US20020081673A1

US20020081673A1 - Novel nucleotide sequences coding for the glbO gene

Info

Publication number: US20020081673A1
Application number: US09/813,932
Authority: US
Inventors: Bettina Mockel; Achim Marx; Walter Pfefferle
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-06-02
Filing date: 2001-03-22
Publication date: 2002-06-27
Also published as: WO2001094569A3; ATE356210T1; DE60127116T2; EP1287143A2; US6759218B2; US20030166173A1; EP1287143B1; CN1432066A; DE60127116D1; WO2001094569A2; AU2001273977A1

Abstract

Isolated polynucleotides containing at least one nucleic acid sequence selected from the group

a) polynucleotides which is at least 70% identical to a polynucleotide,

b) b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to an amino acid sequence o,

c) polynucleotide which is complementary to the polynucleotides of a) or b), and

d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),

and a method for the fermentative production of L-amino acids with amplification of the glb0 gene which codes for the hemoglobin-like protein and the use of the above polynucleotides as a primer or hybridization probe.

Description

The invention provides nucleotide sequences coding for the glb0 gene and a process for the fermentative production of L-amino acids, in particular L-lysine, using coryneform bacteria in which the glb0 gene is amplified. All references cited herein are expressly incorporated by reference. Incorporation by reference is also designated by the term “I.B.R.” following any citation.

PRIOR ART

L-amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but especially in animal nutrition.

It is known that L-amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up to yield the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.

The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant metabolites and produce L-lysine.

For some years, methods of recombinant DNA technology have also been used to improve strains of Corynebacterium which produce L-amino acids by amplifying individual biosynthesis genes for L-amino acids and investigating the effect on L-amino acid production.

Review articles on this subject may be found inter alia in Kinoshita (“Glutamic Acid Bacteria”, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.) I.B.R., Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)) I.B.R., Eggeling (Amino Acids 6:261-272 (1994)) I.B.R., Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)) I.B.R.

OBJECT OF THE INVENTION

An object of the invention is to provide new measures for improved fermentative preparation of L-amino acids, in particular L-lysine. Amino acids, in particular L-lysine, are used in human medicine, in the pharmaceuticals industry and, in particular, in animal nutrition. Therefore, there is a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine. When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e. g. lysine monohydrochloride or lysine sulfate, are also meant by this.

SUMMARY OF THE INVENTION

The new DNA sequence of C. glutamicum which codes for the glb0 gene and which as a constituent of the present invention is SEQ ID NO 1 and related sequences. The amino acid sequence of the corresponding gene product of the glb0 gene has furthermore been derived from the present DNA sequence. The resulting amino acid sequence of the glb0 gene product is SEQ ID NO 2 and related sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood with reference to the drawing offered here for illustration only and not in limitation of this invention. [0009]
FIG. 1 is a map of the plasmid pEC-K18mob2. [0010]
FIG. 2 is a map of the plasmid pEC-K18mob2glb0exp.[0011]

