Classifications

• • 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

• This invention relates to a process for the fermentative preparation of D-pantothenic acid and/or salts thereof or mixtures comprising these using microorganisms of the Bacillus group in which at least one or more of the genes or open reading frames (ORF) chosen from the group consisting of serA, serC, ywpj and glyA is or are enhanced.
• ORF open reading frames
• Pantothenic acid is produced worldwide in an order of magnitude of several thousand tonnes a year. It is used inter alia in human medicine, in the pharmaceuticals industry and in the foodstuffs industry. A large portion of the pantothenic acid produced is used for nutrition of stock animals such as poultry and pigs.
• Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by fermentation of suitable microorganisms in suitable nutrient solutions.
• chemical synthesis DL-pantolactone is an important precursor. It is prepared in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide, and in further process steps, the racemic mixture is separated, D- pantolactone is subjected to a condensation reaction with ⁇ -alanine, and D-pantothenic acid is obtained in this way.
• a typical commercial form is the calcium salt of D- pantothenic acid.
• the calcium salt of the racemic mixture of D,L-pantothenic acid is also customary.
• the advantage of the fermentative preparation by microorganisms lies in the direct formation of the desired stereoisomeric form, that is to say the D-form, which is free from L-pantothenic acid.
• Various species of bacteria such as e.g. Escherichia coli (E. coli) , Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, Corynebacterium glutamicum, Bacillus subtilis and also yeasts, such as e.g. Debaromyces castellii can produce D-pantothenic acid.
• Instructions for improving the fermentative production processes are, for example, EP-A 0 493 060, EP-A-0590857, US-A-5,518,906, WO97/10340, WO01/21772 or US-A-6, 184, 007.
• the D-pantothenic acid or the corresponding salt is isolated from the fermentation broth and purified (EP-A-0590857 and W096/33283) .
• the fermentation broth containing D-pantothenic acid can also be dried with the biomass produced during the fermentation (US-A-6, 238, 714) and then used in particular as a feedstuffs additive.
• the inventors had the object of providing new measures for improved fermentative preparation of D-pantothenic acid and/or salts thereof, and animal feedstuffs additives comprising these.
• D-pantothenic acid or pantothenic acid or pantothenate are mentioned in the following text, this means not only the free acids but also the salts of D-pantothenic acid, such as e.g. the calcium, sodium, ammonium or potassium salt .
• the invention provides a process for the fermentative preparation of D-pantothenic acid and/or salts thereof using microorganisms of the Bacillus group which in particular already produce D-pantothenic acid and in which at least one or more of the nucleotide sequence (s) which code(s) for the serA gene, serC gene, ywpJ-ORF, and glyA gene is or are enhanced, in particular over-expressed.
• the process is a process which comprises carrying out the following steps:
• the invention also provides a process in which, after conclusion of the fermentation, some or all (0 to 100%) of the biomass remains in the fermentation broth, and the broth obtained in this way is processed, optionally after concentration, to a solid mixture which comprises D- pantothenic acid and/or salts thereof and optionally comprises further constituents of the fermentation broth.
• enhancement in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, of the open reading frame (ORF) or ORFs, using a potent promoter or a gene or allele or ORF which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures.
• ORF open reading frame
• Open reading frame describes a section of a nucleotide sequence which codes or can code for a protein or polypeptide or ribonucleic acid to which no function can be assigned according to the prior art. After assignment of a function to the nucleotide sequence section in question, it is in general referred to as a gene.
• the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.
• the microorganisms which the present invention provides can produce D-pantothenic acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They are representatives of the Bacillus group, in particular of the genus Bacillus, preferably the species Bacillus subtilis.
• the Bacillus group includes, inter alia, Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus firmus, Bacillus pantothenticus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurans, Bacillus brevis, Bacillus stearothermophilus Bacillus pulvifaciens and other so-called group 1 Bacillus species which are characterized by the corresponding 16S rRNA type (Priest (1993), In: Bacillus subtilis and Other Gram- Positive Bacteria, eds . Sonenshein et al., ASM, Washington, D.C., USA) .
• Suitable D-pantothenic acid-producing strains of the Bacillus group in particular of the species Bacillus subtilis, are inter alia, for example, the strains mentioned in WO01/21772
• Bacillus subtilis strain PA824-2 Bacillus subtilis strain PA824-2.
• microorganisms of the Bacillus group produce D-pantothenic acid in an improved manner after enhancement, in particular over-expression, of one or more of the genes or ORFs, or of the nucleotide sequences which code for these, chosen from the group consisting of serA, serC, ywpj and glyA.
• nucleic acid sequences can be found in the databanks of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) , the nucleotide sequence databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK) or the DNA databank of Japan (DDBJ, Mishima, Japan) .
• NCBI National Center for Biotechnology Information
• EMBL European Molecular Biologies Laboratories
• DDBJ Mishima, Japan
• genes or open reading frames described in the text references mentioned can be used according to the invention. Alleles of the genes or open reading frames which result from the degeneracy of the genetic code or due to sense mutations of neutral function can furthermore be used.
• nucleic acids or polynucleotides which code for proteins or polypeptides which are identical, homologous or similar to the extent of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, originate from strains of the Bacillus group and have the corresponding function can be used.
• the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated.
• Expression cassettes which are incorporated upstream of the structural gene act in the same way.
• inducible promoters it is additionally possible to increase the expression in the course of fermentative D-pantothenic acid production.
• the expression is likewise improved by measures to prolong the life of the m-RNA.
• the enzyme activity is also increased by preventing the degradation of the enzyme protein.
• the genes or gene constructs can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.
• a strain transformed with one or more plasmid vectors, in particular expression vectors, wherein the plasmid vector (s) carries (carry) at least one of the nucleotide sequences which code for the genes or open reading frames serA, serC, ywpj and glyA, can be employed in a process according to the invention.
• the ribosome binding sites of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA can be optimized.
