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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • 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

• NUCLEOTIDE SEQUENCES WHICH CODE FOR THE FADD15 GENE
• the invention provides genetically modified coryneform bacteria, nucleotide sequences which code for acyl-CoA synthase and a process for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the fadD15 gene, which codes for acyl-CoA synthase, is enhanced.
• Amino acids in particular L-lysine, s used in human medicine and in the pharmaceuticals industry, but in particular 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 processes can relate to fermentation measures, such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
• fermentation measures such as e. g. stirring and supply of oxygen, or the composition of the nutrient media, such as e. g. the sugar concentration during the fermentation, or the working up to the product form by e. g. 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 such as e. g. the lysine analogue S-(2- aminoethyl) -cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e. g. L-lysine, are obtained in this manner.
• the object of the present invention was to provide new aids for improved fermentative preparation of amino acids, in particular L-lysine.
• Amino acids in particular L-lysine
• L-lysine are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
• the invention provides a genetically modified coryneform bacterium, in which its fadD15 gene, which codes for acyl- CoA synthase, is enhanced.
• the number of copies of the corresponding genes can be increased, a potent promoter can be used, 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 or L-glutamate production.
• a gene which codes for a corresponding enzyme with a high activity The expression is likewise improved by measures to prolong the life of the m-RNA.
• the enzyme activity is also increased overall by preventing the degradation of the enzyme. These measures can optionally also be combined as desired.
• Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains
• Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and L-lysine-producing mutants or strains prepared therefrom, such as, for example
• the present invention also provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
• polynucleotide which is homologous to the extent of at least 70% with a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
• polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is homologous to the extent of at least 70% with the amino acid sequence of SEQ ID No. 2,
• polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) .
• a polynucleotide sequence is "homologous" to the sequence according to the invention if it coincides in its base composition and sequence with the sequence according to the invention to the extent of at least 70%, preferably at least 80%, particularly preferably at least 90%.
• a "homologous protein” is to be understood as proteins which have an amino acid sequence which coincides with the amino acid sequence coded by the fadD15 gene (SEQ ID No.l) to the extent of at least 70%, preferably at least 80%, particularly preferably at least 90%, "coincide” being understood as meaning that either the corresponding amino acids are identical or they are amino acids which are homologous to one another. Those amino acids which correspond in their properties, in particular in respect of charge, hydrophobicity, steric properties etc., are called “homologous amino acids”.
• the invention also provides a polynucleotide as described above, this preferably being a DNA which is capable of replication, comprising:
• the invention also provides
• coryneform bacteria serving as the host cell, which contain the vector or in which the fadD15 gene is enhanced.
• the invention also provides polynucleotides which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 1 or fragments thereof, and which are obtainable by screening by means of hybridization of a corresponding gene library with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID No. 1, or a fragment thereof, and isolation of the DNA sequence mentioned.
• Polynucleotide sequences according to the invention are also suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for acyl-CoA synthase and to isolate those cDNA or genes which have a high similarity with the sequence of the acyl-CoA synthase gene.
• Polynucleotide sequences according to the invention are furthermore suitable as primers for the polymerase chain reaction (PCR) , for the preparation of DNA which codes for acyl-CoA synthase.
• PCR polymerase chain reaction
• Such oligonucleotides which serve as probes or primers can comprise more than 30, preferably up to 30, particularly preferably up to 20, especially preferably at lease 15 successive nucleotides. Oligonucleotides which have a length of at Least 40 or 50 nucleotides are also suitable.
• Polynucleotide in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
• Polypeptides is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds .
• polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of the acyl carrier protein (acyl-CoA synthase) , and also those which are homologous to the extent of at least 70% with the polypeptide according to SEQ ID No. 2, and preferably are homologous to the extent of at least 80% and in particular to the extent of at least 90% to 95% with the polypeptide according to SEQ ID No. 2, and have the activity mentioned.
• acyl carrier protein acyl-CoA synthase
• the invention moreover provides a process for the fermentative preparation of amino acids, in particular L- lysine, using coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the fadD15 gene are enhanced, in particular over-expressed.
• strain DH5 ⁇ mcr which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) .
• the long DNA fragments cloned with the aid of cosmids can then in turn be subcloned and subsequently sequenced in the usual vectors which are suitable for sequencing, such as is described e. g. by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
• the amino acid sequence of the corresponding protein has moreover been derived from the present DNA sequence by the methods described above.
• the resulting amino acid sequence of the fadD15 gene product is shown in SEQ ID No. 2.
• Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention.
• DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention.
• Conservative amino acid exchanges such as e. g. exchange of' glycine for alanine or of aspartic acid for glutamic acid in proteins, are moreover known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is moreover known that changes on the N and/or C terminus of a protein cannot substantially impair the function thereof or can even stabilize this.
• the fadD15 gene according to the invention was over-expressed with the aid of plasmids.
• the plasmid vector which contains the gene to be enhanced 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.
• amino acids in particular L-lysine
• the microorganisms prepared according to the invention 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 amino acids, in particular L- lysine.