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group consisting of: [0012]
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID NO: 2, [0013]
b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, [0014]
c) polynucleotide which is complementary to the polynucleotides of a) or b), and [0015]
d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c). [0016]
The invention also provides a polynucleotide which is a preferably recombinant DNA replicable in coryneform bacteria. [0017]
The invention also provides a polynucleotide which is an RNA. [0018]
The invention also provides a polynucleotide as described above, wherein it preferably comprises a replicable DNA containing: [0019]
(i) the nucleotide sequence shown in SEQ ID NO: 1, or [0020]
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or [0021]
(iii) at least one sequence which hybridizes with the complementary sequence to sequence (i) or (ii) and optionally [0022]
(iv) functionally neutral sense mutations in (i). [0023]
The relative degree of substitution or mutation in the polynucleotide or amino acid sequence to produce a desired percentage of sequence identity can be established or determined by well-known methods of sequence analysis. These methods are disclosed and demonstrated in Bishop, et al. “DNA & Protein Sequence Analysis (A Practical Approach”), Oxford Univ. Press, Inc. (1997) I.B.R. and by Steinberg, Michael “Protein Structure Prediction” (A Practical Approach), Oxford Univ. Press, Inc. (1997) I.B.R. Hybridization of complementary sequences can occur at varying degrees of stringency. Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R. [0024]
Hybridization of complementary sequences can occur at varying degrees of stringency. Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R. 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)I.B.R. and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260) I.B.R. [0025]
Comprehensive descriptions 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) I.B.R. [0026]
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, as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity. [0027]
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) I.B.R., that by Winnacker (“Gene und Klone” [Genes and Clones], VCH Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R. or that by Hagemann (“Allgemeine Genetik” [General Genetics], Gustav Fischer Verlag, Stuttgart, 1986) I.B.R. [0028]
The invention also provides a vector containing one of the stated polynucleotides and coryneform bacteria acting as host cell which contain the vector or in which the glb0 gene is amplified. [0029]
The invention also provides polynucleotides which substantially consist of a polynucleotide sequence, which are obtainable by screening by means of hybridization of a suitable gene library, which contains the complete gene having the polynucleotide sequence according to SEQ ID NO: 1, with a probe which contains the sequence of the stated polynucleotide according to SEQ ID NO: 1, or a fragment thereof, and isolation of the stated DNA sequence. [0030]
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA in order to isolate full length cDNA which code for the hemoglobin-like protein (glb0) and to isolate such cDNA or genes, the sequence of which exhibits a high level of similarity with that of the gene for the hemoglobin-like protein. [0031]
Polynucleotide sequences according to the invention are furthermore suitable as primers for the production of DNA of genes which code for the hemoglobin-like protein by the polymerase chain reaction (PCR). [0032]
Such oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides having a length of at least 40 or 50 nucleotides are also suitable. [0033]
“Isolated” means separated from its natural environment. [0034]
“Polynucleotide” generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified. [0035]
“Polypeptides” are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds. [0036]
The polypeptides according to the invention include a polypeptide according to SEQ ID NO: 2, in particular those having the biological activity of the hemoglobin-like protein and also those which are at least 70% identical to the polypeptide according to SEQ ID NO: 2 and which preferably exhibit at least 80% and in particular at least 90% to 95% identity to the polypeptide according to SEQ ID NO: 2 and exhibit the stated activity. [0037]
The invention furthermore relates to a process for the fermentative production of L-amino acids, in particular L-lysine, using coryneform bacteria, which in particular already produce an L-amino acid and in which the nucleotide sequences which code for the glb0 gene are amplified, in particular overexpressed. [0038]
In this connection, the term “amplification” describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme having elevated activity and optionally by combining these measures. [0039]
The microorganisms, provided by the present invention, may produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, the species [0040] Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species [0041] Corynebacterium glutamicum, are for example the known wild type strains
[0042] Corynebacterium glutamicum ATCC13032
[0043] Corynebacterium acetoglutamicum ATCC15806
[0044] Corynebacterium acetoacidophilum ATCC13870
[0045] Corynebacterium thermoaminogenes FERM BP-1539
[0046] Corynebacterium melassecola ATCC17965
[0047] Brevibacterium flavum ATCC14067
[0048] Brevibacterium lactofermentum ATCC13869 and
[0049] Brevibacterium divaricatum ATCC14020
and L-lysine producing mutants or strains produced therefrom, such as for example [0050]
[0051] Corynebacterium glutamicum FERM-P 1709
[0052] Brevibacterium flavum FERM-P 1708
[0053] Brevibacterium lactofermentum FERM-P 1712
[0054] Corynebacterium glutamicum FERM-P 6463
[0055] Corynebacterium glutamicum FERM-P 6464 and
[0056] Corynebacterium glutamicum DSM5715.
The inventors have succeeded in isolating the novel glb0 gene which codes for the hemoglobin-like protein from [0057] C. glutamicum.
