JP4713469B2 - Production of pantothenate using microorganisms that cannot sporulate - Google Patents

Description

  Pantothenate is a member of the vitamin B complex and is required for nutrition of mammals, including humans, for example from food sources, as a water-soluble vitamin supplement, or as a feed additive. In cells, pantothenate is primarily used for biosynthesis of coenzyme A and acyl carrier proteins. These essential coenzymes function in the metabolism of the acyl moiety that forms a thioester with the sulfhydryl group of the 4'-phosphopantethein moiety of these molecules.

  Pantothenate is usually synthesized from a large amount of chemical substances through chemical synthesis. However, the substrates required for chemical synthesis are expensive and the racemic intermediate must be optically resolved. Accordingly, bacterial or microbial systems that produce enzymes useful for pantothenate biosynthesis methods have been employed. In particular, bioconversion methods were evaluated as a means of conveniently producing the preferred isomer of pantothenic acid. More recently, direct synthesis by microorganisms has been investigated as a means of facilitating the production of D-pantothenate.

  Patent applications WO01 / 21772, WO02 / 057474, and WO02 / 0661108 have biosynthesis genes involved in pantothenate production at higher expression levels. A method for producing pantothenate using B. subtilis strain 168 is described. These genes include panB, panC, panD, panE (ylbQ), ilvB, ilvN, ilvC, ilvD, glyA, and serA. In order to achieve higher levels of expression of these genes, standard genetic recombination methods known in the art were used. The production of pantothenate from these engineered strains ranged from 37 g / liter to 85 g / liter in 48 hours of fermentation by fed-batch culture.

In terms of environmental and administrative regulations, if a genetically modified microorganism leaks from a manufacturing facility during the fermentation process or during the disposal of biomass, it is important that the microorganism does not have the ability to survive in the natural environment It is. For this reason B. A sporulation-deficient strain of Subtilis is used in the fermentation process (for example, riboflavin (US5837528), biotin (US6057136)). In general, the spo0A mutation is used to stop sporulation. The spo0A gene encodes a protein that regulates the initiation of sporulation. Inactivation of spo0A, either by point mutations, frameshift mutations or deletion mutations, stops sporulation in the earliest steps, resulting in strains that are no longer resistant to chemical solvents, radiation and / or heat Bring. In addition to mutations in spo0A, WO 97/03185 does not allow sporulation. To obtain bacteria of the genus Bacillus other than subtilis, a mutation in spoIIAC of Bacillus licheniformis that encodes the sigma factor σ ^F of sporulation-specific transcription is used to produce commercially important enzymes Is described.

B. The endospore formation (spore formation) process in subtilis and other related Gram-positive bacteria consists of several steps. Enrollment in sporulation has been determined by phosphorylated spo0A (spo0A-P), a DNA-binding main response regulator (Fawcett et al., 2000). spo0A-P plays two important roles (Phillips and Strauch, 2002). At low intracellular concentrations, spo0A-P represses abrB transcription, thereby increasing the regulation of AbrB-dependent transition state-related gene expression. Next, if spo0A-P concentrations reach even higher limit levels, Spo0A-P activates genes necessary for sporulation entry and orientation (sinI, spoIIG, spoIIE, spoIIA, etc.). Since spo0A-P represses transcription of abrB and AbrB represses transcription of sigH, spo0A-P indirectly activates genes in the σ ^H regulon. σ ^H is an alternative sigma factor that determines the transcription of genes (spo0A, spo0F, spoIIA, phrC, phrE, etc.) involved in the stationary phase of growth and early sporulation (Britton et al., 2002). Examples of genes and gene products that increase or decrease the levels of spo0A-P and AbrB include abrB, kapB, kbaA, kinA, kinB, kinC, kinD, kinE, kipA, kipI, obg, phrC, phrE, rapA, rapB , RapE, sigH, spo0A, spo0B, spo0E and spo0F, but are not limited thereto. Examples of proteins that act on spo0A-P dependent binding at the promoter site to activate gene expression include, but are not limited to, spo0JA and spo0JB. Examples of signals that increase or decrease spo0A-P and AbrB levels include, but are not limited to, nutritional signals, metabolic signals, DNA status signals, cell density (pheromone and cell density sensing molecules) signals, and cell cycle signals. It is not done.

After these steps, the cells divide asymmetrically to produce two compartments of different developmental fate that are unequal in size (Piggot and Losick, 2002). The smaller compartment, the prespore, develops into a spore, while the larger compartment, the mother cell, provides nutrients to the developing spore. When spore morphogenesis is complete, the mother cells lyse and release mature spores. Differentiation requires the action of four cell-specific sigma factors, σ ^F , σ ^E , σ ^G and σ ^K. σ ^F and σ ^E factors are activated immediately after asymmetric division (polar septum formation, phase II), where σ ^F directs gene expression in the prespore (Margolis et al., 1991), and σ ^E is Directs gene expression in mother cells (Dirks and Losick, 1991). Later in sporulation, sigma ^F is replaced by sigma ^{G in the} prespore and sigma ^E is replaced by sigma ^K in the mother cell (Losick and Stragier, 1992; Li and Piggot, 2001).

σ ^E is obtained from an inactive proprotein called proσ ^E (LaBell et al., 1987). Pro-sigma ^E synthesis begins before septum formation (Satola et al., 1992), while pro- lytic processing of pro-sigma ^E to mature sigma ^E occurs after asymmetric division in the mother cell. The reaction is mediated by SpoIIGA a sporulation-specific protease, SpoIIGA cleaves the 27 amino acids from the amino terminus of pro-σ ^E (Stragier et al, 1988; Jonas et, 1988; Peters and Haldenwang, 1994). SpoIIGA before processing is activated by the production to secreted signaling proteins (SpoIIR) before spores under the control of σ ^F (Londono-Vallejo and Stragier, 1995; Hofmeister et al., 1995; Karow et al, 1995). Additional information on the activation of σ ^E and other sigma factors is reviewed by Helmann and Moran (2002).

  It has been shown that introducing sporulation mutations in combination with other mutations into a single bacterium results in unexpected gene activation. For example, expression of ggt, a gene encoding γ-glutamyl transpeptidase that decreases in the absence of spo0A-P, is 3-4 times the wild-type expression level in abrB and spo0A double null mutants. Can increase (Xu and Strauch, 1996). Such strains are also unable to sporulate. However, it was not recognized that these strains could show the same level of pantothenate production as control cells or higher than control cells. In particular, the cells described in Xu and Strauch cannot overproduce pantothenate compounds.

  Interestingly, genetically modified pantothenate production described in WO01 / 21772, WO02 / 057474, and WO02 / 061108 Subtilis 168 strains are all wild type for sporulation. No examples are provided showing the production of pantothenate in sporulation-deficient strains.

  It is an object of the present invention to provide a sporulation-deficient microorganism that can overproduce pantothenate. B. It is a further object of the present invention to provide a method for the preparation of sporulation-deficient microorganisms that are also suitable for subtilis.

  It has been discovered that the commonly used spo0A mutation results in a significant reduction in pantothenate production. However, B. lacks SigE activity or lacks both spo0A and AbrB. Subtilis cells were found to completely lack sporulation and show pantothenate production at levels similar to or higher than control cells.

  Accordingly, the present invention relates to a spore formation-deficient microorganism that can overproduce pantothenate compounds. Microorganisms can be modified to have reduced SigE activity compared to unmodified microorganisms. SigE is a gene product encoded by the spoIIGB gene. Reduction in SigE activity can be caused by a lack of spoIIGB expression, a reduction or absence of expression of a protease encoded by spoIIGA, a reduction or absence of expression of other upstream regulatory genes or proteins, and the like. Preferably the microbial modification comprises a mutation that causes a reduction in SigE activity. More preferably, the mutation acts on one or more genes selected from the group consisting of spoIIGA, spoIIGB, spoIIR and spoIIAC genes.

  B. It was further found that pantothenate production was further increased by increasing the expression of the spo0A gene through the use of a constitutive or inducible promoter in a subtilis spantothenate overproducing strain. Since this strain still forms spores, as a result of the introduction of the sigE mutation, it produces a similar amount of pantothenate compared to the spo0A overproducing parent strain and does not form spores. Subtilis will be brought. Thus, in a preferred embodiment of the present invention, a microorganism having reduced SigE activity with high spo0A-P levels can produce more pantothenate without forming spores.