• batch culture batch culture
• feed process feed process
• repetitive feed process repetition feed process
• a summary of known culture methods is described in the textbook by Chmiel (Bioreatechnik 1. Einf ⁇ hrung in die Biovonstechnik [Bioprocess' Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere bamboo [Bioreactors and Peripheral Equipment] (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
• Sugars and carbohydrates such as e. g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e. g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e. g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e. g. glycerol and ethanol, and organic acids, such as e. g. 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
• 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 moreover comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins, can be employed in addition to the abovementioned substances.
• 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.
• Antifoams such as e. g. fatty acid polyglycol esters, can be employed to control the development of foam.
• Suitable substances having a selective action such as e. g. antibiotics, can be added to the medium to maintain the stability of plasmids.
• oxygen or oxygen-containing gas mixtures such as e. g. air, are introduced into the culture.
• the temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C. Culturing is continued until a maximum of lysine has formed. This target is usually reached within 10 hours to 160 hours.
• L-lysine can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02) .
• the DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250) .
• the DNA of the cosmid vector SuperCosl (Wahl et al.
• the cosmid DNA treated in this manner was mixed with the treated ATCC 13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04) .
• the ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217) .
• Gigapack II XL Packing Extracts Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217
• the cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02) .
• the DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim,
• the ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) . This ligation mixture was then transformed by means of electroporation (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E.
• the raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0.
• the individual sequences of the pZerol derivatives were assembled to a continuous contig.
• the computer-assisted coding region analysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the non- redundant databank of the "National Center for Biotechnology Information” (NCBI, Bethesda, MD, USA) .
• the resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 1857 base pairs, which was called the fadD15 gene. The fadD15 gene codes for a protein of 619 amino acids (SEQ ID No.2) .
• SEQ ID No.2 The fadD15 gene codes for a protein of 619 amino acids.
• Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179). A DNA fragment which carries the fadD15 gene was amplified with the aid of the polymerase chain reaction. The following primers were used for this:
• the primers shown were synthesized by MWG Biotech
• the primers allow amplification of a DNA fragment of 2160 bp in size, which carries the fadDl5 gene from Corynejacteriuin glutamicum .
• the PCR fragment was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
• the vector pUC18 (Norrander et al., Gene (26) 101-106 (1983)) was cleaved completely with the restriction endonuclease Smal and dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
• the PCR fragment of approx. 2160 bp obtained in this manner was mixed with the prepared vector pUC18 and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27- 0870-04) .
• the ligation batch was transformed in the E. coli strain DH5 ⁇ (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA) . Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/1 ampicillin.
• Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and cleaved with the restriction enzyme EcoRI to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was called pUC18fadD15.
• the fadD15 gene was isolated from the plasmid pUC18fadD15 described in Example 3 by complete cleavage with the enzymes EcoRI and Sail.
• the fadDl5 fragment 2201 bp in size was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
• the E. coli - C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478-80) was used as the vector.
• This plasmid was cleaved completely with the restriction enzyme BamHI, treated with Klenow polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
• the fadD15 fragment obtained in this manner was mixed with the prepared vector pJCl and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04) .
• the ligation batch was transformed in the E. coli strain DH5 ⁇ (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA) . Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kanamycin.
• Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and cleaved with the restriction enzyme Xbal to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was called pJClfadD15.
• the strain DSM5715 was transformed with the plasmid pJClfadD15 using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989) ) . Selection of the transfor ants took place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/1 NaCl and 18 g/1 Bacto-agar, which had been supplemented with 25 mg/1 kanamycin. Incubation was carried out for 2 days at 33°C.
• Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927), cleaved with the restriction endonuclease Xbal, and the plasmid was checked by subsequent agarose gel electrophoresis. The resulting strain was called DSM5715/pJClfadD15.
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• the C. glutamicum strain DSM5715/pJClfadDl5 obtained in Example 5 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.
• the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/1) ) for 24 hours at 33°C.
• a preculture was seeded (10 ml medium in a 100 ml conical flask) .
• the complete medium Cglll was used as the medium for the preculture.
• Kanamycin 25 mg/1 was added to this.
• the preculture was incubated for 16 hours at 33°C at 240 rpm on a shaking machine.
• a main culture was seeded from this preculture such that the initial OD (660nm) of the main culture was 0.1.
• Medium MM was used for the main culture.
• MOPS morpholinopropanesulfonic 20 g/1 acid
• the CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved.
• the sterile substrate and vitamin solutions were then added, as well as the CaC0 3 autoclaved in the dry state.
• Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 mg/1) was added. Culturing was carried out at 33 °C and 80% atmospheric humidity.
• the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, M ⁇ nchen) .
• the amount of lysine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
• the plasmid pJCfadD15 obtained in Example 4 was used for transformation of C. glutamicum strain ATCC 13032. This strain was transformed as described in Example 5 and investigated by restriction digestion and agarose gel electrophoresis as described in Example 5. The resulting strain ATCC 13032/pJCfadDl5 was cultured in a nutrient medium suitable for determination of growth and the growth was determined at various temperatures.
• MOPS morpholinopropanesulfonic 42 g/1 acid
• Protocatechuic acid (sterile- 30 mg/1 filtered)
• the MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved.
• the sterile substrate and vitamin solutions were then added.
• the microorganism identified under I. above was accompanied by:

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