The glb0 gene or also other genes from [0058] C. glutamicum are isolated by initially constructing a gene library of this microorganism in E. coli. The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Eine Elnrührung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) I.B.R. or the manual by Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R. One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) I.B.R. in λ-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) I.B.R. describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) I.B.R. in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575) I.B.R. Börmann et al. (Molecular Microbiology 6(3), 317-326, 1992)) I.B.R. also describe a gene library of C. glutamicum ATCC 13032, using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)) I.B.R. A gene library of C. glutamicum in E. coli may also be produced using plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) I.B.R. or pUC9 (Vieira et al., 1982, Gene, 19:259-268) I.B.R. Suitable hosts are in particular those E. coli strains with restriction and recombination defects. One example of such a strain is the strain DH5αMCR, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) I.B.R. The long DNA fragments cloned with the assistance of cosmids may then in turn be sub-cloned in usual vectors suitable for sequencing and then be sequenced, as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977) I.B.R.
The novel DNA sequence from [0059] C. glutamicum which codes for the glb0 gene and, as SEQ ID NO: 1, is provided by the present invention, was obtained in this manner. The amino acid sequence of the corresponding protein was furthermore deduced from the above DNA sequence using the methods described above. SEQ ID NO: 2 shows the resultant amino acid sequence of the product of the glb0 gene.
Coding DNA sequences arising from SEQ ID NO: 1 due to the degeneracy of the genetic code are also provided by the invention. Conservative substitutions of amino acids in proteins, for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known in specialist circles as “sense mutations”, which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. It is furthermore known that changes to the N and/or C terminus of a protein do not substantially impair or may even stabilize the function thereof. The person skilled in the art will find information in this connection inter alia in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)) I.B.R., in O'Regan et al. (Gene 77:237-251 (1989)) I.B.R., in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)) I.B.R., in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) I.B.R. and in known textbooks of genetics and molecular biology. Amino acid sequences arising in a corresponding manner from SEQ ID NO: 2 and DNA sequences which code for these amino acid sequences are also provided by the invention. [0060]
It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)) I.B.R., in O'Regan et al. (Gene 77:237-251 (1989)) I.B.R., in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)) I.B.R., in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) I.B.R. and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID NO 2 are also a constituent of the invention. [0061]
DNA sequences which hybridize with SEQ ID NO: 1 or parts of SEQ ID NO: 1 are similarly provided by the invention. Finally, DNA sequences produced by the polymerase chain reaction (PCR) using primers obtained from SEQ ID NO: 1 are also provided by the invention. Such oligonucleotides typically have a length of at least 15 nucleotides. [0062]
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) I.B.R. and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260) I.B.R. 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 synthesis: a practical approach (IRL Press, Oxford, UK, 1984) I.B.R. and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) I.B.R. [0063]
The inventors discovered that coryneform bacteria produce L-amino acids, in particular L-lysine, in an improved manner once the glb0 has been overexpressed. [0064]
Overexpression may be achieved by increasing the copy number of the corresponding genes or by mutating the promoter and regulation region or the ribosome-binding site located upstream from the structural gene. Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during fermentative L-amino acid production by means of inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover amplified by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions. [0065]
The person skilled in the art will find guidance in this connection inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)) I.B.R., in Guerrero et al. (Gene 138, 35-41 (1994)) I.B.R., Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)) I.B.R., in Eikmanns et al. (Gene 102, 93-98 (1991)) I.B.R., in European patent EPS 0 472 869, in U.S. Pat. No. 4,601,893 I.B.R., in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991) [sic] I.B.R., in Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994) I.B.R. in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)) I.B.R., in patent application WO 96/15246 I.B.R., in Malumbres et al. (Gene 134, 15-24 (1993)) I.B.R., in Japanese published patent application JP-A-10-229891 I.B.R., in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) I.B.R., in Makrides (Microbiological Reviews 60:512-538 (1996)) I.B.R. and in known textbooks of genetics and molecular biology. [0066]
The expert can find additional information on this e.g. in the patent application WO 96/15246 I.B.R., in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)) I.B.R., in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998) I.B.R., in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)) I.B.R., in Pátek et al. (Microbiology 142: 1297 (1996)) I.B.R. 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) I.B.R. or that by Winnacker (“Gene und Klone” [Genes and Clones], VCH Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R. [0067]
By way of example, the glb0 gene according to the invention was overexpressed with the assistance of plasmids. [0068]
Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as for example pZ1 (Menkel et al., Applied and Environmental Microbiology, 64: 549-554(1989)) I.B.R., pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) I.B.R. or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) I.B.R. are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as for example those based on pCG4 (U.S. Pat. No. 4,489,160) I.B.R., or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) I.B.R., or pAG1 (U.S. Pat. No. 5,158,891) I.B.R. may be used in the same manner. [0069]
Further suitable plasmid vectors are those with the assistance of which gene amplification may be performed by integration into the chromosome, as has for example been described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) I.B.R. for the duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which can replicate in a host (typically [0070] E. coli), but not in C. glutamicum. Vectors which may be considered are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)) I.B.R., pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)) I.B.R., pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994) I.B.R. Journal of Biological Chemistry 269:32678-84 I.B.R.; U.S. Pat. No. 5,487,993) I.B.R., pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) I.B.R. or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) I.B.R. The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The conjugation method is described, for example, in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)) I.B.R. Transformation methods are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)) I.B.R., Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) I.B.R. and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)) I.B.R. After homologous recombination by means of “crossing over”, the resultant strain contains at least two copies of the gene in question.
The invention accordingly also provides a process for the fermentative production of L-amino acids, in particular L-lysine, in which a strain transformed with a plasmid vector is used and the plasmid vector bears the nucleotide sequence of the gene which codes for the hemoglobin-like protein. [0071]
It may additionally be advantageous for the production of L-amino acids, in particular L-lysine, to amplify not only the glb0 gene, but also further genes of the biosynthetic pathway of the desired L-amino acid, such that one or more enzymes of the particular biosynthetic pathway, of glycolysis, of anaplerotic metabolism or of amino acid export is/are overexpressed. [0072]
For the production of L-lysine, for example, it is thus possible simultaneously to overexpress one or more genes selected from the group [0073]
the dapA gene which codes for dihydropicolinate synthase (EP-B 0 197 335) I.B.R., [0074]
the gap gene which codes for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) I.B.R., [0075]
the tpi gene which codes for triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) I.B.R., [0076]
the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) I.B.R., [0077]
the pyc gene which codes for pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) I.B.R. and [0078]
the lysE gene which codes for lysine export (DE-A-195 48 222) I.B.R. [0079]
It may furthermore be advantageous for the production of L-amino acids, in particular L-lysine, apart from the glb0 gene, simultaneously to attenuate [0080]
the pck gene which codes for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047) I.B.R. and/or [0081]
the pgi gene which codes for glucose-6-phosphate isomerase (U.S. Ser. No. 09/396,478, DSM 12969) I.B.R. [0082]
the poxB gene which codes for pyruvate oxidase (DE: 19951975.79) I.B.R. [0083]
It may furthermore be advantageous for the production of L-amino acids, in particular L-lysine, in addition to overexpressing the glb0 gene, to suppress unwanted secondary reactions (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) I.B.R. [0084]
For the purposes of amino acid production, in particular of L-lysine, the microorganisms produced according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel ([0085] Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) I.B.R. or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) I.B.R.
The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in “Manual of Methods for General Bacteriology” from the American Society for Bacteriology (Washington D.C., USA, 1981) I.B.R. Carbon sources which may be used are sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose for example, oils and fats, such as soya oil, sunflower oil, peanut oil and coconut oil for example, fatty acids, such as palmitic acid, stearic acid and linoleic acid for example, alcohols, such as glycerol and ethanol for example, and organic acids, such as acetic acid for example. These substances may be used individually or as a mixture. Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium has additionally to contain salts of metals, such as magnesium sulfate or iron sulfate for example, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation. [0086]
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Foaming may be controlled by using antifoaming agents such as fatty acid polyglycol esters for example. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example antibiotics. Oxygen or oxygen-containing gas mixtures, such as air for example, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20° C. to 45° C. and preferably from 25° C. to 40° C. The culture is continued until the maximum quantity of L-lysine has formed. This objective is normally achieved within 10 hours to 160 hours. [0087]
The invention accordingly provides a process for the fermentative production of L-amino acids, in particular L-lysine, in which the following steps are performed: [0088]
a) fermentation of the L-amino acid producing coryneform bacteria in which at least the glb0 gene which codes for the hemoglobin-like protein is amplified, in particular overexpressed. [0089]
b) accumulation of the L-amino acid in the medium or in the cells of the bacteria and [0090]
c) isolation of the L-amino acid. [0091]
Analysis of L-lysine may be performed by anion exchange chromatography with subsequent ninhydrin derivatization, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) I.B.R. [0092]
The purpose of the process according to the invention is the fermentative production of L-amino acids, in particular L-lysine. [0093]