  In another aspect of the invention, the microorganism can be modified such that the activity of both SpoOA and AbrB is reduced compared to an unmodified microorganism. Spo0A and AbrB are gene products encoded by spo0A and abrB genes, respectively. Reduction of Spo0A and AbrB activity is due to lack of gene expression, decreased or absent signals (nutrient signal, metabolic signal, DNA status signal, cell density [pheromone and cell density sensing molecule] signal and cell cycle signal), other upstream May be caused by a decrease or lack of expression of a regulatory gene or protein. Preferably, the microbial modification comprises a mutation that causes a reduction in Spo0A / AbrB activity. More preferably, the mutations are abrB, kapB, kbaA, kinA, kinB, kinC, kinD, kinE, kipA, kipI, obg, phrC, phrE, rapA, rapB, rapE, scoC, sigH, sinR, sinI, spo0A, sp0A, sp0A Act on one, two or more genes selected from the group consisting of spo0E, spo0F, spo0JA and spo0JB genes.

  The microorganism can be eukaryotic or prokaryotic. Preferably the microorganism is a prokaryotic organism. Prokaryotic microorganisms can be gram positive or gram negative. Gram-positive microorganisms include, but are not limited to, those belonging to one of the genera Bacillus, Corynebacterium, Lactobacillus, Lactococci, and Streptomyces. I don't mean. Preferably, the microorganism belongs to the genus Bacillus. Examples are Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus puntis, Bacillus halodurans, and the like. Most preferably the microorganism is Bacillus subtilis. The wild-type form corresponding to the microorganism of the present invention is a spore-forming microorganism.

  The “pantothenate compound” is preferably pantothenate. Pantothenate compounds further include intermediate compounds in the biosynthetic pathway of pantothenate biosynthesis, such as pantoate, α-ketopantoate, α-ketoisovalerate, and the like.

  The present invention encompasses any mutation to a microbial gene that results in reduced function of the sigE gene product (SigE protein). SigE function can be assayed in sporulation assays known to those skilled in the art. The microorganisms of the present invention may result in the absence or reduction of expression of a functional sigE gene product as a result of having a mutation in the spoIIGB (sigE) gene. The present invention further encompasses any mutation in a gene known to activate spoIIGB (sigE). These include, but are not limited to, spoIIGA, spoIIR, and spoIIAC (sigF) (Helmann and Moran, 2002). The microorganisms of the present invention may result in the absence or reduction of functional spoIIGA gene product expression as a result of having a mutation in the spoIIGA gene. The microorganism of the present invention may have a mutation in the spoIIR gene resulting in the absence or reduction of expression of a functional spoIIR gene product. The microorganisms of the present invention may result in the absence or reduction of expression of a functional spoIIAC gene product as a result of having a mutation in the spoIIAC gene.

  The term “expression” refers to transcription of a nucleic acid sequence and / or subsequent translation from a transcribed sequence to an amino acid sequence. Nucleic acid expression is reduced by, for example, gene deletion; nucleotide additions that inactivate or reduce the activity of the encoded protein through the formation of a frameshift in the reading frame or through prematurity termination of translation by introduction of a stop codon Or by nucleotide removal; by introducing mutations into the gene by modifying regulatory sequences such as promoters, ribosome binding sites, and other techniques described herein.

  A “mutation” can be a deletion, substitution or addition in a gene sequence. The mutation can be a disruption, a frameshift mutation, or a nonsense mutation. A mutation can be a disruption that affects the entire sequence of a gene or a portion thereof. Mutations can affect the coding or non-coding sequence of a gene, such as a promoter. Mutations can result in a complete lack of transcription of the gene or prematurity termination of translation. Similarly, a mutation may be located in a preceding (or “upstream”) gene and disrupt the transcription of an adjacent (or “downstream”) gene (s) by a process called polarity. Alternatively, the mutation can have the effect that the translated protein carries a mutation that renders the protein non-functional compared to the wild-type protein.

  Methods for introducing appropriate mutations into genes are known to those skilled in the art. These methods include the introduction of point mutations into the bacterial chromosome (Cutting and Vander Horn, 1990), removal of the chromosomal DNA segment and replacement of that segment with an antibiotic resistance gene (Perego, 1993), transposon Tn917 or mini There are, but are not limited to, direct insertion of transposable elements such as Tn10 (Youngman, 1990; Petit et al., 1990) or plasmids such as pMUTIN (Vagner et al., 1998).

  Preferably, a sporulation-deficient microorganism having a reduced function of the sigE gene product can overexpress spo0A, more preferably the microorganism overexpresses spo0A. Overexpression of spo0A can be achieved as described herein.

  Increased or overexpression of a gene can be achieved in a variety of ways. One approach to obtaining a microorganism that overexpresses one or more genes is to remove regulatory sequences, for example by adding a strong promoter, an inducible promoter or multiple promoters, or so that expression is constitutive By altering or modifying regulatory sequences or sites associated with the expression of a particular gene. Additional procedures are described below.

  A “promoter” is a DNA sequence that is upstream of the start of transcription of a gene and is involved in the recognition and binding of RNA polymerase and / or other proteins to initiate transcription of the gene. Normally, a promoter determines under what conditions a gene is expressed.

An “inducible promoter” is one that temporarily initiates mRNA synthesis of a gene under specific conditions. (I) Promoters can be regulated primarily by cofactors such as repressors or activators. A repressor is a sequence-specific DNA binding protein that suppresses promoter activity. Transcription can be initiated from this promoter in the presence of an inducer that inhibits the repressor from binding to the operator of the promoter. Examples of such promoters from gram positive microorganisms include gnt (gluconate operon promoter); bacillus licheniformis penP; glnA (glutamine synthetase); xylAB (xylose operon); araABD (L-arabinose operon) and There is, but is not limited to, the P _spac promoter (Yansura and Henner, 1984), which is a hybrid _SPO1 / lac promoter that can be controlled by inducers such as isopropyl-β-D-thiogalactopyranoside [IPTG]. . Activators are also sequence-specific DNA binding proteins that induce promoter activity. Examples of such promoters from gram positive microorganisms include, but are not limited to, binary systems (PhoP-PhoR, DegU-DegS, spo0A-phosphate relay), LevR, Mry and GltC. (Ii) The production of secondary sigma factors may be mainly responsible for transcription from specific promoters. Examples from Gram-positive microorganisms include, but are not limited to, promoters activated by the spore formation specific sigma factors σ ^F , σ ^E , σ ^G and σ ^K and the general stress sigma factor σ ^B. is not. The σ ^B mediated response is induced by energy limitation and environmental stress (Hecker and Volker, 1998). (Iii) Attenuation and anti-transcription termination also regulate transcription. Examples from gram positive microorganisms include, but are not limited to, the trp operon and sacB genes. (Iv) Other promoters that are regulated in expression vectors are based on the temperature-sensitive immune repressor from phage Φ105 (Osbourne et al., 1985) and the sacR regulatory system conferring sucrose inducibility (Klier and Rapoport, 1988). Yes.

A “constitutive” promoter is one that allows the gene to be expressed in virtually all environmental conditions, ie, a promoter that directs certain non-specific gene expression. A “strong constitutive promoter” is one that initiates mRNA more frequently than in a natural host cell. Strong constitutive promoters are well known and can be selected appropriately depending on 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. Examples of promoters from gram-negative microorganisms include tac, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-P _R , and λ- there is a P _L, but is not limited to it.

  The invention further encompasses any mutation to one or more genes that results in reduced function of both spo0A and AbrB. The functions of spo0A and AbrB can be assayed by enzymatic assays for γ-glutamyl transpeptidase (Xu and Strauch, 1996) and microarray methods known to those skilled in the art. In addition, spo0A function can be assayed in a sporulation assay, and AbrB function can be assayed with a lacZ reporter gene fused to an AbrB regulated promoter (spo0VG, tycA, abrB, etc.). The microorganism may have at least one mutation in the spo0A gene and at least one mutation in the abrB gene. Mutations result in the absence or decreased expression of a functional spo0A gene product and the absence or decreased expression of a functional abrB gene product.

