WO2003054179A1 - Alleles of the siga gene from coryneform bacteria - Google Patents

Alleles of the siga gene from coryneform bacteria Download PDF

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Publication number
WO2003054179A1
WO2003054179A1 PCT/EP2002/011561 EP0211561W WO03054179A1 WO 2003054179 A1 WO2003054179 A1 WO 2003054179A1 EP 0211561 W EP0211561 W EP 0211561W WO 03054179 A1 WO03054179 A1 WO 03054179A1
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lysine
amino acid
coryneform bacteria
gene
siga
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PCT/EP2002/011561
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French (fr)
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Brigitte Bathe
Stephan Hans
Caroline Reynen
Walter Pfefferle
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Degussa Ag
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Priority to DE60218215T priority Critical patent/DE60218215T2/en
Priority to US10/498,887 priority patent/US7524657B2/en
Priority to AU2002337163A priority patent/AU2002337163A1/en
Priority to EP02772383A priority patent/EP1456363B1/en
Publication of WO2003054179A1 publication Critical patent/WO2003054179A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the invention provides alleles of the sigA gene from coryneform bacteria which code for variants of sigma factor A and a process for the fermentative preparation of L- lysine using bacteria which contain these alleles.
  • the amino acid L-lysine is used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
  • amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply 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 form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
  • fermentation measures such as, for example, stirring and supply of oxygen
  • the composition of the nutrient media such as, for example, the sugar concentration during the fermentation
  • the working up to the product form by, for example, ion exchange chromatography or the intrinsic output properties of the microorganism itself.
  • Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms.
  • Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner.
  • a known antimetabolite is the lysine analogue S- (2-aminoethyl) -L-cysteine (AEC) .
  • Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
  • nucleotide sequence of the gene which codes for sigma factor A from Corynebacterium glutamicum can be found in the patent application EP-A-1108790 as sequence no. 2100 and as sequence no. 7065.
  • nucleotide sequence is also deposited in the databank of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under Accession Number AX122184 and under Accession Number AX127149.
  • NCBI National Center for Biotechnology Information
  • the inventors had the object of providing new measures for improved fermentative preparation of L-lysine.
  • the invention provides replicatable nucleotide sequences (DNA) which originate from coryneform bacteria, in particular Corynebacterium glutamicum, and code for sigma factor A, wherein the associated amino acid sequences in SEQ ID No. 2 contains any proteinogenic amino acid excluding L-alanine at position 414.
  • DNA replicatable nucleotide sequences
  • coryneform bacteria in particular Corynebacterium glutamicum
  • code for sigma factor A wherein the associated amino acid sequences in SEQ ID No. 2 contains any proteinogenic amino acid excluding L-alanine at position 414.
  • the invention furthermore provides a replicatable nucleotide sequence (DNA) which originates from coryneform bacteria, in particular Corynebacterium glutamicum, and codes for sigma factor A, wherein the associated amino acid sequence contains L-valine at position 414, shown in SEQ ID No. 4.
  • the invention furthermore provides a replicatable nucleotide sequence (DNA) which originates from coryneform bacteria, in particular Corynebacterium glutamicum, and codes for sigma factor A, the base sequence of which contains thymine at position 1241, shown in SEQ ID No. 3.
  • the invention furthermore provides plasmids (vectors) which contain the nucleotide sequences according to the invention and optionally replicate in coryneform bacteria.
  • the invention furthermore provides coryneform bacteria which contain the nucleotide sequences according to the invention and in which the nucleotide sequences which code for sigma factor A are optionally present in over-expressed form, wherein the associated amino acid sequences contain another proteinogenic amino acid at position 414 of SEQ ID No. 2.
  • Over-expression is understood as meaning an increase in the intracellular concentration or activity of the sigma factors A according to the invention.
  • 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 the activity or concentration of the protein in the starting microorganism.
  • Sigma factor A is a transcription factor which mediates the binding of RNA polymerase to specific sites (initiation sites) of the DNA and initiates the start (initiation) of transcription. It participates in the initiation of transcription a large number of genes, for example the genes horn, which codes for homoserine dehydrogenase, gap, which codes for glyceraldehyde 3-phosphate dehydrogenase, fda, which codes for fructose bisphosphate aldolase, and pgk, which codes for phosphoglycerate kinase (Patek et al., Microbiology 143: 1297-1309 (1996).
  • 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 L-lysine 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.
  • Plasmids which are replicated in coryneform bacteria are suitable for increasing the number of copies of the sigA alleles according to the invention.
  • Numerous known plasmid vectors such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHMl519, pBLl or pGAl .
  • Other plasmid vectors such as e.g.
  • chromosomal gene amplification such as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon, can furthermore be used to increase the number of copies .
  • the complete gene or allele is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum.
  • Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKl ⁇ mob or pKl9mob (Schafer et al .
  • the plasmid vector which contains the gene or allele to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation.
  • the method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362
  • the increase in protein concentration is detectable via 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel with appropriate evaluation software.
  • a common method for preparation of the protein gels in the case of coryneform bacteria and for identification of the proteins is the procedure described by Hermann et al . (Electrophoresis, 22:1712-23 (2001)).
  • the protein concentration can also be analysed by western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al . , Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989) and subsequent optical evaluation with appropriate software for determination of the concentration (Lohaus and Meyer (1998) Biospektrum 5:32-39; Lottspeich (1999) Angewandte Chemie
  • DNA-binding proteins can be measured by means of DNA band shift assays (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology 183:2151-2155).
