilis 168 (trpC2).
  • the chloramphenicol-resistance gene (cat) cassette was obtained from plasmid pC194 (GeneBank M19465, Cat# 1 E17 Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio 43210 USA).
  • the P 15 promoter of the B. subtilis bacteriophage SPO1 (Lee et al., 1980, Mol. Gen. Genet.
  • BHI [trpC2 pheA1 spoOHAHind ⁇ -EcoR ⁇ ::cat] (BHI is a derivative of BH100 in ref. Healy et al., 1991 , Mol. Microbiol. 5:477-487), 650 [trpC2 ilvB2 leuB16 spollAABC::caf ⁇ (Pragai et al., 2004, J Bacteriol. 186:1182-1190), 731 [trpC2 spolllG .ermC] (Partridge and Errington,
  • Standard minimal medium (MM) for B. subtilis contains 1X Spizizen salts, 0.04% sodium glutamate, and 0.5% glucose.
  • Standard solid complete medium is Tryptone Blood Agar Broth (TBAB, Difco).
  • Standard liquid complete medium is Veal Infusion-Yeast Extract broth (VY). The compositions of these media are described below:
  • TBAB medium 33g Difco Tryptone Blood Agar Base (Catalog # 0232), 1 L water.
  • VY medium 25g Difco Veal Infusion Broth (Catalog # 0344), 5g Difco Yeast Extract
  • 10X Spizizen salts 140g K 2 HPO 4 ; 20g (NH 4 ) 2 SO 4 ; 60g KH 2 PO 4 ; 10g Na 3 citrate.2H 2 O; 2g
  • P-medium 100 ml 10X PAM; 100ml 10X Spizizen salts; 10 ml 50% glucose; 790 ml sterile distilled water.
  • P-agar medium 100 ml 10X PAM; 100ml 10X Spizizen salts; 10 ml 50% glucose; 790 ml sterile distilled water containing 15 g of agar.
  • 10X VFB minimal medium (10X VFB MM): 2.5g Na-glutamate; 15.7g KH 2 PO 4 ; 15.7g
  • Fe solution 0.21 g FeSO 4 .7H 2 0; qsp 10 ml water.
  • Mg/Zn solution 100g MgSO 4 .7H 2 O; 0.4g ZnSO 4 .7H 2 O; qsp 200 ml water.
  • VFB MM medium 100 ml 10X VFB MM; 10 ml 50% glucose; 2 ml Trace elements solution;
  • VFB MMGT medium 100 ml 10X VFB MM; 100 ml 0.5 M Tris (pH 6.8); 44 ml 50% glucose;
  • VF fermentation batch medium Sterilized in place in solution: 0.75g sodium glutamate;
  • VF fermentation feed medium 660g glucose-H 2 O; qsp 1 L. Autoclave. Add 2g MgSO 4 -7H 2 O;
  • Pantothenate assays Pantothenate was assayed using three biological and one physical (HPLC) methods:
  • Pantothenate was assayed on agar plates using an indicator derived from Salmonella typhimurium using known methods.
  • Strain DM3 panC355 (D. Downs, University of Wisconsin at Madison, Madison, Wisconsin USA) responds only to pantothenate.
  • 10 ml of P-agar medium containing 10 7 cells/ml of S. typhimurium DM3 (panC355) indicator strain and 40 ⁇ g/ml of 2,3,5-triphenyltetrazoiium chloride (Fluka; Catalog # 93140) was layered above 20 ml of P-agar medium used as a bottom agar in a standard Petri dish.
  • Biological assay II - test tube bioassay Pantothenate was assayed in liquid using test tube cultures. To assay ⁇ . subtilis cultures, supernatants were filtered sterilized and dilutions prepared using 1X pantothenate assay medium (PAM) in test tubes. The total volume of the dilutions was 5.0 ml. To these dilution test tubes, 0.1 ml of a 200-fold dilution of the DM3 indicator stock (in 1X PAM) was added. The test tubes were then incubated in a roller-drum- type shaker at 37°C for 18-24 hours.