  Mutations to one or more genes that lead to reduced function of spo0A include mutations to genes known to activate spo0A. These include, but are not limited to, spo0B, spo0F, kinA, kinB, kinC, kinD, kinE, and sigH. The microorganisms of the present invention may result in the absence or reduced expression of a functional spo0B gene product as a result of having a mutation in spo0B. The microorganisms of the present invention may result in the absence or reduced expression of a functional spo0F gene product as a result of having a mutation in spo0F.

  In a specific embodiment of the invention, mutations that result in reduced SigE function and mutations that result in reduced function of both spo0A and AbrB may be combined. Thus, the preferred embodiments of the various mutations and modifications described herein may be combined. This applies mutatis mutandis to the method described below.

  The microorganism of the present invention can overexpress pantothenate compounds under appropriate conditions. As used herein, the term “overexpressing pantothenate compounds” refers to a significant increase in the production of pantothenate compounds by the microorganism of the present invention compared to the wild-type form of the microorganism. Preferably 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 of pantothenate per liter of culture.

  In one embodiment, the microbial population of the invention is at least 100 mg pantothenate per liter of culture, preferably at least 125 mg, more preferably at least 140 mg, more preferably at least 200 mg when cultured under conditions as described in Example 2. Still more preferably at least 300 mg, even more preferably at least 400 mg, most preferably at least 500 mg. In this example, culturing in a minimal medium is described. Alternatively, the microbial population of the present invention, when cultured under conditions as described in Example 4 or 5, is at least 100 mg pantothenate per liter of culture, 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.

  In another aspect, the microbial population of the present invention produces at least 5 g, preferably at least 7.5 g, most preferably at least 10 g pantothenate per liter of culture when cultured under conditions as described in Example 3. This example describes fermentation by fed-batch culture.

  The level of pantothenate production by the sporulation-deficient microorganism of the present invention is comparable to the level of pantothenate production by the control cells. Accordingly, the amount of pantothenate compound produced by the microbial population of the present invention exceeds 50% of the amount of pantothenate compound produced by the corresponding microbial population that is not deficient in sporulation when cultured under the same conditions, Preferably it exceeds 75%, more preferably it can exceed 100%. When cultured under the conditions described in Example 2, the amount of pantothenate compound produced by the microbial population of the present invention is 50% of the amount of pantothenate compound produced by the corresponding microbial population that is not deficient in sporulation. More than 75%, preferably more than 75%. When cultured under the conditions described in Example 3, the amount of pantothenate compound produced by the microbial population of the present invention is 50% of the amount of pantothenate compound produced by the corresponding microbial population that is not deficient in sporulation. It is preferred to exceed, preferably more than 75%, most preferably more than 100%. When cultured under the conditions described in Example 4, the amount of pantothenate compound produced by the microbial population of the present invention is 50% of the amount of pantothenate compound produced by the corresponding microbial population that is not deficient in sporulation. It is preferred to exceed, preferably more than 75%, most preferably more than 100%. When cultured under the conditions described in Example 5, the amount of pantothenate compound produced by the microbial population of the present invention is 50% of the amount of pantothenate compound produced by the corresponding microbial population that is not deficient in sporulation. It is preferred to exceed, preferably more than 75%, most preferably more than 100%.

  A “corresponding microorganism that is not deficient in sporulation” is preferably a microorganism that has not been modified to exhibit reduced SigE function or reduced spo0A and ArbB function. For example, if the microorganism of the present invention has a mutation in the spoIIGA gene, the corresponding microorganism that is not deficient in sporulation is substantially the same microorganism except that it does not have a mutation in the spoIIGA gene. The same applies to mutations in other genes.

  Overproduction of pantothenate compounds can be achieved in various ways. Methods for preparing B. subtilis strains that overproduce pantothenate are described, for example, in WO01 / 21772, WO02 / 057474, and WO02 / 061108. One approach to obtaining pantothenate overproducing microorganisms is to overexpress one or more genes involved in the pantothenate biosynthetic pathway. The term “overexpressed” or “overexpression” includes expression of the gene product at a level higher than that expressed in a comparable microorganism that has not been modified or in a comparable microorganism that has not been modified. In one aspect, the microorganism of the present invention is one selected from the group consisting of panB, panC, panD, panE, ilvB, ilvN, ilvC, ilvD, glyA, and serA, ylmA and a gcv gene involved in the glycine cleavage pathway or Overexpress multiple genes.

Overexpression of a gene in a microorganism can be performed by any method described herein including, but not limited to, deregulation of a gene and / or overexpression of at least one gene. In one embodiment, the microorganism can be processed by genetic engineering, such as genetic engineering, to overexpress a level of the gene product that is greater than that expressed in the comparable microorganism that has not been modified or that has not been modified. For genetic manipulation, for example, by adding a strong promoter, an inducible promoter or multiple promoters, or by removing regulatory sequences so that expression is constitutive, regulatory sequences associated with the expression of a particular gene Or change or modify the site, change the location of a specific gene in the chromosome, change the nucleic acid sequence adjacent to a specific gene, such as a ribosome binding site or transcription termination factor, increase the copy number of a specific gene Modifying proteins involved in transcription of specific genes and / or translation of specific gene products, such as regulatory proteins, suppressors, enhancers, transcriptional activators, etc., or are customary in the art (eg, repressors Antisense nucleus that blocks protein expression It includes molecules but not limited thereto) may be any conventional means express addition to deregulation of a specific gene, but is not limited thereto. Examples of 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.

  In another embodiment, the microorganism can be physically or environmentally manipulated to overexpress a level of the gene product prior to manipulation of the microorganism or greater than the level expressed in a comparable microorganism that has not been manipulated. For example, a microorganism can be treated with an agent known or suspected to increase transcription of a specific gene and / or translation of a specific gene product such that transcription and / or translation is increased or increased, or the agent Microorganisms can be cultured in the presence of Alternatively, the microorganism can be cultured at a selected temperature to increase transcription of a specific gene and / or translation of a specific gene product such that transcription and / or translation is increased or increased.

  The term “deregulated” or “deregulated” includes alteration of alteration of at least one gene in the microorganism such that the level or activity of the gene product in the microorganism is altered or altered. Preferably at least one gene is altered or modified such that the gene product is increased or increased.

The present invention further relates to a method for preparing a sporulation-deficient microorganism capable of overproducing a pantothenate compound. Microorganisms that can overproduce pantothenate compounds can be modified to include mutations in genes that regulate SigE function and optionally increased spo0A function. According to this aspect, the method comprises:
(A) providing a microorganism capable of overproducing a pantothenate compound;
(B) optionally introducing a mutation that causes an increase in the function of a DNA sequence (eg promoter) or spo0A into the microorganism, and (c) in step (a) or (b) so as to obtain a sporulation-deficient microorganism. Introducing a mutation that causes a reduction in SigE function in the microorganism.

  The order of steps (b) and (c) may be changed.

Or that way
(A) providing a microorganism capable of overproducing a pantothenate compound, and (b) a mutation that causes the microorganism of step (a) to reduce spo0A function and ArbB function so as to obtain a sporulation-deficient microorganism. May be included.

In another embodiment, sigE gene mutations or spo0A and arbB gene mutations may be introduced prior to performing mutations that result in overproduction of pantothenate compounds. According to this aspect, the method comprises:
(A) providing a microorganism that cannot overproduce pantothenate compounds;
(B) introducing a mutation that causes a decrease in SigE activity in the microorganism of step (a) or causing a decrease in spo0A activity and a decrease in AbrB activity in the microorganism of step (a) so as to obtain a sporulation-deficient microorganism And (c) modifying the sporulation-deficient microorganism obtained in step (b) so that the pantothenate compound can be overproduced.

  If a mutation that causes a reduction in SigE activity is introduced in step (b) of the present invention, the method will allow the microorganism of step (a) or (b) or (c) to have DNA causing an increase in SpoOA function. It may include an optional step of introducing a sequence (eg, a promoter) or mutation.

  Another aspect of the present invention provides a pantothenate compound comprising: (a) culturing the microorganism of the present invention under conditions that produce a pantothenate compound; and (b) optionally recovering the pantothenate compound from a cell culture medium. It is a method for manufacturing.