  • DNA band shift assays also called gel retardation
  • the effect of DNA-binding proteins on the expression of other genes can be detected by various well- described methods of reporter gene assay (Sambrook et al . , Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • the invention provides replicatable, preferably endogenous nucleotide sequences (DNA) which originate from coryneform bacteria and code for the protein sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid, in particular L-valine, shown in SEQ ID No. 4.
  • DNA replicatable, preferably endogenous nucleotide sequences
  • L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid, in particular L-valine, shown in SEQ ID No. 4.
  • the invention also provides replicatable, preferably endogenous nucleotide sequences (DNA) which originate from coryneform bacteria and code for the protein sigma factor A, the associated base sequence of which contains thymine at position 1241, shown in SEQ ID No. 3.
  • DNA replicatable, preferably endogenous nucleotide sequences
  • Endogenous genes or “endogenous nucleotide sequences” are understood as meaning the genes or nucleotide sequences present in the population of a species .
  • the invention also provides vectors (plasmids) which contain the nucleotide sequences mentioned and optionally replicate in coryneform bacteria.
  • Coryneform bacteria which preferably contain the nucleotide sequence (s) mentioned according to the nucleotide sequences which code for sigma factor A in an over-expressed form are also claimed.
  • the invention provides a process for the preparation of L- lysine or feedstuffs additives comprising L-lysine in which in general the following steps are carried out:
  • the alleles of the endogenous sigA gene are over- expressed under conditions suitable for the formation of the sigA gene product sigma factor A,
  • Proteinogenic amino acids are to be understood as meaning all amino acids which are constituents of proteins or polypeptides. These are, in particular: L-aspartic acid, L- asparagine, L-threonine, L-serine, L-glutamic acid, L- glutamine, glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan, L- proline and L-arginine.
  • the wild-type form of the sigA gene is contained in wild- type strains of coryneform bacteria, in particular of the genus corynebacterium. It is shown in SEQ ID No. 1.
  • the wild-type protein is shown in SEQ ID No. 2.
  • thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020.
  • Strains with the designation "ATCC” can be obtained from the American Type Culture Collection (Manassas, VA, USA) . Strains with the designation “FERM” can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) . The strain of Corynebacterium thermoaminogenes mentioned (FERM BP-1539) and others (FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5,250,434.
  • sigA alleles according to the invention which code for variants of sigma factor A characterized by an amino acid exchange at position 414 of SEQ ID No. 2, mutagenesis methods described in the prior art are used.
  • the sigA gene described in the prior art is amplified starting from isolated complete DNA of a wild-type strain with the aid of the polymerase chain reaction, optionally cloned in suitable plasmid vectors, and the DNA is then subjected to the mutagenesis process.
  • Instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) .
  • Suitable sigA alleles are then selected by the processes described above and investigated.
  • the invention provides a new sigA allele which codes for a variant of sigma factor A and is shown in SEQ ID No. 3.
  • the sigA alleles according to the invention can be transferred into suitable strains by the method of gene replacement, such as is described by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)) or Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)).
  • the corresponding sigA allele is cloned here in a vector which is not replicative for C.
  • glutamicum such as, for example, pKl ⁇ mobsacB or pKl9mobsacB (JSger et al., Journal of Bacteriology 174: 5462-65 (1992)) or pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) and this is then transferred into the desired host of C. glutamicum by transformation or conjugation. After homologous recombination by means of a first "cross-over" event which effects integration and a suitable second "cross-over" event which effects excision in the target gene or in the target sequence, the incorporation of the mutation is achieved.
  • L-amino acids may be advantageous for the production of L-amino acids at the same time to enhance, in particular over-express one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the use of the sigA allele according to the invention.
  • the use of endogenous genes is in general preferred.
  • Endogenous genes or “endogenous nucleotide sequences” are understood as meaning the genes or nucleotide sequences and alleles thereof present in the population of a species.
  • the term "enhancement" in this connection describes the increase in the intracellular activity or concentration 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, using a potent promoter or using a gene or allele which codes for a corresponding enzyme or protein having a high activity, and optionally combining these measures.
  • An increase in the activity of the corresponding enzyme protein can also be effected by a reduced sensitivity to inhibitors.
  • 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.
  • 6-phosphogluconate dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-proline for L-serine, L- leucine, L-isoleucine or L-threonine at position 158 of the enzyme protein and/or by exchange of L-serine for L- phenylalanine or L-tyrosine at position 361 of the enzyme protein.
  • amino acid exchanges such as, for example, by exchange of L-proline for L-serine, L- leucine, L-isoleucine or L-threonine at position 158 of the enzyme protein and/or by exchange of L-serine for L- phenylalanine or L-tyrosine at position 361 of the enzyme protein.
  • the enhancement of the glucose 6-phosphate dehydrogenase sub-unit can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-serine by L-phenylalanine or L-tyrosine at position 312 of the enzyme protein.
  • Attenuation in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme or protein, and optionally combining these measures.
  • 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.
  • the attenuation of isopropyl malate dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-glycine for L-aspartate, L- asparagine or L-glutamate at position 131 of the enzyme protein.
  • the attenuation of isopropyl malate dehydratase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-arginine for L-serine at position 451 or L-glycine for L-aspartate at position 456 of the enzyme protein or a combination thereof.
  • homoserine dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-asparagine for L-threonine or L- serine at position 118 or L-leucine for L-proline at position 160 of the enzyme protein or a combination thereof.
  • the attenuation of phosphofructokinase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-leucine for L-alanine, L-glycine or L-proline at position 109 of the enzyme protein.
  • the invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids.
  • the culture medium to be used must meet the requirements of the particular strains in a suitable manner. 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 e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat, 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, can be used as the source of carbon. These substances can be used individually or as a mixture.