  • PAM pantothenate assay medium
  • Turbidity readings were made at 600 nm (OD 60 o) and compared to a standard curve of known quantities of pantothenate.
  • the standard curve was prepared by diluting authentic pantothenate to levels of 0.8, 4, 20, 50, and 100 ⁇ g/liter, adding to each dilution the indicator as prepared above, and measuring turbidity after 18-24 hours at 37°C (same incubation time as the unknown samples). The linear portion of the standard curve was used to generate a logarithmic regression equation. This equation was then used to calculate pantothenate concentrations in unknown samples using the OD 6 oo values of the diluted samples.
  • Biological assay 111 - 96-well microtiter plate bioassay Pantothenate was also assayed in liquid using a 96-well microtiter plate format. 180 ⁇ l of pantothenate assay medium (PAM) containing 5.5x10 5 cell/ml of S. typhimurium DM3 was loaded into each well of the 96-well microtiter plate. To assay B. subtilis cultures, 60 ⁇ l of filtered sterilized supernatant of the cultures were added into the wells in column 1 , rows B-H. After mixing, 60 ⁇ l of the samples were transferred into the next wells in column 2. These 4-fold dilution steps were repeated until column 12.
  • PAM pantothenate assay medium
  • each well contained 180 ⁇ l of samples.
  • Adhesive film was place onto the plate to avoid evaporation and the cultures were incubates at 37°C for 17 h with an agitation of 300 rpm. Turbidity readings were made at 600 nm and compared to a standard curve of known quantities of pantothenate (Pan)
  • Pan standard curve was prepared by adding 60 ⁇ l of Pan standard (100 mg/ml) into the well in column 1 and row A of each microtiter plate. Dilution, incubation and measuring turbidity of the Pan standard was done as described above with the unknown samples.
  • HPLC assay Chromatography of samples was performed on a Phenomenex LUNA C8 column, using an Agilent 1100 HPLC system equipped with a thermostatted autosampler and a diode array detector. The column dimensions are 150 x 4.6 mm, particle size 5 micron. The column temperature was kept constant at 20°C. The mobile phase is a mixture of 0.1 % acetic acid (A) and methanol (B). Gradient elution is applied, ranging from 1 % B to 45% B in 15 minutes. The flow rate is 1 ml/min. Pantothenate was monitored using UV absorption at 220 nm, and is eluted at approximately 9.6 min. The calibration range of the method is from 1 to 100 mg/l pantothenate.
  • Sporulation assay 1 ml of sample was taken from the B. subtilis cultures and 10-fold dilution series was prepared in sterile distilled water. After a 20 min heat-treatment at 80°C, dilutions were plated on TBAB agar, incubated for 20 h at 37°C and then the number of 'heat-resistant' colony forming units (cfu) in 1 ml of the original culture was determined. Sporulation frequency was calculated by dividing the titer (cfu/ml) of heat resistant spores by the titer (cfu/ml) of bacterial cells before heat treatment.
  • Pantothenate producing strains were grown in stirred tank fermentors, for example, in BIOFLO 3000 New Brunswick 2 liter vessels initially containing 1.4 liters of VF fermentation batch medium with glucose/salt solution.
  • Computer control was done by NBS Biocommand 32 commercial software (New Brunswick Scientific Co., Inc., Edison, NJ, USA); Lucullus software (Biospektra AG, Schlieren, Switzerland) was used for data collection and controlling the glucose feeding.
  • the inoculum was prepared for the fermentation.
  • the first seed consisted of inoculating 25 ml of VY medium containing 10 g/liter sorbitol with 50 ⁇ l of a frozen bacterial stock culture and growing the culture for 6 hours at 37°C. 1 OD of cells was then used to inoculate 60 ml of VF Fermentation batch medium containing 10 g/liter sorbitol (no glucose) and this second seed was grown for 8-12 hours at 37°C. This second seed was then used to inoculate the fermentation vessel, the amount of which is usually 4-5% of the initial media volume.