  The method of the present invention includes the step of culturing the modified microorganism under conditions that produce a pantothenate compound. The term “culturing” includes maintaining and / or growing a living microorganism of the present invention. In one embodiment, the microorganism of the present invention is cultured in a liquid medium. In another embodiment, the microorganism is cultured in a solid or semi-solid medium. The microorganism of the present invention is preferably cultured in a liquid medium containing nutrients essential or beneficial for the maintenance and / or growth of the microorganism. Such nutrients include carbon sources or carbon substrates that are complex carbohydrates such as, for example, bean flour or cereal flour, starch, sugar, sugar alcohol, hydrocarbons, oils, fats, fatty acids, organic acids and alcohols; Plant proteins from peas, beans, and tubers; proteins, peptides and peptides derived from animal sources such as peptone, peptides and amino acids, and meat and milk and animal by-products such as peptone, meat extract and casein hydrolysates Amino acids; inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorous sources such as phosphoric acid, its sodium and potassium salts; eg magnesium, iron, manganese, calcium, copper, zinc, boron Trace elements, molybdenum and / or cobalt salts Amino acid not only vitamins, but there is a growth factor, such as growth-promoting agents, but are not limited to it.

  The microorganism is preferably cultured at a controlled pH. In one embodiment, the microorganism is cultured at a pH between 6.0 and 8.5, more preferably at a pH of about 7. The desired pH can be maintained by any method known to those skilled in the art.

  Preferably, the microorganism is further cultured with controlled aeration and controlled temperature. In one aspect, the controlled temperature comprises between 15 and 70 ° C, preferably the temperature is between 20 and 55 ° C, more preferably between 30 and 45 ° C or between 30 and 50 ° C.

  Microorganisms can be cultured by conventional culture methods such as stationary culture, test tube culture, shaking culture, aeration and agitation culture or fermentation, either continuously or intermittently in a liquid medium. Preferably, the microorganism is cultured in a fermentation apparatus. The fermentation methods of the present invention include batch culture methods, fed-batch culture methods and continuous culture methods. A variety of such processes have been developed and are well known in the art.

  Incubation is usually continued for a time sufficient to produce the desired amount of pantothenate compound.

  In another aspect of the invention, the method further comprises recovering the pantothenate compound. The term “recovering” includes isolating, extracting, collecting, separating or purifying the compound from the culture medium. Including, but not limited to, treatment with ordinary resins, treatment with ordinary adsorbents, pH change, solvent extraction, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc. The isolation of the compound can be performed by any conventional isolation or purification method known in the art. For example, the pantothenate compound can be recovered from the culture solution by first removing the microorganisms from the culture. The culture is then passed in or on the cation exchange resin to remove unwanted cations and then in or on the anion exchange resin to remove unwanted inorganic anions and the Compounds, such as organic acids that have a stronger acidity than pantothenate, are removed. The resulting pantothenate can then be converted to a salt as described herein.

  A compound is typically “isolated” when the resulting preparation is substantially free of other compounds. In one embodiment, the preparation has a purity of the desired compound of greater than about 80% (by dry weight) (eg, less than about 20% of all media, components, or fermentation byproducts), more preferably greater than about 90%, still more Preferably the purity of the desired compound is greater than about 95%, and most preferably greater than about 98-99%.

  In another embodiment, the desired compound is not purified from the microorganism or from the culture. The culture or the entire culture supernatant can be used as a source of product. In a specific embodiment, the culture or culture supernatant is used without modification. In further embodiments, the culture or culture supernatant is concentrated, dried and / or lyophilized.

  Various aspects of the invention described herein may be combined with one another.

  The following non-limiting examples further illustrate the invention.

Examples General methods Strains and plasmids. The Bacillus subtilis strain of the present invention is a B. subtilis strain. It was obtained from strain 1A747 (Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio 43210, USA), which is a prototrophic derivative of Subtilis 168 (trpC2). A chloramphenicol resistance gene (cat) cassette was obtained from plasmid pC194 (GeneBank M19465, Cat # 1E17 Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio 43210, USA). Plasmid pX12 (Humbelin et al., 1999, containing the RB50 :: [pRF69] :: [pRF93] -derived SPO1-15 promoter (Perkins et al., 1999, J. Ind. Microbiol. Biotech. 22: 8-18) From plasmid pX123roDTD-SPO1-15, which is a derivative of J. Ind. Microbiol. Biotech. 22: 1-7) P ₁₅ promoter of subtilis bacteriophage SPO1 (.. Lee et al, 1980, Mol Gen. Genet 180:. 57-65) was obtained. SWV215 strain [trpC2 pheA1 spo0A :: kan] (Xu and Strauch, 1996, J. Bacteriol. 178: 4319-4322), BHI strain [trpC2 pheAl spo0HΔHindIII-EcoRI :: cat] (reference document Healy et al., 1991) Mol. Microbiol. 5: 477-487, BHI is a derivative of BH100), 650 strain [trpC2 ilvB2 leuB16 spoIIABC :: cat] (Pragai et al., 2004, J Bacteriol. 186: 1182-1190), 731 strain [TrpC2 spoIIIG :: ermC] (Partridge and Errington, 1993, Mol. Microbiol. 8: 945-955), and 901 strain [trpC2 spoIIGA :: aphA-3] (Wu and Errington, 1994, Science 264: 572-575 ) Obtained a mutant in the sporulation gene. A mutant in the abrB gene was obtained from the strain SWV119 [trpC2 pheA1 abrB :: tet] (Xu and Strauch, 1996). Due to the overexpression of the spo0A gene, a _Pspac spo0A fusion was obtained from the AH1763 strain [trpC2 metC3 spo0A :: neo amyE :: P _spac spo0A (cat)] (Henriques, A, personal information exchange).

Culture medium. B. The standard minimal medium for Subtilis contains 1 × Spizizen salts, 0.04% sodium glutamate, and 0.5% glucose. Standard solid complete medium is tryptone blood agar (TBAB, Difco). The standard liquid complete medium is calf infusion-yeast extract medium (VY). The composition of these media is described below:
TBAB medium: 33 g of Difco tryptone blood agar culture base (catalog # 0232), 1 L of water. Autoclave sterilization.
VY medium: 25 g of Difco calf infusion medium (catalog # 0344), 5 g of Difco yeast extract (catalog # 0127), 1 L of water. Autoclave sterilization.
Minimal medium (MM): 100 ml of 10 × Spizizen salts; 10 ml of 50% glucose; 1 ml of 40% sodium glutamate;
10 × Spizizen salts: 140 g of K ₂ HPO ₄ ; 20 g of (NH ₄ ) ₂ SO ₄ ; 60 g of KH ₂ PO ₄ ; 10 g of Na ₃ .2H ₂ O citrate; ₂ g of MgSO ₄ .7H ₂ O; And
P medium: 10 × PAM 100 ml; 10 × Spizizen salts 100 ml; 50% glucose 10 ml; sterile distilled water 790 ml.
P agar medium: 10 × PAM 100 ml; 10 × Spizizen salts 100 ml; 50% glucose 10 ml; 790 ml sterile distilled water containing 15 g agar.
10 × Pantothenate Assay Medium (10 × PAM): 73 g Difco Pantothenate Assay Medium (Catalog # 260410), 1 L water. Autoclave sterilization.
10 × VFB minimal medium (10 × VFB MM): Na glutamate 2.5 g; KH ₂ PO ₄ 15.7 g; K ₂ HPO ₄ 15.7 g; Na ₂ HPO ₄ · 12H ₂ O 27.4 g; NH ₄ Cl 40 g; citric acid 1 g; (NH 4) ₂ SO ₄ 68 g;
Trace element solution: 1.4 g of MnSO ₄ · H ₂ O; 0.4 g of CoCl ₂ · 6H ₂ O; 0.15 g of (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O; AlCl ₃ · 6H ₂ O 0.1 g; CuCl ₂ .2H ₂ O 0.075 g;
Fe solution: FeSO ₄ . 7H ₂ O 0.21 g; make up to 10 ml with water.
CaCl ₂ solution: 15.6 g of CaCl ₂ .2H ₂ O; make up to 500 ml with water.
Mg / Zn solution: 100 g of MgSO ₄ .7H ₂ O; 0.4 g of ZnSO ₄ .7H ₂ O;
VFB MM medium: 10 × VFB MM 100 ml; 50% glucose 10 ml; trace element solution 2 ml; Fe solution 2 ml; CaCl ₂ solution 2 ml; Mg / Zn solution 2 ml; sterile distilled water 882 ml.
VFB MMGT medium: 10 × VFB MM 100 ml; 0.5 M Tris (pH 6.8) 100 ml; 50% glucose 44 ml; trace element solution 2 ml; Fe solution 2 ml; CaCl ₂ solution 2 ml; Mg / Zn solution 2 ml;
VF fermentation batch culture medium: Sterilize solution in place: sodium glutamate 0.75 g; KH ₂ PO ₄ 4.71 g; K ₂ HPO ₄ 4.71 g; Na ₂ HPO ₄ · 12H ₂ O 8.23 g; 0.23 g of NH ₄ Cl; 1.41 g of (NH ₄ ) ₂ SO ₄ ; 11.77 g of yeast extract (Merck); 0.2 ml of Basildon antifoaming agent;
The solution after autoclaving is put into a fermenter: 27.3 g of glucose / H ₂ O;
The filter sterilized solution is placed in the fermentation apparatus: 2 ml of trace element solution; ₂ ml of CaCl ₂ solution; ₂ ml of Mg / Zn solution; 2 ml of Fe solution;
VF fermentation fed medium: 660 g of glucose / H ₂ O; Autoclave sterilization. ₂ g of MgSO ₄ .7H ₂ O; 14.6 mg of MnSO ₄ .H ₂ O; 4 mg of ZnSO ₄ .H ₂ O; 1 L in total (autoclaved) is added.