  • oils and fats such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat
  • 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
  • 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, for example, 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.
  • Suitable precursors 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.
  • Antifoams such as, for example, fatty acid polyglycol esters, can be employed to control the development of foam.
  • Suitable substances having a selective action such as, for example, antibiotics, can be added to the medium to maintain the stability of plasmids.
  • oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture.
  • the temperature of the culture is usually 20 B C to 45 a C, and preferably 25 a C to 40°C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.
  • the process according to the invention is used for fermentative preparation of L-lysine.
  • the concentration of L-lysine can optionally be adjusted to the desired value by addition of L-lysine.
  • the Corynebacterium glutamicum strain DM1547 was prepared by multiple, non-directed mutagenesis, selection and mutant selection from C. glutamicum ATCC13032.
  • the strain is resistant to the lysine analogue S- (2-aminoethyl) -L- cysteine and methionine-sensitive.
  • chromosomal DNA is isolated by the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA section which carries the sigA gene or allele is amplified.
  • sequence of the sigA gene known for C. glutamicum sequence no. 2100 and sequence no. 7065 from EP1108970
  • primer oligonucleotides are chosen for the PCR:
  • sigA-1 (SEQ ID No. 8):
  • sigA-2 (SEQ ID No. 9) :
  • the primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA section of approx. 1.89 kb in length, which carries the sigA allele.
  • the amplified DNA fragment of approx. 1.89 kb in length which carries the sigA allele of the strain DM1547 is identified by electrophoresis in a 0.8% agarose gel, isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • the nucleotide sequence of the amplified DNA fragment or PCR product is determined by sequencing by MWG Biotech (Ebersberg, Germany) .
  • the sequence of the PCR product is shown in SEQ ID No. 5.
  • the sequence of the coding region is shown again in SEQ ID No. 3.
  • the amino acid sequences of the associated sigma factor A protein resulting with the aid of the Patentin program are shown in SEQ ID No. 6 and 4.
  • amino acid valine SEQ ID No. 6 and 4
  • amino acid alanine SEQ ID No. 2
  • the sigA allele which contains the base thymine at position 1241 of the coding region and accordingly codes for a sigma factor A which contains the amino acid valine at position 414 of the amino acid sequence, is called the sigA_A414V allele in the following.
  • A represents L-alanine
  • V represents L-valine
  • 414 indicates the position of the amino acid exchange (see SEQ ID No. 2 and 4).
  • a DNA section which carries the region of the sigA_A414V allele on which the mutation A414V is located is amplified with the aid of the polymerase chain reaction.
  • the following primer oligonucleotides are chosen for the PCR such that the mutation A414V is located in the central region of the amplification product:
  • sigA_XL-Al (SEQ ID No. 10):
  • sigA_XL-A2 (SEQ ID No. 11):
  • the primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR protocols. A guide to methods and applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA section approx.
  • the primers moreover contain the sequence for a cleavage site of the restriction endonuclease EcoRI, which is marked by underlining in the nucleotide sequence shown above.
  • the amplified DNA fragment of approx. 1.69 kb in length which carries the sigA allele of the strain DM1547 is cleaved with the restriction endonuclease EcoRI, identified by electrophoresis in a 0.8% agarose gel and then isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • the mobilizable cloning vector pKl ⁇ mobsacB is digested with the restriction enzyme EcoRI and the ends are dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) .
  • the vector prepared in this way is mixed with the sigA_A414V fragment of approx. 1.68 kb and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) .
  • the E. coli strain S17-1 (Simon et al., Bio/Technologie [Bio/Technology] 1: 784-791, 1993) is then transformed with the ligation batch (Hanahan, In. DNA cloning. A practical approach. Vol.l. ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor, New York, 1989) , which was supplemented with 25 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage with the enzyme BamHI and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl8mobsacB_sigA_A414V and is shown in figure 1.
  • the vector pKl8mobsacB_sigA_A414V mentioned in example 2.2 is transferred by conjugation by a protocol of Schafer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the C. glutamicum strain DSM5715.
  • the vector cannot replicate independently in DSM5715 and is retained in the cell only if it is present integrated in the chromosome as the consequence of a recombination event. Selection of transconjugants, i.e.
  • clones with integrated pKl8mobsacB_sigA_A414V is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: a laboratory manual. 2 nd Ed., Cold Spring Harbor, New York, 1989) , which is supplemented with 15 mg/1 kanamycin and 50 mg/1 nalidixic acid. Kanamycin-resistant transconjugants are plated out on LB agar plates with 25 mg/1 kanamycin and incubated for 24 hours at 33°C.
  • the clones are cultured unselectively for 30 hours in LB liquid medium and then plated out on LB agar with 10% sucrose and incubated for 16 hours.
  • the plasmid pKl8mobsacB_sigA_A414V contains, in addition to the kanamycin resistance gene, a copy of the sacB gene which codes for levan sucrase from Bacillus subtilis.
  • the expression which can be induced by sucrose leads to the formation of levan sucrase, which catalyses the synthesis of the product levan, which is toxic to C. glutamicum.
  • Only those clones in which the integrated pKl8mobsacB_sigA_A414V has excised as the consequence of a second recombination event therefore grow on LB agar.
  • allele exchange or incorporation of the mutation takes place with the excision, or the original copy remains in the chromosome of the host.
  • Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose” and "non- growth in the presence of kanamycin” .
  • a region of the sigA gene spanning the mutation A414V is sequenced, starting from the sequencing primer sA_l (SEQ ID No. 12) , by GATC Biotech AG (Constance, Germany) to demonstrate that the mutation of the sigA_A414V allele is present in the chromosome.