  • Fermentations can be batch processes but are preferably, carbohydrate-limited, fed-batch processes. Therefore a defined VF fermentation feed solution (see above) was provided to the reactor after consumption of the initial glucose which was usually the case after 6-8 hours process time. At that time, a constant addition of the feed solution was initiated at a rate of 14 g/h.
  • This example describes the construction of pantothenate-overproducing strains of B. subtilis.
  • PCR Polymerase Chain Reaction
  • Two PCR fragment bombardarms were then generated: 0.2 ⁇ l of a 100 mM solution of primer panB/up2/for/R1 and panB/up2/rev/Clal or primers panB/down2/for/Nhel and panB/down2/rev/Bam (Table 1) were added to 0.1 ⁇ g 1A747 chromosomal DNA in a 50 ⁇ l reaction volume containing 1 ⁇ l of 40 mM dNTP's, 5 ⁇ l of 10X buffer and 0.75 ⁇ l PCR enzyme (Taq and Tgo), as described by the manufacturer (Expand High fidelity PCR System-Roche Applied Science).
  • the PCR reaction was performed for 30 cycles, using an annealing temperature of 58 °C and an elongation time of 60 seconds.
  • the resulting fragments called F1 and F2, respectively, were purified, and inserted sequentially, respectively between the EcoRI and C/al sites (for F1) and the Nhe ⁇ and ⁇ a HI (for F2).
  • Ligated DNA was transformed into E. coli TOP10 cells (Invitrogen), selecting for ampicillin- resistance at 100 ⁇ g/ml concentration. This resulted in the E. coli plasmid pPA5.
  • the ApanBp.:cat deletion cassette was then next introduced into the chromosome of B.
  • subtilis 1A747 by DNA transformation, selecting for chloramphenicol-resistance (Cm r ) on TBAB agar plates containing 5 ⁇ g/ml chloramphenicol (Cm) using standard conditions.
  • Cm r chloramphenicol-resistance
  • a single Cm r colony containing a deletion in the panB promoter region was isolated and named PA1 (ApanBp.-.cat).
  • PA1 was also a pantothenate auxotroph (Pan " ), which requires pantothenate for growth on minimal medium.
  • the deletion mutation was confirmed by diagnostic PCR using panB/up2/for/R1 and panB/down2/rev/Bam primers (Table 1 ), again using standard reaction conditions.
  • Nucleotide sequence 5'>3'
  • the next step was to introduce a strong constitutive promoter upstream of the panB gene.
  • a strong constitutive promoter upstream of the panB gene Any number of such promoters are described in the literature, including those derived from the SP01 bacteriophage of B. subtilis, P 15 and P 26 (Lee et al., 1980). Long Flanking Homology Polymerase Chain Reaction (LFH-PCR) was used to generate DNA fragments containing P 15 upstream of the ribosome binding site (RBS) of panB.
  • LDH-PCR Long Flanking Homology Polymerase Chain Reaction
  • PCR fragment bombardments were first created: 0.2 ⁇ l of a 100 mM solution of primer PlpanBCD and P2panBCD or primers P3panBCD and P4panBCD (Table 2) were added to 0.1 ⁇ g 1A747 chromosomal DNA in a 50 ⁇ l reaction volume containing 1 ⁇ l of 40 mM dNTP's, 5 ⁇ l of 10X buffer and 0.75 ⁇ l PCR enzyme (Taq and Tgo), as described by the manufacturer (Expand High fidelity PCR System-Roche Applied Science). The PCR reaction was performed for 30 cycles using an annealing temperature of 55.7 °C and an elongation time of 45 seconds.
  • F3 and F4 were purified and next used as primers in a second round of PCR.
  • F3 and F4 fragments were diluted 50-fold and 1 ⁇ l of each was added to 0.1 ⁇ g of linearized plasmid pXI23roDTD-SPO1-15 (containing the P 15 promoter) in a 50 ⁇ l reaction volume.
  • an annealing temperature of 63 °C and an elongation time of 6 minutes was used.
  • the elongation time was extended by 20 seconds after each cycle.
  • the resulting products were then used in a third round of PCR as a template.