Pantothenate assay. Pantothenate was assayed by three biological methods and one physical (HPLC) method:
Biological Assay I-Plate Bioassay: Pantothenate was assayed on an agar plate using an indicator strain from Salmonella typhimurium using known methods. The DM3 (panC355) strain (D. Downs, University of Wisconsin-Madison, Madison, Wis., USA) responds only to pantothenate. 10 ⁷ cells / ml of S. pylori on 20 ml of P agar medium used as bottom agar in a standard Petri dish. 10 ml of P agar medium containing Tyhumulium DM3 (panC355) indicator strain and 40 μg / ml 2,3,5-triphenyltetrazolium chloride (Fluka; catalog # 93140) was overlaid. Bacteria were transferred from single colonies grown on TBAB agar to a bioassay P agar plate using a sterile pipette tip. B. produced more than about 10 mg / l of Pan after overnight incubation at 37 ° C. Around S. subtilis colonies An observable purple halo formed of Tyhumulium DM3. The size of the halo is B. Correlated with Pan production by subtilis strains.
Biological Assay II-Tube Bioassay: Pantothenate was assayed in a liquid state using a tube culture. B. To assay a subtilis culture, the supernatant was sterilized by filtration and a dilution was prepared in 1 × pantothenate assay medium (PAM) in a test tube. The total volume of the diluted solution was 5.0 ml. 0.1 ml of DM3 indicator stock solution diluted 200-fold (in 1 × PAM) was added to the test tube containing these dilutions. Next, the test tube was cultured at 37 ° C. for 18 to 24 hours using a roller drum type shaker. Turbidity readings were taken at 600 nm (OD ₆₀₀ ) and compared to a standard curve of known amounts of pantothenate. Specifically, the standard pantothenate was diluted to levels of 0.8, 4, 20, 50, and 100 μg / l, and the indicator strain prepared as described above was added to each diluted solution, and the mixture was incubated at 37 ° C. for 18 to 24 hours. A standard curve was prepared by measuring turbidity later (same incubation time as unknown sample). The linear portion of the standard curve was used to create a log regression equation. The pantothenate concentration in the unknown sample was then calculated from the OD ₆₀₀ value of the diluted sample using this equation.
Biological Assay III-96-well Microtiter Plate Bioassay: Pantothenate was also assayed in liquid state using a 96-well microtiter plate format. In each well of a 96-well microtiter plate, 5.5 × 10 ⁵ cells / ml of S.P. 180 μl of pantothenate assay medium (PAM) containing Tyhumulium DM3 was added. B. To assay the subtilis culture, 60 μl of filter sterilized culture supernatant was added to the wells in the first row, rows BH. After mixing, 60 μl of sample was transferred to the next hole in the second row. These 4-fold dilution steps were repeated up to the 12th column. After mixing the sample in the twelfth row, 60 μl was discarded, thus each hole contained 180 μl of sample. An adhesive film was placed on the plate to avoid evaporation and the culture was incubated at 37 ° C. for 17 h with stirring at 300 rpm. Turbidity readings were taken at 600 nm and compared to a standard curve of known amounts of pantothenate (Pan). A standard curve was prepared by adding 60 μl of Pan standard solution (100 mg / ml) to the hole in row 1 row A of each microtiter plate. Dilution, incubation and Pan standard turbidity measurements were performed as described above for unknown samples.
HPLC assay: Samples were chromatographed on a Phenomenex LUNA C8 column using an Agilent 1100 HPLC system equipped with a thermostat autosampler and a diode array detector. The column dimensions are 150 x 4.6 mm and the particle size is 5 microns. The column temperature was kept constant at 20 ° C. The mobile phase is a mixture of 0.1% acetic acid (A) and methanol (B). Apply gradient elution from 1% B to 45% B over 15 minutes. The flow rate is 1 ml / min. Pantothenate was monitored from UV absorption at 220 nm. Pantothenate elutes at about 9.6 minutes. The calibration range for this method is 1-100 mg / l pantothenate.

  Molecular and genetic methods. Standard genetic and molecular biology techniques are generally known in the art and have been previously described. DNA transformation, PBS1 universal transduction, and other standard B.P. Subtilis genetic techniques are also generally known in the art and have been previously described (Harwood and Cutting, 1992).

  Sporulation assay. B. A 1 ml sample was taken from the subtilis culture and a 10-fold dilution series was prepared with sterile distilled water. After heat treatment at 80 ° C. for 20 minutes, they were placed on TBAB agar plates and incubated at 37 ° C. for 20 h before determining the number of “heat-resistant” colony forming units (cfu) in 1 ml of the original culture. The frequency of spore formation was calculated by dividing the titer of heat-resistant spores (cfu / ml) by the titer of bacterial cells before heat treatment (cfu / ml).

fermentation. Pantothenate producing strains were grown in a stirred fermentor initially containing 1.4 liters of VF fermentation batch culture medium containing glucose / salt solution, for example, a 2 liter container of BIOFLO 3000 New Brunswick. Computer controlled with NBS Biocommand 32 commercial software (New Brunswick Scientific Co., Inc., Edison, NJ, USA); using Lucullus software (Biospektra AG, Schlieren, Switzerland) to control data collection and glucose feed did.

Two seed culture protocols were used to prepare the inoculum for fermentation. The first species consists of inoculating 50 μl of a frozen bacterial stock culture in 25 ml of VY medium containing 10 g / liter sorbitol and allowing the culture to grow at 37 ° C. for 6 hours. Next, 1 OD cells were used to inoculate 60 ml of VY fermentation batch culture medium (without glucose) containing 10 g / liter sorbitol, and this second species was grown at 37 ° C. for 8-12 hours. It was. This second species is used to inoculate the fermentation vessel, and the amount of inoculation is typically 4-5% of the initial media volume. Bacteria are grown in VY medium until late logarithmic growth (OD ₆₀₀ = 0.8-1.0), sterilized glycerol is added to a final concentration of 20%, then 1 ml aliquots are frozen on dry ice and frozen Frozen bacteria stock solutions were prepared by storing the resulting bacteria at −80 ° C.

During the fermentation, the pH in the reactor was kept constant at 6.8 by automatically adding ammonium hydroxide solution (28% aqueous solution). The fermentation temperature was 39 ° C. The minimum concentration of dissolved oxygen (pO ₂ ) of 15% was achieved by automatically adjusting the stirrer (in the range of 400 rpm to 1000 rpm) and manually maintaining the aeration rate at 1-2 vvm. Antifoam (Basildon) was added manually at any time.

  Fermentation can be a batch culture process, but is preferably a fed-batch process with limited carbohydrates. Therefore, the prescribed VF fermentation fed-batch solution (see above) was fed into the reactor after consumption of the initial glucose, which is usually the situation after 6-8 hours of process time. At that time, steady addition of the feed solution was started at a rate of 14 g / h.

Example 1
In this embodiment, B.I. Describes the construction of a pantothenate overproducing strain of Subtilis.