  • the primer sA_l used is synthesized for this by GATC:
  • SA_1 (SEQ ID No. 12) :
  • the C. glutamicum strain DSM5715sigA_A414V obtained in example 2 is cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant is determined.
  • the strain is first incubated on an agar plate for 24 hours at 33 a C.
  • a preculture is seeded (10 ml medium in a 100 ml conical flask) .
  • the medium MM is used as the medium for the preculture.
  • the preculture is incubated for 24 hours at 33 a C at 240 rpm on a shaking machine.
  • a main culture is seeded from this preculture such that the initial OD (660 nm) of the main culture is 0.1.
  • the Medium MM is also used for the main culture.
  • the CSL corn steep liquor
  • MOPS morpholinopropanesulfonic acid
  • the salt solution is brought to pH 7 with aqueous ammonia and autoclaved.
  • Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Culturing is carried out at 33 a C and 80% atmospheric humidity.
  • the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Kunststoff) .
  • the amount of lysine formed is determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
  • Figure 1 Map of the plasmid pKl8mobsacB_sigA_A414V.
  • Kan Kanamycin resistance gene
  • EcoRI Cleavage site of the restriction enzyme EcoRI
  • sacB sacB gene
  • RP4-mob mob region with the replication origin for the transfer (oriT)

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Abstract

The invention relates to alleles of the sigA gene from coryneform bacteria which code for sigma factors A and a process for the fermentative preparation of L-lysine using bacteria which contain these alleles.

Description

Alleles of the sigA gene from coryneform bacteria
Field of the Invention
The invention provides alleles of the sigA gene from coryneform bacteria which code for variants of sigma factor A and a process for the fermentative preparation of L- lysine using bacteria which contain these alleles.
Prior art
The amino acid L-lysine is used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply 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 form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner. A known antimetabolite is the lysine analogue S- (2-aminoethyl) -L-cysteine (AEC) .
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
The nucleotide sequence of the gene which codes for sigma factor A from Corynebacterium glutamicum can be found in the patent application EP-A-1108790 as sequence no. 2100 and as sequence no. 7065.
The nucleotide sequence is also deposited in the databank of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under Accession Number AX122184 and under Accession Number AX127149.
Object of the invention
The inventors had the object of providing new measures for improved fermentative preparation of L-lysine.
Summary of the invention
When L-lysine or lysine are mentioned in the following, not only the bases but also the salts, such as e.g. lysine monohydrochloride or lysine sulfate, are meant by this.
The invention provides replicatable nucleotide sequences (DNA) which originate from coryneform bacteria, in particular Corynebacterium glutamicum, and code for sigma factor A, wherein the associated amino acid sequences in SEQ ID No. 2 contains any proteinogenic amino acid excluding L-alanine at position 414.
The invention furthermore provides a replicatable nucleotide sequence (DNA) which originates from coryneform bacteria, in particular Corynebacterium glutamicum, and codes for sigma factor A, wherein the associated amino acid sequence contains L-valine at position 414, shown in SEQ ID No. 4. The invention furthermore provides a replicatable nucleotide sequence (DNA) which originates from coryneform bacteria, in particular Corynebacterium glutamicum, and codes for sigma factor A, the base sequence of which contains thymine at position 1241, shown in SEQ ID No. 3.
The invention furthermore provides plasmids (vectors) which contain the nucleotide sequences according to the invention and optionally replicate in coryneform bacteria.
The invention furthermore provides coryneform bacteria which contain the nucleotide sequences according to the invention and in which the nucleotide sequences which code for sigma factor A are optionally present in over-expressed form, wherein the associated amino acid sequences contain another proteinogenic amino acid at position 414 of SEQ ID No. 2.
Over-expression is understood as meaning an increase in the intracellular concentration or activity of the sigma factors A according to the invention.
By over-expression measures, 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 the activity or concentration of the protein in the starting microorganism.
Sigma factor A is a transcription factor which mediates the binding of RNA polymerase to specific sites (initiation sites) of the DNA and initiates the start (initiation) of transcription. It participates in the initiation of transcription a large number of genes, for example the genes horn, which codes for homoserine dehydrogenase, gap, which codes for glyceraldehyde 3-phosphate dehydrogenase, fda, which codes for fructose bisphosphate aldolase, and pgk, which codes for phosphoglycerate kinase (Patek et al., Microbiology 143: 1297-1309 (1996).
To achieve an over-expression, 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. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative L-lysine production. The expression is likewise improved by measures to prolong the life of the m- RNA. Furthermore, 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.
Plasmids which are replicated in coryneform bacteria are suitable for increasing the number of copies of the sigA alleles according to the invention. Numerous known plasmid vectors, such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHMl519, pBLl or pGAl . Other plasmid vectors, such as e.g. those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119- 124 (1990)), or pAGl (US-A 5,158,891) can be used in the same manner.
The method of chromosomal gene amplification, such as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon, can furthermore be used to increase the number of copies . In this method, the complete gene or allele is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al . , Gene 145, 69- 73 (1994)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269:32678-84 (1994); US-A 5, 487 , 993 ) , pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEMl (Schrumpf et al., Journal of Bacteriology 173:4510-4516 (1991)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)). The plasmid vector which contains the gene or allele to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation 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 "cross over" event, the resulting strain contains at least two copies of the gene or allele in question.
