WO2003023044A2 - Process for the production of l-amino acids using coryneform bacteria - Google Patents
Process for the production of l-amino acids using coryneform bacteria Download PDFInfo
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- WO2003023044A2 WO2003023044A2 PCT/EP2002/009043 EP0209043W WO03023044A2 WO 2003023044 A2 WO2003023044 A2 WO 2003023044A2 EP 0209043 W EP0209043 W EP 0209043W WO 03023044 A2 WO03023044 A2 WO 03023044A2
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- 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
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
Definitions
- the present invention provides a process for the production of L-amino acids, in particular L-methionine, using coryneform bacteria, in which the metK gene coding for S- adenosylmethionine synthetase and/or the brnQ gene coding for a transporter for branched-chain amino acids is attenuated.
- L-amino acids in particular L-methionine, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry and most particularly in animal nutrition.
- Process improvements may relate to fermentation technology measures, such as for example stirring and provision of oxygen, or the composition of the nutrient media, such as for example the sugar concentration during the fermentation, or the working-up to the product for by for example ion exchange chro atography, or the intrinsic performance properties of the microorganism itself.
- strains are obtained that are resistant to anti etabolites such as for example the methionine analogues ⁇ -methylmethionine, ethionine, norleucine, N-acetylnorleucine, S- trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1- aminocyclopentanecarboxylic acid or are auxotrophic for regulatorily important metabolites, and produce amino acids such as for example L-methionine.
- the inventors have been involved in devising new principles and procedures for improved processes for the fermentative production of L-amino acids, in particular L-methionine, using coryneform bacteria.
- the present invention provides a process for the fermentative production of L-amino acids using coryneform bacteria in which at least the nucleotide sequence coding for S-adenosylmethionine synthetase and/or the nucleotide sequence coding for a transporter for branched-chain amino acids is/are attenuated, in particular switched off or expressed at a low level.
- the present invention also provides a process for the fermentative production of L-amino acids, in which the following steps are carried out:
- the coryneform bacteria that are employed already preferably produce L-amino acids, in particular L- methionine, before the attenuation of the metK gene coding for S-adenosylmethionine synthetase and/or of the brnQ gene coding for a transporter for branched-chain amino acids.
- coryneform bacteria after attenuation of the gene coding for S-adenosylmethionine synthetase (EC: 2.5.1.6) and/or of the gene coding for a transporter for branched-chain amino acids produce L-amino acids, in particular L-methionine, in an improved manner.
- nucleotide sequence of the metK gene coding for the S-adenosylmethionine synthetase of Corynebacterium glutamicum has been deposited in the Gene Bank under
- nucleotide sequence may furthermore be obtained as SEQ ID No. 241 from patent application WO01/00843 under the Identification Code AX063959.
- the nucleotide sequence of the brnQ gene of Corynebacterium glutamicum has been published by Tauch et al . (Archives of Microbiology 169 (4): 303-312 (1998)) and may likewise be obtained from the Gene Bank under the Accession Number M89931. It may furthermore be obtained as SEQ ID 423 from patent application WOOl/00805 under the Identification Code AX066841.
- the sequences described in the specified references coding for S-adenosylmethionine synthetase or for a transporter for branched-chain amino acids may be used according to the invention.
- L-amino acids or amino acids are understood to mean one or more amino acids including their salts selected from the group comprising L-asparagine, threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L- leucine, L-tyrosine, L-phenylalanine, L-histidine, L- lysine, L-tryptophan and L-arginine.
- L-methionine is particularly preferred.
- L-methionine or methionine are understood to mean also the salts, such as for example methionine hydrochloride or methionine sulfate.
- the term "attenuation” describes in this connection the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism that are coded by the corresponding DNA, by using for example a weak promoter or a gene or allele that codes for a corresponding enzyme with a low activity or inactivating the corresponding gene or enzyme (protein) , and optionally combining these measures .
- the activity or concentration of the corresponding protein is generally 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, and/or the activity or concentration of the protein in the initial microorganism.
- the microorganisms that are provided by the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and etha ⁇ ol .