  • the PCR products were diluted 50-fold and 1 ⁇ l was combined with 0.2 ⁇ l of a 100 mM solution of primer PlpanBCD and P4panBCD in a 50 ⁇ l reaction volume containing dNTP's, buffer, and enzyme as described above.
  • the PCR reaction parameters were identical to those used in the second round PCR.
  • the finished PCR fragments were next transformed into the panB promoter-deleted strain PA1 by DNA transformation, selecting for pantothenate prototrophy (Pan + ) on minimal medium agar plates using standard conditions. These Pan + colonies were also chloramphenicol sensitive (Cm s ), confirming the insertion of the promoter cassette.
  • a single Pan + Cm s colony containing a panBCD operon expressed from the P ⁇ 5 promoter was isolated and named PA12 (P 15 panBCD).
  • the presence of the P 15 promoter upstream of panB gene was confirmed by diagnostic PCR using P15seq and P4panBCD primers (Table 2), again using standard reaction conditions.
  • PA12 produced approximately 100 mg/liter pantothenate in VFB MM medium and 250 mg/liter pantothenate in VFB MMGT medium (based on HPLC/MS assays) whereas the 1A747 control produced less than 1 mg/liter pantothenate.
  • PA12 In standard fed-batch fermentation using VF medium and growth conditions, PA12 produced around 10-14 g/liter pantothenate at 48 hours.
  • Table 2 Primers used to generate a B. subtilis strain containing a P 15 panBCD expression cassette.
  • a deletion mutation was first constructed. Inspection of the panE gene reveals two potential start sites: Start Site 1 (5' - AAATTGGGTG - 3' (RBS)- 7 nt - ATG) that overlaps a ⁇ spHI site and is 33 bp upstream from Start Site 2 (5' - GGAGG - 3' (RBS) - 5 nt - TTG) that overlaps a ⁇ saXI site. Consequently, a 219 bp deletion of the panE/ylbQ promoter region was constructed by LFH-PCR using a S.
  • aureus erythromycin resistance (Em r ) gene GeneBank V012778.
  • Em r aureus erythromycin resistance
  • two PCR fragment bombardarms were first created: 0.2 ⁇ l of a 100 mM solution of primer Pl panE and P2panE/Er or primers P3panE/Er/2 and P4panE (Table 3) were added to 0.1 ⁇ g 1A747 chromosomal DNA in a 50 ⁇ l reaction volume containing 1 ⁇ i of 40 mM dNTP's, 5 ⁇ l of 10X buffer and 0.75 ⁇ l PCR enzyme (Taq and Tgo), as described by the manufacture (Expand High fidelity PCR System-Roche Applied Science).
  • the PCR reaction was performed for 30 cycles using an annealing temperature of 55.7°C and an elongation time of 45 seconds.
  • the resulting fragments called F1 and F2 respectively, were purified and next used as primers in a second round of PCR.
  • F1 and F2 fragments were diluted 50-fold and 1 ⁇ l of each was added to 0.1 ⁇ g of linearized plasmid pDG646 (containing the erm cassette; Guerout-Fleury et al., 1995) in a 50 ⁇ l reaction volume.
  • an annealing temperature of 63°C and an elongation time of 6 minutes was used.
  • the elongation time was extended by 20 seconds after each cycle.
  • the resulting products were then used in a third round of PCR as a template.
  • the PCR products were diluted 50-fold and 1 ⁇ l was combined with 0.2 ⁇ l of a 100 mM solution of primer Pl panE and P4panE in a 50 ⁇ l reaction volume containing dNTP's, buffer, and enzyme as described above.
  • the PCR reaction parameters were identical to those used in the second round PCR.
  • the finished PCR fragments were next transformed into the PA4 (Trp + colonies obtained from B. subtilis CU550 trpC2 ilvC4 leu-124 by transformation with 1A747 chromosomal DNA) resulted in Em r colonies that were pantothenate auxotrophs.
  • panE p ApanE p ::erm ilvC leuC
  • Diagnostic PCR was used to confirm the structure of the deletion.
  • panB promoter deletion was introduced into PA5 by transformation of PA1 chromosomal DNA at non- legislative concentration to generate PA6 (ilvC leuC ApanB p ::cat ApanE p ::erm).