The increase in protein concentration is detectable via 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel with appropriate evaluation software. A common method for preparation of the protein gels in the case of coryneform bacteria and for identification of the proteins is the procedure described by Hermann et al . (Electrophoresis, 22:1712-23 (2001)). The protein concentration can also be analysed by western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al . , Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989) and subsequent optical evaluation with appropriate software for determination of the concentration (Lohaus and Meyer (1998) Biospektrum 5:32-39; Lottspeich (1999) Angewandte Chemie
111:2630-2647). The activity of DNA-binding proteins can be measured by means of DNA band shift assays (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology 183:2151-2155). The effect of DNA-binding proteins on the expression of other genes can be detected by various well- described methods of reporter gene assay (Sambrook et al . , Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
The invention provides replicatable, preferably endogenous nucleotide sequences (DNA) which originate from coryneform bacteria and code for the protein sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid, in particular L-valine, shown in SEQ ID No. 4.
The invention also provides replicatable, preferably endogenous nucleotide sequences (DNA) which originate from coryneform bacteria and code for the protein sigma factor A, the associated base sequence of which contains thymine at position 1241, shown in SEQ ID No. 3.
"Endogenous genes" or "endogenous nucleotide sequences" are understood as meaning the genes or nucleotide sequences present in the population of a species .
The invention also provides vectors (plasmids) which contain the nucleotide sequences mentioned and optionally replicate in coryneform bacteria.
Coryneform bacteria which preferably contain the nucleotide sequence (s) mentioned according to the nucleotide sequences which code for sigma factor A in an over-expressed form are also claimed.
The invention provides a process for the preparation of L- lysine or feedstuffs additives comprising L-lysine in which in general the following steps are carried out:
a) fermentation of coryneform bacteria which contain endogenous nucleotide sequences which code for sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 is replaced by another proteinogenic amino acid, preferably L-valine,
the alleles of the endogenous sigA gene are over- expressed under conditions suitable for the formation of the sigA gene product sigma factor A,
b) concentration of the L-lysine in the fermentation broth,
c) isolation of the L-lysine or feedstuffs additive comprising L-lysine from the fermentation broth, optionally
d) with constituents from the fermentation broth and/or the biomass (> 0 to 100%) .
Proteinogenic amino acids are to be understood as meaning all amino acids which are constituents of proteins or polypeptides. These are, in particular: L-aspartic acid, L- asparagine, L-threonine, L-serine, L-glutamic acid, L- glutamine, glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan, L- proline and L-arginine.
The wild-type form of the sigA gene is contained in wild- type strains of coryneform bacteria, in particular of the genus corynebacterium. It is shown in SEQ ID No. 1. The wild-type protein is shown in SEQ ID No. 2.
Of the genus Corynebacterium, the species Corynebacterium glutamicum known to experts is to be mentioned in particular. Known wild-type strains of the species Corynebacterium glutamicum are, for example
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.
Strains with the designation "ATCC" can be obtained from the American Type Culture Collection (Manassas, VA, USA) . Strains with the designation "FERM" can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) . The strain of Corynebacterium thermoaminogenes mentioned (FERM BP-1539) and others (FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5,250,434.
To produce the sigA alleles according to the invention which code for variants of sigma factor A characterized by an amino acid exchange at position 414 of SEQ ID No. 2, mutagenesis methods described in the prior art are used.
Conventional in vivo mutagenesis methods using mutagenic substances, such as, for example, N-methyl-N' -nitro-N- nitrosoguanidine, or ultraviolet light can be used for the mutagenesis .
In vitro methods, such as, for example, a treatment with hydroxylamine (Miller, J. H. : A Short Course in Bacterial Genetics . A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) or mutagenic oligonucleotides (T. A. Brown: Gentechnologie fur Einsteiger [Genetic Engineering for Beginners] , Spektrum Akademischer Verlag, Heidelberg, 1993) or the polymerase chain reaction (PCR) such as is described in the handbook by Newton and Graham (PCR, Spektrum Akademischer Verlag, Heidelberg, 1994) can furthermore be used for the mutagenesis.
Further instructions on generation of mutations can be found in the prior art and in known textbooks of genetics and molecular biology, such as e.g. 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) .
If in vitro methods are used, the sigA gene described in the prior art is amplified starting from isolated complete DNA of a wild-type strain with the aid of the polymerase chain reaction, optionally cloned in suitable plasmid vectors, and the DNA is then subjected to the mutagenesis process. Instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) . Suitable sigA alleles are then selected by the processes described above and investigated.
The invention provides a new sigA allele which codes for a variant of sigma factor A and is shown in SEQ ID No. 3. The sigA alleles according to the invention can be transferred into suitable strains by the method of gene replacement, such as is described by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)) or Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)). The corresponding sigA allele is cloned here in a vector which is not replicative for C. glutamicum, such as, for example, pKlδmobsacB or pKl9mobsacB (JSger et al., Journal of Bacteriology 174: 5462-65 (1992)) or pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) and this is then transferred into the desired host of C. glutamicum by transformation or conjugation. After homologous recombination by means of a first "cross-over" event which effects integration and a suitable second "cross-over" event which effects excision in the target gene or in the target sequence, the incorporation of the mutation is achieved.
In addition, it may be advantageous for the production of L-amino acids at the same time to enhance, in particular over-express one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the use of the sigA allele according to the invention. The use of endogenous genes is in general preferred.
"Endogenous genes" or "endogenous nucleotide sequences" are understood as meaning the genes or nucleotide sequences and alleles thereof present in the population of a species.
The term "enhancement" in this connection describes the increase in the intracellular activity or concentration 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, using a potent promoter or using a gene or allele which codes for a corresponding enzyme or protein having a high activity, and optionally combining these measures. An increase in the activity of the corresponding enzyme protein can also be effected by a reduced sensitivity to inhibitors.