- These microorganisms may be representatives of coryneform bacteria, in particular of the genus Corynebacterium.
- Corynebacterium there should in particular be mentioned the species Corynebacterium glutamicum, which is known to the specialists in this field for its ability to produce L-amino acids .
- Suitable strains of the genus Corynebacterium in particular of the species Corynebacterium glutamicum, are in particular the following known wild type strains
- thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020
- L-amino acid-producing mutants and/or strains produced therefrom such as for example the L-methionine-producing strain
- either the expression of the gene coding for S-adenosylmethionine synthetase and/or of the gene coding for a transporter for branched- chain amino acids or the catalytic properties of the gene products may be reduced or switched off.
- both measures are combined
- the gene expression may be reduced by suitable culture conditions or by genetic alteration (mutation) of the signal structures of the gene expression.
- Signal structures of the gene expression include for example repressor genes, activator genes, operators, promoters, attentuators, ribosome binding sites, the start codon and terminators.
- repressor genes for example repressor genes, activator genes, operators, promoters, attentuators, ribosome binding sites, the start codon and terminators.
- a conventional method of mutating genes of C. glutamicum is the method of gene disruption and of gene replacement described by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991) ) .
- a central part of the coding region of the gene that is of interest is cloned in a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum.
- a host typically E. coli
- the following are for example suitable as vectors: pSUP301 (Simon et al . , Bio/Technology 1, 784-791 (1983)), pKl ⁇ mob or pKl9mob (Schafer et al . , Gene 145, 69-73 (1994)), pKl ⁇ mobsacB or pKl9mobsacB (Jager et al .
- a mutation such as for example a deletion, insertion or base replacement, is produced in vi tro in the gene that is of interest.
- the allele produced is in turn cloned in a non-replicative vector for C. glutamicum and this is then transferred by transformation or conjugation to the desired host of C. glutamicum.
- a suitable second crossover event producing an excision, the incorporation of the mutation or allele in the target gene or in the target sequence is achieved.
- This method was used for example by Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)) to switch off the pyc gene of C. glutamicum by means of a deletion.
- a deletion, insertion or a base replacement can be incorporated in this way into the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids .
- L-amino acids apart from attenuating the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids, also to enhance, in particular overexpress, one or more enzymes of the relevant biosynthesis pathway, glycolysis, anaplerosis, citric acid cycle, pentose phosphate cycle, amino acid export and optionally regulatory proteins.
- the activity or concentration of the corresponding protein is generally raised by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000%, referred to the activity or concentration of the wild type protein and/or the activity or concentration of the protein in the starting microorganism.
- one or more of the genes selected from the following group may be enhanced, in particular overexpressed: • the gene lysC coding for a feedback-resistant aspartate kinase (Accession No.P26512, EP-B-0387527; EP-A-0699759; WO 00/63388) ,
- amino acids in particular L-methionine
- amino acids in particular L-methionine
- the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids also at the same time to attenuate, in particular to reduce the expression, of one or more of the genes selected from the following group:
- microorganisms produced according to the invention are also covered by the invention and may be cultivated continuously or discontinuously in a batch process (batch cultivation) or in a fed-batch process (feed process) or repeated fed-batch process (repetitive feed process) for the purposes of producing L-amino acids .
- the culture medium to be used must satisfy in a suitable manner the requirements of the respective strains. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C. , USA, 1981).
- sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as for example soy bean oil, sunflower oil, groundnut oil and coconut oil, fatty acids such as for example palmitic acid, stearic acid and linoleic acid, alcohols such as for example glycerol and ethanol, and organic acids such as for example acetic acid.
- sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
- oils and fats such as for example soy bean oil, sunflower oil, groundnut oil and coconut oil
- fatty acids such as for example palmitic acid, stearic acid and linoleic acid
- alcohols such as for example glycerol and ethanol
- organic acids such as for example acetic acid.
- nitrogen source there may be used organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
- organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea
- inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
- the nitrogen sources may be used individually or as a mixture.
- phosphorus source there may be used phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
- the culture medium must furthermore contain salts of metals, such as for example magnesium sulfate or iron sulfate, that are necessary for growth.