• an additional promoter can be placed in front of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA.
• At least one further copy of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA can be added to the chromosome of the host.
• D- pantothenic acid with strains of the Bacillus group, in addition to enhancement of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA, for one or more of the genes chosen from the group consisting of
• panE gene which codes for ketopantoate reductase WO/0121772
• polypeptide coded by the open reading frame ylbQ or the apbA gene Kanunst et . al . Nature 20; 390 (6657) : 249- 256 (1997); Accession No. Z99111
• panB gene which codes for ketopantoate hydroxymethyltransferase (Sorokin et al., Microbiology 142:2005-2016 (1996); WO01/21772; Accession No. : L47709)
• panD gene which codes for aspartate 1-decarboxylase (Sorokin et al., Microbiology 142:2005-2016 (1996);
• panC gene which codes for pantothenate synthetase
• D- pantothenic acid with strains of the Bacillus group, in addition to enhancement of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA, or nucleotide sequences which code for these, for one or more of the genes or open reading frames chosen from the group consisting of
• the term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele or ORF which codes for a corresponding enzyme (protein) with a low activity or inactivates the corresponding gene or ORF or enzyme (protein) and optionally combining these measures.
• the reduction in gene expression can take place by suitable culturing, by genetic modification (mutation) of the signal structures of gene expression or also by the antisense-RNA technique.
• Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators.
• Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, " issense mutations” or "nonsense mutations” are referred to.
• Insertions or deletions of at least one base pair in a gene lead to "frame shift mutations", which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. If a stop codon is formed in the coding region as a consequence of the mutation, this also leads to a premature termination of the translation Deletions of several codons typically lead to a complete loss of the enzyme activity.
• the activity or concentration of the corresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.
• D- pantothenic acid in addition to over-expression of one or more of the genes or open reading frames chosen from the group consisting of serA, serC, ywpJ and glyA, to eliminate undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
• Bacteria in which the metabolic pathways which reduce the formation of D- pantothenic acid are at least partly eliminated can be employed in the process according to the invention.
• the microorganisms produced according to the invention can be cultured in the batch process (batch culture) , the fed batch (feed process) or the repeated fed batch process (repetitive feed process) .
• batch culture the fed batch
• feed process the fed batch
• repetitive feed process the repeated fed batch process
• the culture medium to be used must meet the requirements of the particular strains in a suitable manner.
• Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea
• inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate., ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen.
• the sources of nitrogen can be used individually or as a mixture.
• Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus.
• the culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins, can be employed in addition to the abovementioned substances.
• Precursors of pantothenic acid, such as aspartate, ⁇ - alanine, ketoisovalerate, ketopantoic acid or pantoic acid and optionally salts thereof, can moreover be added to the culture medium.
• the starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
• Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture.
• alkaline earth metal salts of pantothenic acid in particular the calcium salt or magnesium salt
• an inorganic compound containing an alkaline earth metal such as, for example, calcium hydroxide or MgO
• an organic compound such as the alkaline earth metal salt of an organic acid, for example calcium acetate
• the cation necessary for preparation of the desired alkaline earth metal salt of D- pantothenic acid is introduced into the fermentation broth directly in the desired amount, preferably in an amount of 0.95 to 1.1 equivalents.
• the salts can also be formed after conclusion of the fermentation by addition of the inorganic or organic compounds to the fermentation broth, from which the biomass has optionally been removed beforehand.
• Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids.
• oxygen or oxygen- containing gas mixtures such as e.g. air, are introduced into the culture.
• the temperature of the culture is usually 15°C to 95°C, in particular 15°C to 70°C, , referably 20°C to 55°C, very particularly preferably 30°C to 50°C or 30°C to 45°C. Culturing is continued until a maximum of D-pantothenic acid has formed. This target is usually reached within 10 hours to 160 hours.
• the D-pantothenic acid or the corresponding salts of D- pantothenic acid contained in the fermentation broth can then be isolated and purified in accordance with the prior art.
• the fermentation broths comprising D-pantothenic acid and/or salts thereof preferably first to be freed from all or some of the biomass by known separation methods, such as, for example, centrifugation, filtration, decanting or a combination thereof.
• separation methods such as, for example, centrifugation, filtration, decanting or a combination thereof.
• the biomass in its entirety in the fermentation broth.
• the suspension or solution is preferably concentrated and then worked up to a powder, for example with the aid of a spray dryer or a freeze-drying unit. This powder in then in general converted by suitable compacting or granulating processes, e. g.
• auxiliary substances or carriers such as starch, gelatine, ' cellulose derivatives or similar substances, such as are conventionally used as binders, gelling agents or thickeners in foodstuffs or feedstuffs processing, or further substances, such as, for example, silicas, silicates or stearates.
• the fermentation product with or without further of the conventional fermentation constituents, can be absorbed on to an organic or inorganic carrier substance which is known and conventional in feedstuffs processing, such as, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or stabilized with conventional thickeners or binders.
• feedstuffs processing such as, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or stabilized with conventional thickeners or binders.
• D-Pantothenic acid and/or the desired salt of D-pantothenic acid or a formulation comprising these compounds is optionally added in a suitable process stage during or after the fermentation in order to achieve or establish the content of pantothenic acid desired in the product or the desired salt.
• the desired content of pantothenic acid and/or the desired salt is in general in the range from 20 to 80 wt.% (based on the total dry weight) .
• pantothenic acid can be determined with known chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) or microbiological methods, such as e.g. the .Lactobacillus plantarum test (DIFCO MANUAL, 10 th Edition, p. 1100-1102; Michigan, USA) .

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