By enhancement measures, in particular over-expression, 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.
Thus, for the preparation of L-lysine, in addition to the use of the variants of the sigA gene, at the same time one or more of the endogenous genes chosen from the group consisting of
• the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335),
• the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
• the eno gene which codes for enolase (DE: 19947791.4),
• the tpi gene which codes for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
• the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
• the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661, EP-A-1108790),
• the pyc gene which codes for pyruvate carboxylase (DE-A- 198 31 609; EP-A1108790) , • the mqo gene which codes for malate-quinone oxidoreductase (Molenaar et al . , European Journal of Biochemistry 254, 395-403 (1998)),
• the lysC gene which codes for a feed-back resistant aspartate kinase (Accession NO.P26512; EP-B-0387527; EP- A-0699759; WO 00/63388),
• the lysE gene which codes for the lysine export protein (DE-A-195 48 222) ,
• the zwal gene which codes for the Zwal protein (DE: 19959328,0, DSM 13115)
• the gnd gene which codes for 6-phosphogluconate dehydrogenase (WO 01/71012),
• the opcA gene which codes for a sub-unit of glucose 6- phosphate dehydrogenase (sequence no. 79 from WO 01/00844; WO 01/04322),
can be enhanced, in particular over-expressed.
The enhancement of 6-phosphogluconate dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-proline for L-serine, L- leucine, L-isoleucine or L-threonine at position 158 of the enzyme protein and/or by exchange of L-serine for L- phenylalanine or L-tyrosine at position 361 of the enzyme protein.
The enhancement of the glucose 6-phosphate dehydrogenase sub-unit can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-serine by L-phenylalanine or L-tyrosine at position 312 of the enzyme protein.
It may be furthermore advantageous for the production of L- lysine, in addition to the use of the variants of the sigA gene, at the same time for one or more of the endogenous genes chosen from the group consisting of
• the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047),
• the pgi gene which codes for glucose 6-phosphate isomerase (US 09/396,478, DSM 12969),
• the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7, DSM 13114),
• the zwa2 gene which codes for the Zwa2 protein (DE: 19959327,2, DSM 13113),
• the fda gene which codes for fructose 1, 6-bisphosphate aldolase (Accession No. X17313; von der Osten et al., Molecular Microbiology 3 (11), 1625-1637 (1989)),
• the horn gene which codes for homoserine dehydrogenase (EP-A -0131171) ,
• the leuB gene which codes for isopropyl alate dehydrogenase (Patek et al., Applied Environmental Microbiology 50:43-47(1989)), Accession No. Y09578) ,
• the leuC gene which codes for isopropyl malate dehydratase (Accession No. AX121536, sequence no. 1452 from patent EP1108790, Accession No. AX063983, sequence no. 265 from patent WO0100843),
• the thrB gene which codes for homoserine kinase (Peoples, O.W., et al., Molecular Microbiology 2:63-72(1988)) and
• the pfkB gene which codes for phosphofructokinase (SEQ ID No. 57 from WO 01/00844)
to be attenuated, in particular for the expression thereof to be reduced. The term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme or protein, and optionally combining these measures.
By attenuation measures, 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.
The attenuation of isopropyl malate dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-glycine for L-aspartate, L- asparagine or L-glutamate at position 131 of the enzyme protein.
The attenuation of isopropyl malate dehydratase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-arginine for L-serine at position 451 or L-glycine for L-aspartate at position 456 of the enzyme protein or a combination thereof.
The attenuation of homoserine dehydrogenase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-asparagine for L-threonine or L- serine at position 118 or L-leucine for L-proline at position 160 of the enzyme protein or a combination thereof.
The attenuation of phosphofructokinase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by exchange of L-leucine for L-alanine, L-glycine or L-proline at position 109 of the enzyme protein.
The invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids. A summary of known culture 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, Braunschweig/Wiesbaden, 1994) ) .
The culture medium to be used must meet the requirements of the particular strains in a suitable manner. 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 e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat, 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, can be used as the source of carbon. These substances can be used individually or as a mixture.
Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or 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, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances.
Suitable precursors 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. Antifoams, such as, for example, fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as, for example, antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as, for example, air, are introduced into the culture. The temperature of the culture is usually 20BC to 45aC, and preferably 25aC to 40°C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.
Methods for the determination of L-amino acids are known from the prior art. The analysis can thus be carried out, for example, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography with subsequent ninhydrin derivatization, or it can be carried out by reversed phase HPLC, for example as described by Lindroth et al . (Analytical Chemistry (1979) 51: 1167-1174) .
The process according to the invention is used for fermentative preparation of L-lysine.
The concentration of L-lysine can optionally be adjusted to the desired value by addition of L-lysine.
The present invention is explained in more detail in the following with the aid of embodiment examples.
Example 1
Amplification and sequencing of the DNA of the sigA allele of strain DM1547
The Corynebacterium glutamicum strain DM1547 was prepared by multiple, non-directed mutagenesis, selection and mutant selection from C. glutamicum ATCC13032. The strain is resistant to the lysine analogue S- (2-aminoethyl) -L- cysteine and methionine-sensitive.
From the strain DM1547, chromosomal DNA is isolated by the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA section which carries the sigA gene or allele is amplified. On the basis of the sequence of the sigA gene known for C. glutamicum (sequence no. 2100 and sequence no. 7065 from EP1108970) , the following primer oligonucleotides are chosen for the PCR:
sigA-1 (SEQ ID No. 8):
5 tgatcggctgaccaactcta 3N
sigA-2 (SEQ ID No. 9) :
5 aaggtctcgaatccgagaac 3N
The primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA section of approx. 1.89 kb in length, which carries the sigA allele.