EXAMPLES

The present invention is illustrated in greater detail by the following practical examples. [0094]

Example 1

Production of a genomic cosmid gene library from [0095] Corynebacterium glutamicum ATCC13032
Chromosomal DNA from [0096] Corynebacterium glutamicum ATCC 13032 was isolated as described in Tauch et al., (1995, Plasmid 33:168-179) I.B.R. and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, code no. 27-0913-02) I.B.R. The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, code no. 1758250) I.B.R. The DNA of cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164) I.B.R., purchased from Stratagene (La Jolla, USA, product description SuperCos1 Cosmid Vector Kit, code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, code no. 27-0948-02) I.B.R. and also dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04) I.B.R. 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) I.B.R. The ligation mixture was then packed in phages using Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, code no. 200217). E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) I.B.R. was infected by suspending the cells in 10 mM MgSO₄and mixing them with an aliquot of the phage suspension. The cosmid library was infected and titred as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) I.B.R., the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) I.B.R. with 100 μg/ml of ampicillin. After overnight incubation at 37° C., individual recombinant clones were selected.

Example 2

Isolation and sequencing of the glb0 gene. [0097]
Cosmid DNA from an individual colony was isolated in accordance with the manufacturer's instructions using the Qiaprep Spin Miniprep Kit (product no. 27106, Qiagen, Hilden, Germany) and partially 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, Germany, product description SAP, product no. 1758250). Once separated by gel electrophoresis, the cosmid fragments of a size of 1500 to 2000 bp were isolated using the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, product no. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, product no. 27-0868-04). Ligation of the cosmid fragments into the sequencing vector pZero-1 was performed as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) I.B.R., the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated into the [0098] E. coli strain DH5aMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) I.B.R. (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) I.B.R. and plated out onto LB agar (Lennox, 1955, Virology, 1:190) I.B.R. with 50 μg/ml of Zeocin. Plasmids of the recombinant clones were prepared using the Biorobot 9600 (product no. 900200, Qiagen, Hilden, Germany, Germany). Sequencing was performed using the dideoxy chain termination method according to Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) I.B.R. as modified by Zimmermann et al. (1990, Nucleic Acids Research, 18:1067) I.B.R. The “RR dRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems (product no. 403044, Weiterstadt, Germany) was used. Separation by gel electrophoresis and analysis of the sequencing reaction was performed in a “Rotiphorese NF” acrylamide/bisacrylamide gel (29:1) (product no. A124.1, Roth, Karlsruhe, Germany) using the “ABI Prism 377” sequencer from PE Applied Biosystems (Weiterstadt, Germany).