B. Subtilis strain PA12
Using the polymerase chain reaction (PCR), A deletion mutation in the promoter region of the panBCD operon of Subtilis prototrophic strain 1A747 was generated. In that mutation, the 215 bp long nucleotide region between birA and panB was replaced with a chloramphenicol resistance (cat) cassette from Staphylococcus aureus (GeneBank M58515). In order to do this, a cat cassette was first introduced between the NheI and ClaI sites of the pBR322 plasmid (GeneBank J01749) in the direction opposite to the transcription direction of panB. Next, “arms” of two PCR fragments were generated: 1 μl of 40 mM dNTP, 5 μl of 10 × buffer and PCR enzyme (Taq) as described by the manufacturer (Expand High fidelity PCR System-Roche Applied Science). And Tgo) in 0.1 μg of 1A747 chromosomal DNA in a reaction volume of 50 μl containing 0.75 μl, primers panB / up2 / for / R1 and panB / up2 / rev / ClaI or primers panB / down2 / for / NheI and panB 0.2 μl of a 100 mM solution of / down2 / rev / Bam (Table 1) was added. The PCR reaction was performed for 30 cycles using an annealing temperature of 58 ° C. and an extension time of 60 seconds. The resulting fragments, called F1 and F2, respectively, were purified and sequential insertions were made between EcoRI and ClaI sites (in F1) and between NheI and BamHI (in F2), respectively. The ligated DNA was introduced into E. coli TOP10 cells (Invitrogen) by transformation and selected for ampicillin resistance at a concentration of 100 μg / ml. This resulted in the E. coli plasmid pPA5. Next, by B. Select for chloramphenicol resistance (Cm ^r ) using standard conditions on a TBAB agar plate containing a ΔpanB _p :: cat deletion cassette into the subtilis 1A747 chromosome and containing 5 μg / ml chloramphenicol (Cm) Went. A single Cm ^r colony containing a deletion in the panB promoter region was isolated and named PA1 (ΔpanB _p :: cat). As expected, PA1 was also a pantothenate auxotroph (Pan ⁻ ) that required pantothenate for growth on minimal medium. Using the panB / up2 / for / R1 primer and the panB / down2 / rev / Bam primer (Table 1), the deletion mutation was again confirmed by diagnostic PCR using standard reaction conditions.

The next step was to introduce a strong constitutive promoter upstream of the panB gene. B. P ₁₅ and P ₂₆ (Lee et al., 1980) derived from SP01 bacteriophage subtilis any number of such promoters, including described in the literature. Long flanking homology polymerase chain reaction (LFH-PCR) was used to generate a DNA fragment containing P ₁₅ upstream of the ribosome binding site (RBS) of panB. To do this, two PCR fragments, “arms”, were first generated: 1 μl of 40 mM dNTP and 5 μl of 10 × buffer and PCR as described by the manufacturer (Expand High fidelity PCR System-Roche Applied Science). 0.2 μl of a 100 mM solution of primers P1panBCD and P2panBCD or primers P3panBCD and P4panBCD (Table 2) was added to 0.1 μg of 1A747 chromosomal DNA in which the enzyme (Taq and Tgo) was dissolved in 50 μl of a reaction volume containing 0.75 μl. The PCR reaction was performed for 30 cycles using an annealing temperature of 55.7 ° C. and an extension time of 45 seconds. The resulting fragments, called F3 and F4, respectively, were purified and then used as primers in the second round of PCR. The F3 and F4 were diluted 50-fold, respectively 1 [mu] l, plasmid pX123roDTD-SPO1-15 was linearized dissolved in the reaction volume 50 [mu] l (including P ₁₅ promoter) was added to 0.1 [mu] g. An annealing temperature of 63 ° C. and an extension time of 6 minutes was used for the first 10 cycles. The extension time was extended for 20 seconds per cycle for the next 20 cycles. The resulting product was then used as a template for the third round of PCR. The PCR product was diluted 50-fold and 1 μl was mixed with 0.2 μl of a 100 mM solution of primers P1panBCD and P4panBCD dissolved in a reaction volume of 50 μl containing dNTPs, buffer and enzyme as described above. The parameters of the PCR reaction were the same as those used in the second round PCR. The completed PCR fragment was then introduced into the panB promoter-deficient strain PA1 by DNA transformation and selected for pantothenate prototrophy (Pan ⁺ ) on minimal medium agar plates using standard conditions. These Pan ⁺ colonies is also the chloramphenicol sensitive (Cm ^s), to confirm the insertion of the promoter cassettes. Isolated single ^Pan + Cm ^s colonies containing PanBCD operon expressed from P ₁₅ promoter, was designated PA12 (P ₁₅ panBCD). By diagnostic PCR using P15seq primers and P4panBCD primers using standard reaction conditions again (Table 2), it was confirmed that the presence of P ₁₅ promoter panB gene upstream. In shake flask culture, PA12 produced approximately 100 mg / liter pantothenate in VFB MM medium and 250 mg / liter pantothenate in VFB MMGT medium (based on HPLC / MS assay), whereas the 1A747 control was less than 1 mg / liter. Only pantothenate was produced. In fermentation by standard fed-batch culture using VF medium and growth conditions, PA12 produced approximately 10-14 g / liter pantothenate in 48 hours.

B. In order to construct a strain that also contains a strong constitutive promoter (ylbQ) upstream of the subtilis PA49 strain panE gene, a deletion mutation was first constructed. Investigation of the panE gene revealed two potential start sites: start site 1 (5′-AAATTGGGGTG-3 ′ (RBS) -7nt-ATG) overlaps the BspHI site and overlaps the BsaXI site. 2 (5′-GGAGGG-3 ′ (RBS) -5nt-TTG) 33 bp upstream. As a result, S. A 219 bp deletion of the panE / ylbQ promoter region was constructed by LFH-PCR using the Aureus erythromycin resistance (Em ^r ) gene (GeneBank V01278). To do this, “arms” of two PCR fragments were first made: 1 μl of 40 mM dNTPs, 5 μl of 10 × buffer and PCR as described by the manufacturer (Expand High fidelity PCR System-Roche Applied Science). 0.1 μg of 1A747 chromosomal DNA dissolved in a reaction volume of 50 μl containing 0.75 μl of enzymes (Taq and Tgo), 0.2 μl of 100 mM solution of primers P1panE and P2panE / Er or primers P3panE / Er / 2 and P4panE (Table 3) Was added. The PCR reaction was performed for 30 cycles using an annealing temperature of 55.7 ° C. and an extension time of 45 seconds. The resulting fragments, called F1 and F2, respectively, were purified and then used as primers in the second round of PCR. F1 and F2 fragments were diluted 50-fold and 1 μl each was added to 0.1 μg of linearized plasmid pDG646 (containing erm cassette; Guerout-Fleury et al., 1995) dissolved in 50 μl reaction volume. The first 10 cycles used an annealing temperature of 63 ° C. and an extension time of 6 minutes. In the next 20 cycles, the extension time was extended by 20 seconds per cycle. The resulting product was then used as a template for the third round of PCR. The PCR product was diluted 50-fold and 1 μl was mixed with 0.2 μl of a 100 mM solution of primers P1panE and P4panE dissolved in 50 μl of reaction volume containing dNTPs, buffer, and enzyme as described above. The PCR reaction parameters were the same as those used for the second round PCR. Next, as a result of introducing the completed PCR fragment into PA4 (Trp ⁺ colony obtained from B. subtilis CU550 trpC2 ilvC4 leu-124 by transformation using chromosomal DNA of 1A747) by transformation, pantothenate auxotrophic strain Resulting in an Em ^r colony. This strain was called PA5 (ΔpanE _p :: erm ilvC leuC). Diagnostic PCR was used to confirm the structure of the deletion. Thereafter, the panB promoter deletion was introduced into PA5 by transformation of PA1 chromosomal DNA at a non-discussed concentration to generate PA6 (ilvC leuC ΔpanB _p :: catΔpanE _p :: erm).

The next step was to simultaneously introduce a strong constitutive P ₁₅ promoter upstream of both panB and panE. LFH-PCR and using again contains an open reading frame upstream P ₁₅ of panB the open reading frame upstream of the panE caused a DNA fragment containing the P _15. To do this, using the same PCR protocol used to construct PA12 (see above), primers P1panB and P2panB / P15 (F1) and primers P3panB / P15 and P4panB (F2) (Table 1). and and 2, the construction of P ₁₅ panB), P1panE and P2panE / P15 (F1) and P3panE / P15 and P4panE (F2) (tables 3 and 4, P ₁₅ construction of panE) for panB and panE two using An “arm” of the PCR fragment was made.