The amplified DNA fragment of approx. 1.89 kb in length which carries the sigA allele of the strain DM1547 is identified by electrophoresis in a 0.8% agarose gel, isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
The nucleotide sequence of the amplified DNA fragment or PCR product is determined by sequencing by MWG Biotech (Ebersberg, Germany) . The sequence of the PCR product is shown in SEQ ID No. 5. The sequence of the coding region is shown again in SEQ ID No. 3. The amino acid sequences of the associated sigma factor A protein resulting with the aid of the Patentin program are shown in SEQ ID No. 6 and 4.
At position 1241 of the nucleotide sequence of the coding region of the sigA allele of strain DM1547, that is to say at position 1466 of the nucleotide sequence shown in SEQ ID No. 5, is the base thymine. At the corresponding position of the wild-type gene is the base cytosine (SEQ ID No. 1) .
At position 414 of the amino acid sequence of sigma factor A of strain DM1547 is the amino acid valine (SEQ ID No. 6 and 4) . At the corresponding position of the wild-type protein is the amino acid alanine (SEQ ID No. 2) .
The sigA allele, which contains the base thymine at position 1241 of the coding region and accordingly codes for a sigma factor A which contains the amino acid valine at position 414 of the amino acid sequence, is called the sigA_A414V allele in the following. In the designation "sigA_A414V", A represents L-alanine, V represents L-valine and 414 indicates the position of the amino acid exchange (see SEQ ID No. 2 and 4).
Example 2
Replacement of the sigA wild-type gene of strain DSM5715 by the sigA_A414V allele
2.1. Production of a DNA fragment which carries the region of the sigA_A414V allele on which the mutation A414V is located
From the strain DM1547, chromosomal DNA is isolated by the conventional methods (Eikmanns et al., Microbiology 140:
1817 - 1828 (1994)). A DNA section which carries the region of the sigA_A414V allele on which the mutation A414V is located is amplified with the aid of the polymerase chain reaction. On the basis of the sequence of the sigA gene known for C. glutamicum (sequence no. 2100 and sequence no. 7065 from EP-A-1108790), the following primer oligonucleotides are chosen for the PCR such that the mutation A414V is located in the central region of the amplification product:
sigA_XL-Al (SEQ ID No. 10):
5V ac gaa ttc-cga egg cga tga ctt cgt ag 3N
sigA_XL-A2 (SEQ ID No. 11):
5V tg gaa ttc-cgt tec ace teg etc cat tc 3
The primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR protocols. A guide to methods and applications, 1990, Academic Press) .
The primers allow amplification of a DNA section approx.
1.69 kb in length which carries a region of the sigA_A414V allele (SEQ ID No. 7) . The primers moreover contain the sequence for a cleavage site of the restriction endonuclease EcoRI, which is marked by underlining in the nucleotide sequence shown above.
The amplified DNA fragment of approx. 1.69 kb in length which carries the sigA allele of the strain DM1547 is cleaved with the restriction endonuclease EcoRI, identified by electrophoresis in a 0.8% agarose gel and then isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
2.2. Construction of the exchange vector pKl8mobsacB_sigA_A414V
The approx. 1.68 kb long DNA fragment cleaved with the restriction endonuclease EcoRI, which contains a region of the sigA_A414V allele which carries the mutation A414V, is incorporated by means of replacement mutagenesis with the aid of the sacB system described by Schafer et al. (Gene, 14, 69-73 (1994)) into the chromosome of the C. glutamicum strain DSM5715. This system enables preparation and selection of allele exchanges which take place by homologous recombination.
The mobilizable cloning vector pKlδmobsacB is digested with the restriction enzyme EcoRI and the ends are dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) . The vector prepared in this way is mixed with the sigA_A414V fragment of approx. 1.68 kb and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) .
The E. coli strain S17-1 (Simon et al., Bio/Technologie [Bio/Technology] 1: 784-791, 1993) is then transformed with the ligation batch (Hanahan, In. DNA cloning. A practical approach. Vol.l. ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, New York, 1989) , which was supplemented with 25 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage with the enzyme BamHI and subsequent agarose gel electrophoresis. The plasmid is called pKl8mobsacB_sigA_A414V and is shown in figure 1.
2.3 Allele exchange
The vector pKl8mobsacB_sigA_A414V mentioned in example 2.2 is transferred by conjugation by a protocol of Schafer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the C. glutamicum strain DSM5715. The vector cannot replicate independently in DSM5715 and is retained in the cell only if it is present integrated in the chromosome as the consequence of a recombination event. Selection of transconjugants, i.e. clones with integrated pKl8mobsacB_sigA_A414V, is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed., Cold Spring Harbor, New York, 1989) , which is supplemented with 15 mg/1 kanamycin and 50 mg/1 nalidixic acid. Kanamycin-resistant transconjugants are plated out on LB agar plates with 25 mg/1 kanamycin and incubated for 24 hours at 33°C. For selection of mutants in which excision of the plasmid has taken place as a consequence of a second recombination event, the clones are cultured unselectively for 30 hours in LB liquid medium and then plated out on LB agar with 10% sucrose and incubated for 16 hours.
The plasmid pKl8mobsacB_sigA_A414V, like the starting plasmid pKlδmobsacB, contains, in addition to the kanamycin resistance gene, a copy of the sacB gene which codes for levan sucrase from Bacillus subtilis. The expression which can be induced by sucrose leads to the formation of levan sucrase, which catalyses the synthesis of the product levan, which is toxic to C. glutamicum. Only those clones in which the integrated pKl8mobsacB_sigA_A414V has excised as the consequence of a second recombination event therefore grow on LB agar. Depending on the position of the second recombination event with respect to the mutation site, allele exchange or incorporation of the mutation takes place with the excision, or the original copy remains in the chromosome of the host.
Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . In 4 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin", a region of the sigA gene spanning the mutation A414V is sequenced, starting from the sequencing primer sA_l (SEQ ID No. 12) , by GATC Biotech AG (Constance, Germany) to demonstrate that the mutation of the sigA_A414V allele is present in the chromosome. The primer sA_l used is synthesized for this by GATC:
SA_1 (SEQ ID No. 12) :
5" aag ttc tec ace tac gca ac 3
A clone which contains the base thymine at position 1241 of the sigA gene and thus has the sigA_A414V allele was identified in this manner. This clone was called strain DSM5715sigA_A414V.
Example 3
Preparation of lysine
The C. glutamicum strain DSM5715sigA_A414V obtained in example 2 is cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant is determined. For this, the strain is first incubated on an agar plate for 24 hours at 33 aC. Starting from this agar plate culture, a preculture is seeded (10 ml medium in a 100 ml conical flask) . The medium MM is used as the medium for the preculture. The preculture is incubated for 24 hours at 33aC at 240 rpm on a shaking machine. A main culture is seeded from this preculture such that the initial OD (660 nm) of the main culture is 0.1. The Medium MM is also used for the main culture.
Medium MM
CSL 5 g/1
MOPS 20 g/1
Glucose (autoclaved separately) 50 g/1
Salts:
(NH4)2S04 25 g/1
KH2P04 0.1 g/1
MgS04 * 7 H20 1.0 g/1
CaCl2 * 2 H20 10 mg/1
FeS04 * 7 H20 10 mg/1
MhS04 * H20 5.0mg/l
Biotin (sterile-filtered) 0.3 mg/1
Thiamine * HCl (sterile-filtered) 0.2 mg/1
L-Leucine (sterile-filtered) 0.1 g/1
CaC03 25 g/1
The CSL (corn steep liquor) , MOPS (morpholinopropanesulfonic acid) and the salt solution are brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions, as well as the CaC03 autoclaved in the dry state, are then added.
Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Culturing is carried out at 33 aC and 80% atmospheric humidity.
After 72 hours, the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The amount of lysine formed is determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
The result of the experiment is shown in table 1.
Tabelle 1
Figure imgf000026_0001
Brief description of the figure:
Figure 1: Map of the plasmid pKl8mobsacB_sigA_A414V.
The abbreviations and designations used have the following meaning. The base pair numbers stated are approximate values obtained in the context of reproducibility of measurements . Kan: Kanamycin resistance gene
EcoRI : Cleavage site of the restriction enzyme EcoRI
BamHI: Cleavage site of the restriction enzyme BamHI
'sigA: Cloned DNA fragment containing a 3 ' terminal region of the sigA allele (= sigA_A414V allele) and the downstream region
sacB: sacB gene
RP4-mob: mob region with the replication origin for the transfer (oriT)
oriV: Replication origin V

Claims

WHAT IS CLAIMED IS:
1. Replicatable nucleotide sequences (DNA) which originate from coryneform bacteria and code for the protein sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid.
2. Replicatable nucleotide sequence (DNA) according to claim 1 which originates from coryneform bacteria and codes for the protein sigma factor A, wherein the associated amino acid sequence contains the L-valine at position 414, shown in SEQ ID No. 4.
3. Replicatable nucleotide sequence (DNA) according to claim 1 which originates from coryneform bacteria and codes for the protein sigma factor A, the base sequence of which contains thymine at position 1241, shown in SEQ ID No. 3.
4. Plasmids (vectors) which contain the nucleotide sequences according to claims 1 to 3 and optionally replicate in coryneform bacteria.
5. Coryneform bacteria which contain nucleotide sequences according to claims 1 to 4 and in which the nucleotide sequences which code for sigma factor A are preferably present in over-expressed form.
6. Coryneform bacteria which contain nucleotide sequences which code for sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid.
7. Process for the preparation of L-lysine or feedstuffs additives comprising L-lysine, in which the following steps are carried out (comprising) : a) fermentation of coryneform bacteria in which alleles of the endogenous sigA gene are over- expressed under conditions which are suitable for the formation of the sigA gene product sigma factor A; and
b) isolation of the L-lysine or the feedstuffs additive comprising L-lysine from the fermentation broth, the coryneform bacteria forming the L-lysine.
8. Process according to claim 7, wherein coryneform bacteria which contain an allele of the sigA gene are employed, wherein in the associated amino acid sequences the L-alanine at position 414 of SEQ ID No. 2 is replaced by another proteinogenic amino acid.
9. Process according to claim 7, wherein microorganisms in which further genes of the biosynthesis pathway of L-lysine are additionally over-expressed are employed.
10. Process according to claim 7, wherein microorganisms in which the metabolic pathways which reduce the formation of L-lysine are at least partly eliminated are employed.
11. Process for the preparation of L-lysine or feedstuffs additives comprising L-lysine, in which the following steps are carried out:
a) fermentation of coryneform bacteria which contain endogenous nucleotide sequences which code for the protein sigma factor A, wherein in the associated amino acid sequences the L-alanine at position 414 is replaced by another proteinogenic amino acid, preferably L-valine,
b) concentration of the L-lysine in the fermentation broth, c) isolation of the L-lysine or feedstuffs additive comprising L-lysine from the fermentation broth, optionally
d) with constituents from the fermentation broth and/or the biomass (> 0 to 100%) .
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