The resultant raw sequence data were then processed using the Staden software package (1986, Nucleic Acids Research, 14:217-231) I.B.R., version 97-0. The individual sequences of the [0099] pZero 1 derivatives were assembled into a cohesive contig. Computer-aided coding range analysis was performed using XNIP software (Staden, 1986, Nucleic Acids Research, 14:217-231) I.B.R. Further analysis was performed using the “BLAST search programs” (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402) I.B.R., against the non-redundant database of the “National Center for Biotechnology Information” (NCBI, Bethesda, Md., USA).
The resultant nucleotide sequence is stated in SEQ ID NO: 1. Analysis of the nucleotide sequence revealed an open reading frame of 393 base pairs, which was designated the glb0 gene. The glb0 gene codes for a protein of 131 amino acids. [0100]
1 2 1 840 DNA Corynebacterium glutamicum CDS (186)..(578) 1 atggtggttt taccggcgcc atttggacca agtacggccc agtgcgaccc ttgcggaatt 60 gagagggaga tatcgtcgag aagcaatttc tcgccgcgcc gcacggtgac gccggccatg 120 ttgagtgcta gttcgcgcat gggtaacacc ttactggcgt aaggccaggg cttagactgg 180 taccc atg aca acc tca gaa aat ttt tat gat tct gtg ggc ggc gag gaa 230 Met Thr Thr Ser Glu Asn Phe Tyr Asp Ser Val Gly Gly Glu Glu 1 5 10 15 acg ttt tcc ctc atc gtc cac cgt ttt tat gaa cag gtc ccc aac gac 278 Thr Phe Ser Leu Ile Val His Arg Phe Tyr Glu Gln Val Pro Asn Asp 20 25 30 gat att tta ggc ccg atg tat ccg ccg gat gat ttt gag ggc gcc gag 326 Asp Ile Leu Gly Pro Met Tyr Pro Pro Asp Asp Phe Glu Gly Ala Glu 35 40 45 cag cgt cta aag atg ttc ctc agc cag tac tgg ggc ggc ccg aag gat 374 Gln Arg Leu Lys Met Phe Leu Ser Gln Tyr Trp Gly Gly Pro Lys Asp 50 55 60 tat cag gag cag cgt gga cac cct cgt ctg cgc atg cgt cac gtc aat 422 Tyr Gln Glu Gln Arg Gly His Pro Arg Leu Arg Met Arg His Val Asn 65 70 75 tac ccc atc ggc gtc acc gca gcg gag cgt tgg ctg cag ctc atg tcc 470 Tyr Pro Ile Gly Val Thr Ala Ala Glu Arg Trp Leu Gln Leu Met Ser 80 85 90 95 aat gca ctc gac ggc gtg gat ttg acc gcg gag cag cgt gaa gcg att 518 Asn Ala Leu Asp Gly Val Asp Leu Thr Ala Glu Gln Arg Glu Ala Ile 100 105 110 tgg gag cat atg gtg cgc gcg gcc gat atg ctg atc aat tcc aac ccc 566 Trp Glu His Met Val Arg Ala Ala Asp Met Leu Ile Asn Ser Asn Pro 115 120 125 gat ccg cac gct taacttctgc caaaaagtcg ttttgaccat aagctaagcg 618 Asp Pro His Ala 130 attgtgaatc gaattgcaga aatcgcacgc agtttcggcg tgctgggctt cagcgctttc 678 ggcggcccca ccgcgcacct cggatatttc cgcacggaat tcgtggagcg gcggcgctgg 738 ctggatgatc gccaatattc cgagatcgta gcgctcagcc aactacttcc cggacctgga 798 tcgtcgcagg tcggtatgat gctgggctac caccgcgccg gt 840 2 131 PRT Corynebacterium glutamicum 2 Met Thr Thr Ser Glu Asn Phe Tyr Asp Ser Val Gly Gly Glu Glu Thr 1 5 10 15 Phe Ser Leu Ile Val His Arg Phe Tyr Glu Gln Val Pro Asn Asp Asp 20 25 30 Ile Leu Gly Pro Met Tyr Pro Pro Asp Asp Phe Glu Gly Ala Glu Gln 35 40 45 Arg Leu Lys Met Phe Leu Ser Gln Tyr Trp Gly Gly Pro Lys Asp Tyr 50 55 60 Gln Glu Gln Arg Gly His Pro Arg Leu Arg Met Arg His Val Asn Tyr 65 70 75 80 Pro Ile Gly Val Thr Ala Ala Glu Arg Trp Leu Gln Leu Met Ser Asn 85 90 95 Ala Leu Asp Gly Val Asp Leu Thr Ala Glu Gln Arg Glu Ala Ile Trp 100 105 110 Glu His Met Val Arg Ala Ala Asp Met Leu Ile Asn Ser Asn Pro Asp 115 120 125 Pro His Ala 130