- essential growth promoters such as amino acids and vitamins may be used in addition to the aforementioned substances.
- suitable precursors may be added to the culture medium.
- the aforementioned substances may be added to the culture in the form of a single batch or may be fed in an appropriate manner during the cultivation.
- basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are used as appropriate.
- acidic compounds such as phosphoric acid or sulfuric acid are used.
- antifoaming agents such as for example fatty acid polyglycol esters may be used.
- suitable selectively acting substances for example antibiotics, may be added to the medium.
- oxygen or oxygen-containing gas mixtures such as for example air are fed into the culture.
- the temperature of the culture is normally 20°C to 45°C, and preferably 25°C to 40°C. Cultivation is continued until a maximum amount of desired product has been formed. This target is normally achieved within 10 hours to 160 hours.
Abstract
The invention relates to a process for the production of L-amino acids, in which the following steps are carried out: a) fermentation of the coryneform bacteria producing the desired L-amino acid, in which at least the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids is/are attenuated, b) enrichment of the desired L-amino acid in the medium or in the bacterial cells, and c) isolation of the L-amino acid, and optionally bacteria are used in which in addition further genes of the biosynthesis pathway of the desired L-amino acid are enhanced, or bacteria are used in which the metabolic pathways that reduce the formation of the desired L-amino acid are at least partially switched off.
Description
Process for the Production of -Amino Acids using Coryneform Bacteria
The present invention provides a process for the production of L-amino acids, in particular L-methionine, using coryneform bacteria, in which the metK gene coding for S- adenosylmethionine synthetase and/or the brnQ gene coding for a transporter for branched-chain amino acids is attenuated.
Prior Art
L-amino acids, in particular L-methionine, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry and most particularly in animal nutrition.
It is known to produce amino acids by fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum. On account of their great importance efforts are constantly being made to improve the production processes . Process improvements may relate to fermentation technology measures, such as for example stirring and provision of oxygen, or the composition of the nutrient media, such as for example the sugar concentration during the fermentation, or the working-up to the product for by for example ion exchange chro atography, or the intrinsic performance properties of the microorganism itself.
In order to improve the performance properties of these microorganisms methods involving mutagenesis, selection and choice of mutants are employed. In this way strains are obtained that are resistant to anti etabolites such as for example the methionine analogues α-methylmethionine, ethionine, norleucine, N-acetylnorleucine, S- trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1-
aminocyclopentanecarboxylic acid or are auxotrophic for regulatorily important metabolites, and produce amino acids such as for example L-methionine.
For some years recombinant DNA technology methods have also been employed to improve L-amino acid producing strains of Corynebacterium glutamicum, by amplifying individual amino acid biosynthesis genes and investigating the effect on L-amino acid production.
Object of the Invention
The inventors have been involved in devising new principles and procedures for improved processes for the fermentative production of L-amino acids, in particular L-methionine, using coryneform bacteria.
Summary of the Invention
The present invention provides a process for the fermentative production of L-amino acids using coryneform bacteria in which at least the nucleotide sequence coding for S-adenosylmethionine synthetase and/or the nucleotide sequence coding for a transporter for branched-chain amino acids is/are attenuated, in particular switched off or expressed at a low level.
The present invention also provides a process for the fermentative production of L-amino acids, in which the following steps are carried out:
a) fermentation of the L-amino acid-producing coryneform bacteria, in which at least the nucleotide sequence coding for S-adenosylmethionine synthetase and/or the nucleotide sequence coding for a transporter for branched-chain amino acids is/are attenuated, in particular switched off or expressed at a low level;
b) enrichment of the L-amino acids in the medium or in the bacterial cells; and
c) isolation of the desired L-amino acids, in which optionally constituents of the fermentation broth and/or of the biomass remain in part or in total in the end product .
Detailed Description of the Invention
The coryneform bacteria that are employed already preferably produce L-amino acids, in particular L- methionine, before the attenuation of the metK gene coding for S-adenosylmethionine synthetase and/or of the brnQ gene coding for a transporter for branched-chain amino acids.