  • the next step was to introduce simultaneously strong constitutive P ⁇ 5 promoters upstream of the both panB and panE.
  • LFH-PCR was used again to generate DNA fragments containing P 15 upstream of open reading frame of panB and containing P 15 upstream of the open reading frame of panE.
  • panB and P ⁇ 5 panE PCR fragments were then transformed together into the panB and panE promoters deleted strain PA6 (ilvC leuC ApanB p ::cat ApanE p ::erm) by DNA transformation, selecting for pantothenate prototrophy (Pan + ) on minimal medium agar plates using standard conditions. Recovered Pan + colonies were also Cm s and erythromycin sensitive (Em s ), confirming the insertion of the promoter cassettes.
  • a single Pan + Cm s Em s colony containing both panBCD operon and panE gene expressed from the P 15 promoter was isolated and named PA32.
  • panB gene The presence of the P 15 promoter upstream of panB gene was confirmed by diagnostic PCR using P15seq and P4panB primers (Tables 1 and 2), again using standard reaction conditions. An identical control was performed on the panE gene using P15seq and P4panE primers (Tables 3 and 4). Subsequent sequencing of the Pis promoter in front of panE, however, revealed a partial deletion of the P « promoter.
  • the ApanE p ::erm mutation was re-introduced into PA32 by DNA transformation using chromosomal DNA from PA5 (ApanE p ::erm ilvC leuC) and selecting for erythromycin- resistance.
  • PA41 P 15 panBCD ApanE p ::erm HvC leuC
  • PA49 produced on average approximately 400 mg/liter pantothenate in VFB MMGT medium (based on HPLC/MS assays).
  • This example describes the construction and testing of B. subtilis pantothenate overproducing strains containing sporulation-deficient mutations spoOA, spoOH, spollA (sigF) spollG (sigE) and spolllG (sigG). Construction of B. subtilis mutants deficient in spore formation.
  • Sporulation-deficient mutations were introduced into 1A747 (wild-type strain) and PA12, a pantothenate-overproducing strain by transformation of chromosomal DNA from strains SWV215 (spoOAr.kan), BHI (spo0H ⁇ H//7dlll-EcoRI::cat), 650 (spollAABCwcaf), 901 (spollGA::aphA-3) and 731 (spolllGwermC), respectively.
  • Extraction of chromosomal DNA and transformation of B. subtilis strains by the 'Groningen' method was according to Bron (1990).
  • Transformants were selected on TBAB agar medium containing 0.3 ⁇ g/ml of erythromycin (Em) and 25 ⁇ g/ml of lincomycin (Lm) for ermC gene; 6 ⁇ g/ml of Cm for cat gene; and 10 ⁇ g/ml of kanamycin (Km) for kan and aphA-3 genes.
  • Em erythromycin
  • Lm lincomycin
  • Km kanamycin
  • Kanamycin-resistant (Km r ) transformants from SWV215 DNA were named
  • pantothenate production of mutants 1A747_spoO/4, PA12_spoO ⁇ , 1A747_s/gH and PA12_s/o7- decreased 2 to 3 fold compared to the parent strains (Table 5), indicating that the lack of SpoOA and ⁇ H functions negatively affected the pantothenate production.
  • production of pantothenate was similar to control levels within experimental error, indicating that mutations blocking sporulation in later stages had little or no effect on pantothenate production.
  • the cell biomass of all mutants was similar to that of the parental controls.
  • the sporulation frequency of the parent strains 1A747 and PA12 was approximately 0.3-1%. Spores were never detected in mutants lacking SpoOA, ⁇ H , and ⁇ E .
  • a small number of heat-resistant cells was detected in the cultures of 1A747_s/ ' gFand PA12_s/gF (Table 5), showing that mutations in the spollAABC genes did not result in complete loss of spore formation. Based on these results, only strains containing mutations in the sigma E gene both produced control levels of pantothenate and were completely devoid of spore-forming bacteria.