Next, the PCR fragments of P ₁₅ panB and P ₁₅ panE completed into the panB and panE promoter-deficient strain PA6 (ilvC IeuC ΔpanB _p :: cat ΔpanE _p :: erm) by DNA transformation were introduced together, and the standard Were selected for pantothenic acid prototrophy (Pan ⁺ ) on minimal medium agar plates using typical conditions. Recovered Pan ⁺ colonies are Cm ^s and erythromycin sensitive (Em ^s), to confirm the insertion of the promoter cassettes. Single ^Pan + ^{Cm s} Em s colony containing both panBCD operon and panE gene expressed from P ₁₅ promoter was designated PA32 isolated. Again under standard reaction conditions to diagnostic PCR using P15seq and P4panB primers were used to confirm the presence of P ₁₅ promoter panB gene upstream (Tables 1 and 2). The same control for the panE gene was performed using P15seq and P4panE primers (Tables 3 and 4). However, the subsequent sequencing of the front P ₁₅ promoter panE, revealed partial deletion of the P ₁₅ promoter.

To replace the partially deleted P ₁₅ panE gene with the correct construction, the ΔpanE _p :: erm mutation was reintroduced into PA32 by DNA transformation using chromosomal DNA from PA5 (ΔpanE _p :: erm ilvC leuC), Selected for erythromycin resistance. This resulted in the PA41 strain (P ₁₅ panBCDΔpanE _p :: erm ilvC leuC). Introducing DNA fragment P ₁₅ panE generated by LFH-PCR as previously discussed next PA41 by transformation were selected Pan ⁺ prototrophy strains. This resulted in the strain PA43 (ilvC leuC P ₁₅ panBCD P ₁₅ panE). This Ilv ⁻ Leu ⁻ auxotroph is then transformed into wild type B. cerevisiae using standard methods. PBS1 phage lysate prepared with Subtilis 1A747 was used to transduce to Ilv ⁺ Leu ⁺ prototrophy. This resulted in the PA49 strain (P ₁₅ panBCD P ₁₅ panE).

  In shake flask culture, PA49 produced on average approximately 400 mg / liter pantothenate in VFB MMGT medium (based on HPLC / MS assay).

Example 2
This example includes a sporulation deficient mutation spo0A, spo0H, spoIIA (sigF) spoIIG (sigE) and spoIIIG (sigG). Describe the construction and testing of a subtilispantothenate overproducing strain.

B. deficient in sporulation Construction of a subtilis mutant.
From the strain SWV215 (spo0A :: kan), BHI (spo0HΔHindIII-EcoRI :: cat), 650 (spoIIAABC :: cat), 901 (spoIIGA :: aphA-3) and 731 (spoIIIG :: ermC), respectively A spore formation-deficient mutation was introduced into 1A747 (wild-type strain) and PA12, a pantothenate-overproducing strain, by transformation of the chromosomal DNA. Chromosomal DNA extraction and “Groningen” method Subtilis strains were transformed according to Bron (1990). 0.3 μg / ml erythromycin (Em) and 25 μg / ml lincomycin (Lm) for the ermC gene; 6 μg / ml Cm for the cat gene; and 10 μg / ml kanamycin (Km) for the kan and aphA-3 genes. Transformants were selected on the TBAB agar medium contained. The transformation efficiency was approximately 5 × 10 ⁴ transformants per μg of chromosomal DNA, resulting in the development of the following strains:
(I) SWA215 DNA-derived kanamycin resistant (Km ^r ) transformants were named 1A747_spo0A and PA12_spo0A;
(Ii) BHI DNA-derived Cm ^r transformants were named 1A747_sigH and PA12_sigH;
(Iii) 650 DNA-derived Cm ^r transformants were named 1A747_sigF and PA12_sigF;
(Iv) 901 DNA-derived Km ^r transformants were named 1A747_sigE and PA12_sigE;
(V) 731 DNA-derived erythromycin / lincomycin resistance (Em ^r / Lm ^r ) transformants were named 1A747_sigG and PA12_sigG.

In addition to being a major regulator of the early steps of sporulation, spo0A and sigma H (σ ^H ) are surface related B. It also plays a critical role in the formation of aerial structures that act as sporulation sites in surface associated B. subtilis communities (Brenda et al., 2001). Thus, 1A747_spo0A and PA12_spo0A lacking spo0A produced unstructured mucoid colonies on TBAB agar plates, while 1A747_sigH and PA12_sigH lacking σ ^H were similar but less intense (less on the agar surface) Phenotype that does not spread strongly). In contrast, other mutants lacking sigma F (σ ^F ), sigma E (σ ^E ) and sigma G (σ ^G ) formed colonies with colony morphology very similar to the wild type strain.

B. Effects of sporulation-deficient mutations on pantothenate production and sporulation in subtilis.
From each of the transformation experiments described above, 25 independently isolated colonies were tested for pantothenate production from a plate halo test. Two colonies of each mutant with a halo size representing the majority of test colonies were analyzed in shake flask cultures. A single colony grown on a TAB plate supplemented with the appropriate antibiotic was used to inoculate 20 ml of VFB MM in a 250 ml Meyer flask with baffle. Bacteria were cultured at 37 ° C. with stirring at 250 rpm. When the culture reached late stationary growth (after about 18 hours in culture), the turbidity of the cells was measured at 600 nm (OD ₆₀₀ ) and the number of “heat-resistant” cfu was determined by a sporulation assay. The cells were then removed by filtration through a 0.45 μm pore size filter and the pantothenate content in the post-culture medium was assayed using both biological and HPLC assays. The results showed that the total pantothenate production of mutants 1A747_spo0A, PA12_spo0A, 1A747_sigH and PA12_sigH was reduced from one-half to one-third compared to the parent strain (Table 5). This indicates that the lack of spo0A and σ ^H functions negatively affected pantothenate production. In mutants lacking σ ^F or σ ^E , pantothenate production is similar to the control level within experimental error, which indicates that mutations that block sporulation later have little or no effect on pantothenate production. It shows that there was not. The cell biomass of all mutants, determined by measuring the OD ₆₀₀ of the culture, was similar to the parent control biomass. The sporulation frequency of parent strains 1A747 and PA12 was approximately 0.3-1%. No spores were detected in mutants lacking spo0A, σ ^H and σ ^E. However, a few heat resistant cells were detected from the cultures of 1A747_sigF and PA12_sigF (Table 5). This indicates that the mutation in the spoIIABC gene did not result in a complete lack of sporulation. Based on these results, only strains containing mutations in the Sigma E gene produced control levels of pantothenate and were completely devoid of spore-forming bacteria.

strain containing sigma ^G mutation, other spo mutant produced a distinct phenotype not expressed. When this is introduced into 1A747 wild type cells, the resulting mutant produces normal levels of pantothenate and no spores. However, with the engineered PA12 strain, the resulting strain produces very low levels of pantothenate and no spores (Table 5). These results depend on either inhibiting the expression of genes transcribed by Sigma G whose protein product is involved in pantothenate production (or excretion) or blocking the transcription of downstream genes by polarity. , disruption of sigma ^G by erm gene, suggesting that blocked the expression of high levels of pantothenate. Investigation of the DNA sequence downstream of spoIIIG revealed the presence of a single gene ylmA that can be co-transcribed with spoIIIG. The ylmA gene potentially encodes an unknown ATP binding protein that can function as one of the components of the ABC transporter. As a result, overproduction of proteins combined with individual or engineered pan and / or ilv biosynthetic genes could lead to higher pantothenate production. Furthermore, a Spo ^- Pan ^- phenotype suppressor host mutation that restores pantothenate production could result in strains with higher pantothenate production.

Example 3
This example shows that a B. cerevisiae containing a sporulation-deficient mutation spo0A or spoIIG (sigE). The fermentation of the subtilis spantothenate overproducing strain is described. The sigE mutant (PA12_sigE), spo0A mutant (PA12_spo0A) and their parent strain PA12 were grown for 48 hours in a standard fed-batch fermentation using a laboratory scale 2 liter fermenter. Both pantothenate levels and bacterial sporulation frequency were measured during the fermentation process. As shown in Table 6, pantothenate production was similar in both the parent strain and the sigE mutant, but was reduced by approximately 85% in the spo0A mutant. The pantothenate yield relative to glucose was similar between the sigE mutant and the parent strain. Spores were detected in the parent strain but not in either mutant.