It has been found that coryneform bacteria after attenuation of the gene coding for S-adenosylmethionine synthetase (EC: 2.5.1.6) and/or of the gene coding for a transporter for branched-chain amino acids produce L-amino acids, in particular L-methionine, in an improved manner.
The nucleotide sequence of the metK gene coding for the S-adenosylmethionine synthetase of Corynebacterium glutamicum has been deposited in the Gene Bank under
Accession Number AJ290443. The nucleotide sequence may furthermore be obtained as SEQ ID No. 241 from patent application WO01/00843 under the Identification Code AX063959.
The nucleotide sequence of the brnQ gene of Corynebacterium glutamicum has been published by Tauch et al . (Archives of Microbiology 169 (4): 303-312 (1998)) and may likewise be obtained from the Gene Bank under the Accession Number M89931. It may furthermore be obtained as SEQ ID 423 from patent application WOOl/00805 under the Identification Code AX066841.
The sequences described in the specified references coding for S-adenosylmethionine synthetase or for a transporter for branched-chain amino acids may be used according to the invention. There may furthermore be used alleles of S-adenosylmethionine synthetase or of the transporter for branched-chain amino acids that are formed as a result of the degeneracy of the genetic code or due to function- neutral sense mutations .
References hereinafter to L-amino acids or amino acids are understood to mean one or more amino acids including their salts selected from the group comprising L-asparagine, threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L- leucine, L-tyrosine, L-phenylalanine, L-histidine, L- lysine, L-tryptophan and L-arginine. L-methionine is particularly preferred.
References hereinafter to L-methionine or methionine are understood to mean also the salts, such as for example methionine hydrochloride or methionine sulfate.
Preferred embodiments are disclosed in the claims.
The term "attenuation" describes in this connection the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism that are coded by the corresponding DNA, by using for example a weak promoter or a gene or allele that codes for a corresponding enzyme with a low activity or inactivating the corresponding gene or enzyme (protein) , and optionally combining these measures .
By means of these attenuation measures, including the reduction in the gene expression, the activity or concentration of the corresponding protein is generally 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, and/or the activity or concentration of the protein in the initial microorganism.
The microorganisms that are provided by the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethaηol . These microorganisms may be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Among the genus Corynebacterium there should in particular be mentioned the species Corynebacterium glutamicum, which is known to the specialists in this field for its ability to produce L-amino acids .
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the following known wild type strains
Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020
or L-amino acid-producing mutants and/or strains produced therefrom, such as for example the L-methionine-producing strain
Corynebacterium glutamicum ATCC21608.
In order to achieve an attenuation, either the expression of the gene coding for S-adenosylmethionine synthetase and/or of the gene coding for a transporter for branched- chain amino acids or the catalytic properties of the gene
products may be reduced or switched off. Optionally both measures are combined
The gene expression may be reduced by suitable culture conditions or by genetic alteration (mutation) of the signal structures of the gene expression. Signal structures of the gene expression include for example repressor genes, activator genes, operators, promoters, attentuators, ribosome binding sites, the start codon and terminators. The person skilled in the art may find relevant details in for example patent application WO
96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996)) and in known textbooks of genetics and molecular biology, such as for example the textbook by Knippers ("Molekulare Genetik", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or the textbook by Winnacker ("Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) .
Mutations that lead to a change and/or reduction of the catalytic properties of enzyme proteins are known from the prior art; as examples there may be mentioned the work by Qiu and Goodman (Journal of Biological Chemistry 272: 8611- 8617 (1997)), Sugimoto et al . (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mδckel ("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms") , reports published by Forschungszentrum Jϋlich, Jϋl-2906, ISSN09442952, Jϋlich, Germany, 1994). Overviews may be found in known textbooks of genetics and molecular biology, such as for example the textbook by Hage ann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .
Suitable mutations include transitions, transversions, insertions and deletions . Depending on the effect of the amino acid replacement on the enzyme activity, one talks of "missense mutations" or "nonsense mutations". Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a result of which false amino acids are incorporated or the translation is terminated prematurely. Deletions of several codons typically lead to a complete breakdown of enzyme activity. Instructions for producing such mutations are known in the prior art and may be obtained from known textbooks of genetics and molecular biology, such as for example the textbook by Knippers ("Molekulare Genetik", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) , that by Winnacker ("Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .
A conventional method of mutating genes of C. glutamicum is the method of gene disruption and of gene replacement described by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991) ) .
In the method of gene disruption a central part of the coding region of the gene that is of interest is cloned in a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum. The following are for example suitable as vectors: pSUP301 (Simon et al . , Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al . , Gene 145, 69-73 (1994)), pKlδmobsacB or pKl9mobsacB (Jager et al . , Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-T0P0 (Shuman (1994), Journal of Biological Chemistry 269:32678-84; US-Patent 5,487,993), pCR®Blunt (Fir a Invitrogen, Groningen, Netherlands; Bernard et al . , Journal of Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-
4516) . The plasmid vector that contains the central part of the coding region of the gene is then transferred by conjugation or transformation to the desired strain of C. glutamicum. The method of conjugation is described for example in Schafer et al . (Applied and Environmental
Microbiology 60, 756-759 (1994) ) . Transformation methods are described for example by Thierbach et al . (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event the coding region of the relevant gene is disrupted by the vector sequence and two incomplete alleles are obtained, in which in each case the 3' end and the 5' end are missing. This method was used for example by
Fitzpatrick et al . (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to switch off the recA gene of C. glutamicum.
In the gene replacement method a mutation, such as for example a deletion, insertion or base replacement, is produced in vi tro in the gene that is of interest. The allele produced is in turn cloned in a non-replicative vector for C. glutamicum and this is then transferred by transformation or conjugation to the desired host of C. glutamicum. After homologous recombination by means of a first crossover event producing integration and a suitable second crossover event producing an excision, the incorporation of the mutation or allele in the target gene or in the target sequence is achieved. This method was used for example by Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)) to switch off the pyc gene of C. glutamicum by means of a deletion.
A deletion, insertion or a base replacement can be incorporated in this way into the gene coding for
S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids .
In addition it may be advantageous for the production of L-amino acids , apart from attenuating the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids, also to enhance, in particular overexpress, one or more enzymes of the relevant biosynthesis pathway, glycolysis, anaplerosis, citric acid cycle, pentose phosphate cycle, amino acid export and optionally regulatory proteins.
The expressions "enhancement" and "to enhance" describe in this connection the increase of the intracellular activity of one or more enzymes or proteins in a microorganism that are coded by the corresponding DNA, by for example increasing the number of copies of the gene or genes, employing a strong promoter or a gene that codes for a corresponding enzyme or protein having a high activity, and optionally combining these measures.
By means of these enhancement, in particular overexpression measures, the activity or concentration of the corresponding protein is generally raised by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000%, referred to the activity or concentration of the wild type protein and/or the activity or concentration of the protein in the starting microorganism.
Accordingly, for the production of L-methionine, in addition to the attenuation of the gene coding for S-adenosylmethionine synthetase and/or of the gene coding for a transporter for branched-chain amino acids, one or more of the genes selected from the following group may be enhanced, in particular overexpressed:
• the gene lysC coding for a feedback-resistant aspartate kinase (Accession No.P26512, EP-B-0387527; EP-A-0699759; WO 00/63388) ,
• the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992) . Journal of Bacteriology 174:6076-6086) ,
• the gene pyc coding for pyruvat carboxylase (DE-A-198 31 609) ,
• the gene zwf coding for glucose-6-phosphate dehydrogenase (JP-A-09224661.) ,
• the gene mqo coding for malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),
• the gene zwal coding for the Zwal protein (DE: 19959328.0, DSM 13115),
• the gene tpi coding for triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
• the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
• the gene horn coding for homoserine dehydrogenase (EP-A 0131171) ,
• the gene metA coding for homoserine 0-acetyltransferase (ACCESSION Number AF052652),
• the gene metB coding for cystathionine γ-synthase (ACCESSION Number AF126953) ,
• the gene metE coding for homocysteine methyltransferase
I (DE: 10038023.9),
• the gene metH coding for homocysteine methyltransferase
II (DE: 10038050.6) ,
• the gene aecD coding for cystathionine γ-lyase (ACCESSION Number M89931) ,
• the gene glyA coding for serine hydroxymethyltransferase (JP-A-08107788) ,
• the gene metY coding for O-acetylhomoserine sulfhydrylase (DSM 13556) .