  • PA12_spoOA (spoOAr.kan) 50 0
  • PA12_s/gH (spoOHAHindW- 60 0
  • This example describes the fermentation of ⁇ . subtilis pantothenate-overproducing strains containing sporulation-deficient mutations spoOA or spollG (sigE).
  • the sigE mutant (PA12_s/gE), the spoOA mutant (PA12_spoOA), and their parent, PA12, were grown in standard fed-batch fermentations using 2-liter lab scale fermentors for 48 hours. Both pantothenate levels and spore formation frequency of bacteria were measured during the course of the fermentation. As shown in Table 6, pantothenate production was similar in both the parent and the sigE mutant, but was reduced approximately 85% in the spoOA mutant. The yield of pantothenate on glucose was again similar between the sigE mutant and the parent strain. Spores were detected in the parent, but not in either mutant. Table 6. Sporulation and pantothenate production of ⁇ . subtilis strains PA12, PA12_spoOA, and PA12_s/g£ grown in 2-liter bench scale fermentation for 48 hours.
  • PA12_spoOA (spoOAr.kan) 1.4 0
  • This example describes the construction and testing of B. subtilis pantothenate overproducing strains containing null-mutations in spoOA and abrB.
  • Single and double abrB and spoOA mutations were introduced into PA49, a pantothenate- overproducing strain by transformation of chromosomal DNA from strains SWV215 (spoOAr.kan) and SWV119 (abrBrtet).
  • Transformants were selected on TBAB agar medium containing 10 ⁇ g/ml of Km for kan gene; and 10 ⁇ g/ml of tetracycline (Tc) for tet gene.
  • the transformation efficiency was 2x10 3 transformants/ ⁇ g DNA for spoOAr.kan, 4x10 2 transformants/ ⁇ g DNA for abrBr.tet and 4 transformants/ ⁇ g DNA for double spoOAr.kan and abrBrtet mutant.
  • the resulting strains were:
  • Tetracycline-resistant (Tc r ) transformant from SWV119 DNA was named PA1037;
  • PA1051 (spoOAr.kan abrBr.tet) 600 0
  • This example describes the construction and testing of B. subtilis pantothenate overproducing strain that overexpresses spoOA and contains a sporulation-deficient mutation (spollGA).
  • Example II it was shown that the lack of SpoOA decreased pantothenate production 2 to 3 fold compared to the parent strains.
  • an IPTG-inducible P spac -spoOA fusion was introduced into amyE gene of PA49 by transformation of chromosomal DNA from strain AH 1763 (amyEr.Pspa c spoOA[caf ⁇ ). Transformants were selected on TBAB agar medium containing 6 ⁇ g/ml of Cm and the resulted Cm r strain was named PA1025.
  • a sporulation-deficient mutation in spollGA was introduced into PA1025 by transformation of chromosomal DNA from strain 901 (spollGA::aphA-3). Transformants were selected on TBAB agar medium containing 6 ⁇ g/ml of Cm for cat gene and 10 ⁇ g/ml of Km for aphA-3 gene and the resulted Cm r and Km r strain was named PA1064.
  • PA1025 (P spac spoOA) 0 400 2.5x10 2
  • PA1025 (P spac spo0A) 10 670 3.7x10 3
  • Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, p31. Proc. Natl Acad. Sci. USA 84: 1784-1788.

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Abstract

L'invention concerne des micro-organismes inaptes à la sporulation mais capables de produire du pantothénate en excès. Une mutation dans un gène influençant la fonction SigE ou des mutations dans des gènes influençant la fonction Spo0A et la fonction AbrB rendent ces micro-organismes inaptes à la sporulation mais gardent sensiblement leur capacité à produire du pantothénate en excès. Les micro-organismes de l'invention sont particulièrement utiles pour la production industrielle du pantothénate.
PCT/EP2004/006619 2003-06-18 2004-06-18 Production de pantothenate au moyen de micro-organismes inaptes a la sporulation Ceased WO2004113510A2 (fr)

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CN2004800171442A CN1809631B (zh) 2003-06-18 2004-06-18 用不能形成芽孢的微生物来生产泛酸盐/酯的方法
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