  While the invention has been illustrated by way of examples, the examples are merely illustrative of specific embodiments of the invention. Since many embodiments will be apparent to those of ordinary skill in the art reading this, this should not be construed as limiting the invention thereto.

Example 4
This example demonstrates that B. cerevisiae containing null mutations in spo0A and abrB. Describes the construction and testing of subtilispantothenate overproducing strains.

Single and double mutations of abrB and spo0A were introduced into PA49, a pantothenate overproducing strain, by transformation using chromosomal DNA from the SWV215 strain (spo0A :: kan) and the SWV119 strain (abrB :: tet). . Transformants were selected on TBAB agar medium containing 10 μg / ml Km for the kan gene and 10 μg / ml tetracycline (Tc) for the tet gene. The transformation efficiency was 2 × 10 ³ transformants / μg DNA for spo0A :: kan, 4 × 10 ² transformants / μg DNA for abrB :: tet, and spo0A :: kan and abrB :: tet double suddenly. The mutant was 4 transformants / μg DNA. The resulting strains are as follows:
(I) Km ^r transformant from SWV215 DNA was named PA1030;
(Ii) a tetracycline resistant (Tc ^r ) transformant from SWV119 DNA was named PA1037;
(Iii) a Km ^r and Tc ^r transformants from the SWV215 DNA and SWV119 DNA of was named PA1051.

Four colonies of each mutant were analyzed for pantothenate production and sporulation in shake flask cultures. A single colony grown on a TBAB plate supplemented with the appropriate antibiotic was used to inoculate 10 ml of VY medium supplemented with the appropriate antibiotic. Bacteria were grown overnight (about 17 h) at 37 ° C. with stirring at 250 rpm. The culture after overnight culture in 20 ml of VFB MMGT medium was diluted (1: 100) and grown to mid-logarithmic growth (OD ₆₀₀ = 0.6 to 0.8). These cultures were inoculated into 30 ml of VFB MMGT medium with an initial turbidity OD ₆₀₀ = 0.03, and the bacteria were grown at 37 ° C. with stirring at 250 rpm in a 250 ml Meyer flask with baffle. After 18 h of growth, cell turbidity was measured at 600 nm (OD ₆₀₀ ) and the number of “heat-resistant” cfu was determined by a sporulation assay. Next, the cells were removed by filtration through a filter having a pore size of 0.45 μm, and the pantothenate content in the culture medium after the culture was assayed by HPLC assay. The results showed that the cell biomass of all mutants determined by measuring the OD ₆₀₀ of the culture was similar to the parent control. Total pantothenate production of PA1030 (spo0A :: kan) and PA1037 (abrB :: tet) was reduced to 1/3 to 1/4 compared to the parental PA49 strain (Table 7). This indicates that lack of spo0A and AbrB function negatively affects pantothenate production (as also demonstrated in Example 2). However, the double mutant PA1051 (spo0A :: kan abrB :: tet) produced 40% more pantothenate and no spores than the PA49 control. Based on these results, pantothenate production can be increased beyond wild-type expression levels in abrB and spo0A double null mutants.

Example 5
This example shows that B. overexpressing spo0A contains a sporulation-deficient mutation (spoIIGA). Describes the construction and testing of subtilispantothenate overproducing strains.

B. carrying _Pspac- spo0A and sigE-null mutations. Construction of a subtilis strain In Example 2, the absence of spo0A was shown to reduce pantothenate production from one-half to one-third compared to the parent strain. In order to examine the effect of overexpression of spo0A gene on pantothenate production, IPTG-inducible P _spac − was expressed in the amyE gene of PA49 by transformation using chromosomal DNA derived from AH1763 strain (amyE :: P _spac spo0A [cat]). A spo0A fusion was introduced. Transformants were selected on TBAB agar containing 6 μg / ml Cm and the resulting Cm ^r strain was named PA1025. Next, a sporulation-deficient mutation in spoIIGA was introduced into PA1025 by transformation using chromosomal DNA derived from strain 901 (spoIIGA :: aphA-3). Transformants were selected on TBAB agar medium containing 6 μg / ml Cm for the cat gene and 10 μg / ml Km for the aphA-3 gene, and the resulting Cm ^r and Km ^r strains were named PA1064. It was.

B. Effect of spo0A overexpression on pantothenate production in Subtilis.
Single colonies of PA49, PA1025 and PA1064 were used to inoculate 10 ml of VY medium supplemented with appropriate antibiotics. Bacteria were grown overnight (about 17 h) at 37 ° C. with stirring at 250 rpm. The culture after overnight culture was diluted (1: 100) with 20 ml of VFB MMGT medium and grown to mid-logarithmic growth (OD ₆₀₀ = 0.6 to 0.8). 30 ml of VFB MMGT medium containing 30 ml of VFB MMGT and 10 mM IPTG were inoculated with these cultures at an initial turbidity of OD ₆₀₀ = 0.03 at 37 ° C. with stirring at 250 rpm in a 250 ml Meyer flask with baffle. Bacteria were grown. After 18 h of growth, cell turbidity was measured at 600 nm (OD ₆₀₀ ) and the number of “heat-resistant” cfu was determined by a sporulation assay. Next, the cells were removed by filtration through a filter having a pore size of 0.45 μm, and the pantothenate content in the culture medium after the culture was assayed by HPLC assay. The results showed that the cell biomass of all mutants determined by measuring the OD ₆₀₀ of the culture was similar to the parent control. In the absence of inducer IPTG, total pantothenate production from PA1025 ( _Pspac spo0A) and PA1064 ( _Pspac spo0AspoIIGA :: aphA-3) was similar to the PA49 parental strain (Table 8). When 10 mM IPTG was used to induce the expression of spo0A from the P _spac promoter, PA1025 produced 70% more pantothenate and 10 times more spores compared to the parental control. On the other hand, PA1064 produced 70% more pantothenate than the PA49 control, but lacked sporulation.

Claims (6)

A sporulation-deficient microorganism belonging to the genus Bacillus, which overproduces a pantothenate compound ,
Exhibit low Hesi was SigE activity, and to overexpress spo0A gene; or,
A microorganism that has been modified to exhibit reduced SpoOA activity and reduced AbrB activity.   The microorganism according to claim 1, wherein one or more genes selected from the group consisting of panB, panC, panD, panE, ilvB, ilvN, ilvC, ilvD, glyA, serA, ylmA and gcv genes are overexpressed. . A process for producing a pantothenate compound comprising:
Process the pantothenate compound is cultured microorganism under conditions produced, seen including a
The microorganism is a sporulation-deficient microorganism belonging to the genus Bacillus, which overproduces a pantothenate compound,
To show reduced SigE activity;
Exhibit reduced SigE activity and overexpress the spo0A gene; or
A method that is a microorganism that has been modified to exhibit reduced SpoOA activity and reduced AbrB activity .   The method according to claim 3, further comprising the step of recovering the pantothenate compound from a cell culture medium. A method for the preparation of a sporulation-deficient microorganism belonging to the genus Bacillus, which can overproduce pantothenate compounds, comprising the following steps:
(A) a microorganism belonging to the genus Bacillus, overexpressing one or more genes selected from the group consisting of panB, panC, panD, panE, ilvB, ilvN, ilvC, ilvD, glyA, serA, ylmA and gcv genes And (b) so as to obtain a sporulation-deficient microorganism of the genus Bacillus ,
Microorganism in or introducing a mutation that causes a reduction in reducing and AbrB activity Spo0A activity Engineering as (a),
Or introducing a mutation that causes a reduction in SigE activity and overexpression of the spo0A gene in the microorganism of step (a);
Including methods. Use of a sporulation-deficient microorganism belonging to the genus Bacillus for the preparation of a pantothenate compound, the microorganism comprising:
To show reduced SigE activity;
Exhibit reduced SigE activity and overexpress the spo0A gene; or
Use, which is a microorganism that has been modified to exhibit reduced SpoOA activity and reduced AbrB activity .