Furthermore, it may be advantageous for the production of amino acids, in particular L-methionine, in addition to the attenuation of the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids, also at the same time to attenuate, in particular to reduce the expression, of one or more of the genes selected from the following group:
• the gene thrB coding for homoserine kinase (ACCESSION Number P08210) ,
• the gene ilvA coding for threonine dehydratase (ACCESSION Number Q04513),
• the gene thrC coding for threonine synthase (ACCESSION Number P23669) ,
• the gene ddh coding for meso-diaminopimelate D- dehydrogenase (ACCESSION Number Y00151) ,
• the gene ccpAl coding for a catabolite control protein A (DE: 10042054.0),
• the gene pck coding for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047),
• the gene pgi coding for glucose-6-phosphate isomerase (US 09/396,478, DSM 12969),
• the gene poxB coding for pyruvate oxidase (DE:1995 1975.7, DSM 13114),
• the gene fda coding for fructose-bisphosphate aldolase (Mol. Microbiol. 3 (11), 1625-1637 (1989); ACCESSION
Number X17313) ,
• the gene zwa2 coding for the Zwa2 protein (DE: 19959327.2, DSM 13113).
Finally, for the production of amino acids it may be advantageous, apart from attenuating the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids, also to switch off undesirable secondary reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
The microorganisms produced according to the invention are also covered by the invention and may be cultivated continuously or discontinuously in a batch process (batch cultivation) or in a fed-batch process (feed process) or repeated fed-batch process (repetitive feed process) for the purposes of producing L-amino acids . A summary of known cultivation methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Brunswick/ Wiesbaden, 1994)).
The culture medium to be used must satisfy in a suitable manner the requirements of the respective strains. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C. , USA, 1981).
As carbon source there may be used sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose,
maltose, molasses, starch and cellulose, oils and fats such as for example soy bean oil, sunflower oil, groundnut oil and coconut oil, fatty acids such as for example palmitic acid, stearic acid and linoleic acid, alcohols such as for example glycerol and ethanol, and organic acids such as for example acetic acid. These substances may be used individually or as a mixture.
As nitrogen source there may be used organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture.
As phosphorus source there may be used phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium must furthermore contain salts of metals, such as for example magnesium sulfate or iron sulfate, that are necessary for growth. Finally, essential growth promoters such as amino acids and vitamins may be used in addition to the aforementioned substances. Apart from these, suitable precursors may be added to the culture medium. The aforementioned substances may be added to the culture in the form of a single batch or may be fed in an appropriate manner during the cultivation.
In order to regulate the pH of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are used as appropriate. In order to control foam formation antifoaming agents such as for example fatty acid polyglycol esters may be used. In order to maintain the stability of plasmids, suitable selectively acting substances, for example antibiotics, may be added to the medium. In order to maintain aerobic conditions,
oxygen or oxygen-containing gas mixtures such as for example air are fed into the culture. The temperature of the culture is normally 20°C to 45°C, and preferably 25°C to 40°C. Cultivation is continued until a maximum amount of desired product has been formed. This target is normally achieved within 10 hours to 160 hours.
Methods for the determination of L-amino acids are known from the prior art. The analysis may be carried out as described by Spackman et al . (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography followed by ninhydrin derivation, or by reversed phase HPLC as described by Lindroth et al . (Analytical Chemistry (1979) 51: 1167-1174) .
Claims
1. Process for the production of L-amino acids by fermentation of coryneform bacteria, wherein bacteria are used in which the nucleotide sequence (metK) coding for S-adenosylmethionine synthetase and/or the nucleotide sequence (brnQ) coding for a transporter for branched-chain amino acids is/are attenuated, in particular switched off or expressed at a low level.
