The invention provides nucleotide sequences from coryneform bacteria which code for the pck gene and a process for the fermentative preparation of L-amino acids, in particular L-Lysine and L-threonine, by attenuation of the pck gene.
Amino acids, in particular lysine and threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine.
It is known that these substances are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of its 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.
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 metabolism products of regulatory importance and produce the desired amino acid are obtained in this manner.
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 [sic].
OBJECT OF THE INVENTION
The inventors had the object of providing the general public with new measures for improved fermentative preparation of amino acids.
DESCRIPTION OF THE INVENTION
Amino acids, in particular L-lysine and L-threonine, are used in animal nutrition, in the foodstuffs industry, in the pharmaceuticals industry and in human medicine. There is therefore a general interest in providing new improved processes for the preparation of these products.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
b) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for the polypeptide mentioned and is contained on the plasmid pEK-pckA (FIG. 1 ) or pEK-pckB (FIG. 2 ),
c) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,
d) polynucleotide which is complementary to the polynucleotides of a), b) or c), and
e) polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b), c) or d).
The invention also provides a preferably recombinant DNA with Corynebacterium origin which is capable of replication in coryneform microorganisms and contains at least the nucleotide sequence which codes for the pck gene, shown in SEQ ID No. 1.
The invention also provides a DNA according to claim 1 which is capable of replication, comprising
(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and/or optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide according to claim 2, comprising the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide according to claim 2, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2,
a vector containing the polynucleotide according to claim 1, in particular pEK-pckA or pEK-pckB, shown in FIGS. 1 and 2
and coryneform bacteria serving as the host cell, into which the Δpck deletion has been incorporated.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprise the complete gene with the polynucleotide sequence corresponding to SEQ ID no. 1, 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 suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for phosphoenol pyruvate carboxykinase and to isolate those cDNA or genes which have a high similarity of sequence with that of the phosphoenol pyruvate carboxykinase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA of genes which code for phosphoenol pyruvate carboxykinase by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, especially preferably at lease 15 successive bases. Oligonucleotides which have a length of at least 40 or 50 base pairs are also suitable.
“Isolated” means separated out of its natural environment.
“Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA and DNA or modified RNA and DNA.
“Polypeptides” is understood as meaning peptides or proteins which obtain [sic] two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include the polypeptide according to SEQ ID No. 2, in particular those with the biological activity of PEP carboxykinase, and also those which are identical to the extent of at least 70% to the polypeptide according to SEQ ID No. 2, preferably to the extent of at least 80%, and in particular those which are identical to the extent of at least 90% to 95% to the polypeptide according to SEQ ID no. 2, and have the activity mentioned.
The invention also provides a process for the fermentative preparation of L-amino acids, in particular L-lysine and L-threonine, using coryneform bacteria which in particular already produce the L-amino acids and in which the nucleotide sequence(s) which code(s) for the pck gene are attenuated, in particular expressed at a low level.
The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding enzyme (protein), and optionally combining these measures.
The microorganisms which the present invention provides can produce L-amino acids, in particular lysine and threonine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among specialists for its ability to produce L-amino acids. Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium melassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-amino acid-producing mutants or strains prepared therefrom,
such as, for example, the lysine-producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM5714 or
such as, for example, the L-threonine-producing strains
Corynebacterium glutamicum ATCC21649
Brevibacterium flavum B369
Brevibacterium flavum DSM5399
Brevibacterium lactofermentum, FERM-BP 269
Brevibacterium lactofermentum TBB-10
Corynebacterium glutamicum MH20-22B-DR17.
The inventors have succeeded in isolating the new pck gene of C. glutamicum which codes for the enzyme phosphoenol pyruvate carboxykinase (PEP carboxykinase) (EC 22.214.171.124).
To isolate the pck gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in E. coli. The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einführung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bormann et al. (Molecular Microbiology 6(3), 317-326)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).
To prepare a gene library of C. glutamicum in E. coli, it is also possible to use plasmids or plasmid vectors, such as, for example, pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)), pUC9 (Vieira et al., 1982, Gene, 19:259-268), pACYC177 (Chang and Cohen, Journal of Bacteriology 134, 1141-1156 (1978)) or pSC101 (Cohen and Chang, Journal of Bacteriology 132, 734-737 (1977)). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective.
The gene library is then incorporated into an indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580, 1983) or electroporation (Tauch et.al., 1994, FEMS Microbiological Letters, 123:343-347). The indicator strain is distinguished in that it has a mutation in the gene of interest which causes a detectable phenotype. In the context of the present invention, the E. coli mutant HG4 described by Goldie and Sanwal (Journal of Bacteriology 141: 1115-1121 (1980)) is of importance. This strain carried a mutation in the pck gene, as a result of which growth on succinate as the sole source of carbon is severely impaired. By transformation with a vector which contains the pck gene, growth on succinate can be reestablished.