2. Process according to claim 1, wherein L-methionine is produced.
3. Process for the fermentative production of L-amino acids, in particular L-methionine, wherein the following steps are carried out:
a) fermentation of the coryneform bacteria producing the desired L-amino acid, in which at least the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids is/are attenuated,
b) enrichment of the desired product in the medium or in the bacterial cells, and
c) isolation of the desired L-amino acids, in which optionally constituents of the fermentation broth and/or of the biomass remain in part or in total in the end product .
4. Process according to claim 1 or 3 , wherein bacteria are used in which in addition further genes of the biosynthesis pathway of the desired L-amino acid are enhanced.
5. Process according to claim 1 or 3 , wherein bacteria are used in which the metabolic pathways that reduce the formation of the desired L-amino acid are at least partially switched off.
6. Process according to claim 1 or 3, wherein the expression of the polynucleotide(s) coding for S- adenosylmethionine synthetase and/or for a transporter for branched-chain amino acids is reduced.
7. Process according to claim 1 or 3 , wherein the catalytic properties of the polypeptide(s) (enzyme protein (s)) for the nucleotide sequence (metK) coding for S-adenosylmethionine synthetase and/or the nucleotide sequence (brnQ) coding for a transporter for branched-chain amino acids, are reduced.
8. Process according to claim 1 or 3 , wherein for the production of L-methionine coryneform microorganisms are fermented in which at the same time one or more of the genes selected from the following group
8.1 the gene lysC coding for a feedback-resistant aspartate kinase,
8.2 the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase,
8.3 the gene pyc coding for pyruvate carboxylase,
8.4 the gene zwf coding for glucose-6-phosphate dehydrogenase,
8.5 the gene mqo coding for alate : quinone oxidoreductase,
8.6 the gene zwal coding for the Zwal protein,
8.7 the gene tpi coding for triosephosphate isomerase,
8.8 the gene pgk coding for 3-phosphoglycerate kinase,
8.9 the gene horn coding for homoserine dehydrogenase,
8.10 the gene metA coding for homoserine 0- acetyltransferase,
8.11 the gene metB coding for cystathionin γ-synthase,
8.12 the gene metE coding for homocysteine methyltransferase I,
8.13 the gene metH coding for homocysteine methyltransferase II,
8.14 the gene aecD coding for cystathionine γ-lyase,
8.15 the gene glyA coding for serine hydroxymethy1transferase,
8.16 the gene metY coding for O-acetylhomoserine sulfhydrylase
is/are enhanced, in particular overexpressed.
9. Process according to the claim 1 or 3 , wherein for the production of L-amino acids coryneform microorganisms are fermented in which at the same time one or more of the genes selected from the following group
9.1 the gene thrB coding for homoserine kinase,
9.2 the gene ilvA coding for threonine dehydratase,
9.3 the gene thrC coding for threonine synthase,
9.4 the gene ddh coding for meso-diaminopimelate D-dehydrogenase,
9.5 the gene ccpAl coding for a catabolite control protein A,
9.6 the pck gene coding for phosphoenolpyruvate carboxykinase,
9.7 the pgi gene coding for glucose-6-phosphate isomerase,
9.8 the gene poxB coding for pyruvate oxidase,
9.9 the gene fda coding for fructose-bisphosphate aldolase,
9.10 the gene zwa2 coding for the Zwa2 protein
is/are attenuated.
10. Process according to one or more of claims 1-9, wherein microorganisms of the species Corynebacterium glutamicum are used.