The long DNA fragments cloned with the aid of cosmids or other vectors can than be subcloned into known plasmid vectors in the form of shorter DNA fragments. Assignment of the gene according to the invention to a specific DNA section is made possible as a result. For this, plasmid vectors known from the prior art, such as e. g. pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or the pSU vectors described by Bartolomé et al. (Gene 102, 75-78 (1991)) are used. However, shuttle vectors which replicate both in Eschericnia coli and in Corynebacterium glutamicum, such as e. g. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or pEK0 (Eikmanns et al., Gene 102 (1991)) are preferably used, so that Investigations can be carried out in both species. Examples of these are the plasmids pEK-pckA (FIG. 1 ) and pEK-pckB FIG. 2 ), which were prepared starting from the plasmid vector pEK0 and carry the pck gene according to the invention.
The DNA sections characterized in this manner are then again subcloned into the usual vectors suitable for DNA sequencing. Alternatively, the long DNA sections cloned in cosmids can be subcloned directly into sequencing vectors. Examples of such vectors which are suitable for DNA sequencing are the plasmids pGEM-5zf(−) or pGEM-5zf(+) from the company Promega Corporation (Promega Protocols and Application Guide, Second Edition, 1991, part number Y981, Promega Corporation, Madison, Wis., USA).
Methods of DNA sequencing are described, inter alia, by Sanger et al. (Proceedings of the National of Sciences of the United States of America USA, 74:5463-5467, 1977).
The DNA sequences obtained can then be investigated with known algorithms or sequence analysis programs, such as e. g. that of Staden (Nucleic Acids Research 14, 217-232(19B6)),the CCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithm of Pearson and Tipman (Proceedings of the National Academy of Sciences USA 85,2444-2448 (1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries which exist in databanks accessible to the public. Databanks for nucleotide sequences which are accessible to the public are, for example, that of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) of that of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).
The new DNA sequence of C. glutamicum which codes for the pck gene and which, as SEQ ID No. 1, is a constituent of the present invention was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the pck 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. In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID no. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 base pairs.
Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). 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: Oligonukleotide [sic] synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
The inventors have found that coryneform bacteria produce L-amino acids, in particular lysine and threonine, in an improved manner after attenuation of the pck gene.
To achieve an attenuation, either the expression of the pck gene or the catalytic properties of the enzyme protein can be reduced or eliminated. The two measures can optionally be combined.
The reduction in gene expression can take place by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. The expert can find information on this e. g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996) and in known textbooks of genetics and molecular biology, such as e. g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genes and Clones”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).
Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]”, Reports from the Julich Research Centre, Jül-2906, ISSN09442952, Jütlich, Germany, 1994). Comprehensive description can be found in known textbooks of genetics and molecular biology, such as e. g. that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).
Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e. g. the textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986).
An example of a mutated pck gene is the Δpck allele contained in the plasmid pK19mobsacBΔpck (FIG. 3 +L) . The Δpck allele contains only the 5′ and the 3′ flanks of the pck gene; a section of the coding region 1071 bp long is missing (deletion). This Δpck allele can be incorporated into coryneform bacteria by integration mutagenesis. The abovementioned plasmid pK19mobsacBΔpck, which is not capable of replication in C. glutamicum, is used for this. After transfer by conjugation or transformation and homologous recombination by means of a first “cross-over” event which effects integration and a second “cross-over” event which effects excision in the pck gene, the incorporation of the Δpck allele is achieved and a total loss of the enzyme function in the particular strain is achieved.
Instructions and explanations on integration mutagenesis are to be found, for example, in Schwarzer and Puhler (Bio/Technology 9,84-87 (1991)) or Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)).
Examples of amino acid-producing strains of coryneforme bacteria with an attenuated pck gene are the lysine-producing strain MH20-22BΔpck and the threonine-producing strain DM368-2Δpck.
In addition, it may be advantageous for the production of L-amino acids to over-express one or more enzymes of the particular biosynthesis route, in addition to attenuation of the pck gene.
Thus, for example, for the preparation of L-lysine
at the same time the dapA gene which codes for dihydrodipicolinate synthase can be over-expressed (EP-B 0 197 335), or
at the same time a DNA fragment which imparts S-(2-aminoethyl)-cysteine resistance can be amplified (EP-A 0 088 166).
Thus, for example, for the preparation of L-threonine
at the same time the horn gene which codes for homoserine dehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72 (1988)) or the homdr or homFBR allele which codes for a “feed back resistant” homoserine dehydrogenase (Archer et al., Gene 107, 53-59 (1991); Reinscheid et al., Journal of Bacteriology 173, 3228-3230 (1991)) can be over-expressed.
In addition to attenuation of the pck gene it may furthermore be advantageous, for the production of L-amino acids, in particular lysine and threonine, to eliminate undesirable side reactions (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
The microorganisms 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 L-amino acids, in particular L-lysine and L-threonine. A summary of known culture methods are [sic] described in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [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 n 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 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, or inorganic compounds, such as ammonium sulphate, 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 he corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth. 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 culturing 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, e.g. 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 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 the desired L-amino acid has formed. This target is usually reached within 10 hours to 160 hours.
The following microorganism has been deposited at the Deutsche Sammlung für Mikrorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:
Escherichia coli strain DH5α/pK19mobsacBΔpck as DSM 13047