11. Coryneforme bacteria in which at least the gene coding for S-adenosylmethionine synthetase and/or the gene coding for a transporter for branched-chain amino acids is/are present in attenuated form.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2002325942A AU2002325942A1 (en) | 2001-09-11 | 2002-08-13 | Process for the production of l-amino acids using coryneform bacteria |
| EP02760318A EP1425406A2 (en) | 2001-09-11 | 2002-08-13 | Process for the production of l-amino acids using coryneform bacteria |
Applications Claiming Priority (2)
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|---|---|---|---|
| DE10144493.1 | 2001-09-11 | ||
| DE2001144493 DE10144493A1 (en) | 2001-09-11 | 2001-09-11 | Process for the fermentative production of L-amino acids using coyneform bacteria |
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| WO2003023044A2 true WO2003023044A2 (en) | 2003-03-20 |
| WO2003023044A3 WO2003023044A3 (en) | 2003-08-14 |
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| PCT/EP2002/009043 WO2003023044A2 (en) | 2001-09-11 | 2002-08-13 | Process for the production of l-amino acids using coryneform bacteria |
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|---|---|
| EP (1) | EP1425406A2 (en) |
| AU (1) | AU2002325942A1 (en) |
| DE (1) | DE10144493A1 (en) |
| WO (1) | WO2003023044A2 (en) |
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|---|---|---|---|---|
| WO2003100072A3 (en) * | 2002-05-23 | 2005-01-27 | Basf Ag | Method for the production of sulphur-containing fine chemicals by fermentation |
| EP1611232A1 (en) * | 2003-04-04 | 2006-01-04 | CJ Corp. | tdcBC/pckA GENE-INACTIVATED MICROORGANISM AND METHOD OF PRODUCING L-THREONINE USING THE SAME |
| WO2006008152A1 (en) * | 2004-07-20 | 2006-01-26 | Basf Ag | Microorganisms for producing sulphur-containing compounds |
| WO2007011845A2 (en) * | 2005-07-18 | 2007-01-25 | Basf Ag | Use of a bacillus meti gene to improve methionine production in microorganisms |
| WO2007012078A1 (en) * | 2005-07-18 | 2007-01-25 | Basf Ag | Methionine producing recombinant microorganisms |
| EP1801117A1 (en) * | 2005-12-21 | 2007-06-27 | Friesland Brands B.V. | Means and methods for regulating amino acid pools and/or transport |
| WO2010020681A1 (en) * | 2008-08-22 | 2010-02-25 | Metabolic Explorer | Producing methionine without n-acyl-methionine |
| US8143031B2 (en) | 2008-08-22 | 2012-03-27 | Metabolic Explorer | Production of N-acylated sulphur-containing amino acids with microorganisms having enhanced N-acyltransferase enzymatic activity |
| JP2014504876A (en) * | 2011-01-18 | 2014-02-27 | シージェイ チェイルジェダン コーポレーション | Microorganism having improved L-amino acid production ability, and method for producing L-amino acid using the same |
| US10208323B2 (en) | 2013-12-13 | 2019-02-19 | Basf Se | Recombinant microorganism for improved production of fine chemicals |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000139471A (en) * | 1998-11-17 | 2000-05-23 | Ajinomoto Co Inc | Production of l-methionine by fermentation |
-
2001
- 2001-09-11 DE DE2001144493 patent/DE10144493A1/en not_active Withdrawn
-
2002
- 2002-08-13 AU AU2002325942A patent/AU2002325942A1/en not_active Abandoned
- 2002-08-13 WO PCT/EP2002/009043 patent/WO2003023044A2/en not_active Application Discontinuation
- 2002-08-13 EP EP02760318A patent/EP1425406A2/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000139471A (en) * | 1998-11-17 | 2000-05-23 | Ajinomoto Co Inc | Production of l-methionine by fermentation |
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| WO2006008152A1 (en) * | 2004-07-20 | 2006-01-26 | Basf Ag | Microorganisms for producing sulphur-containing compounds |
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| JP2009501550A (en) * | 2005-07-18 | 2009-01-22 | ビーエーエスエフ ソシエタス・ヨーロピア | Methionine-producing recombinant microorganism |
| WO2007011845A3 (en) * | 2005-07-18 | 2007-04-12 | Basf Ag | Use of a bacillus meti gene to improve methionine production in microorganisms |
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Also Published As
| Publication number | Publication date |
|---|---|
| AU2002325942A1 (en) | 2003-03-24 |
| WO2003023044A3 (en) | 2003-08-14 |
| DE10144493A1 (en) | 2003-07-03 |
| EP1425406A2 (en) | 2004-06-09 |
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