Claims

What is claimed is:

1. An isolated polynucleotide from coryneform bacteria containing one of the following polynucleotide sequences:

a) a polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID NO: 2,

b) a polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2,

c) a polynucleotide which is complementary to the polynucleotides of a) or b), and

d) a polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c).

2. The polynucleotide of claim 1, wherein the polynucleotide is a recombinant DNA replicable in coryneform bacteria.

3. The polynucleotide of claim 1, wherein the polynucleotide is an RNA.

4. The polynucleotide of claim 2, wherein the replicable DNA comprises:

(i) the nucleotide sequence shown in SEQ ID NO: 1, or

(ii) at least one sequence which matches the sequence of (i) within the degeneration range of the genetic code, or

(iii) at least one sequence which hybridizes with the complementary sequence to sequence (i) or (ii), or

(iv) functionally neutral sense mutations in (i).

5. The polynucleotide of claim 2 and having a sequence which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID NO: 2.

6. A vector containing the polynucleotide sequence of claim 1.

7. A coryneform bacterium containing the vector as claimed in claim 6.

8. A method for the fermentative production of L-amino acids, comprising:

a) fermenting L-amino acid producing coryneform bacteria in which at least the gene which codes for the hemoglobin-like protein is amplified.

9. The method of claim 8, wherein bacteria are used in which further genes of the biosynthetic pathway of the desired L-amino acid are additionally amplified.

10. The method of claim 8, wherein bacteria are used in which the metabolic pathways which reduce the formation of the L-amino acid are at least partially suppressed.

11. The method of claim 8, wherein bacteria transformed with a plasmid vector are used and the plasmid vector bears the nucleotide sequence of the gene which codes for the hemoglobin-like protein.

12. The method of claim 8, wherein coryneform bacteria are used which produce L-lysine.

13. The method claim 9, wherein one or more genes selected from the group consisting of the dapA gene which codes for dihydropicolinate synthase,the pyc gene which codes for pyruvate carboxylase,the tpi gene which codes for triosephosphate isomerase, the gap gene which codes for glyceraldehyde-3-phosphate dehydrogenase,the pgk gene which codes for 3-phosphoglycerate kinase, and the lysE gene which codes for lysine export is/are simultaneously amplified.

14. The method of claim 10, wherein bacteria are fermented for the production of L-lysine in which one or more of the genes selected from the group consisting of the pck gene which codes for phosphoenolpyruvate carboxykinase and the pgi gene which codes for glucose-6-phosphate isomerase is/are simultaneously attenuated.

15. The method of claim 8, wherein microorganisms of the genus Corynebacterium glutamicum are used.

16. The method of claim 8 further comprising:

b. accumulating L-amino acid in the medium or in the cells of the bacteria.

17. The method of claim 16 further comprising:

c. isolating the L-amino acid.

18. A primer which comprises a polynucleotide sequence of claim 1, wherein the primer is used for the production of DNA of genes which code for the gene product of Glb0 by the polymerase chain reaction.

19. A hybridization probe comprising a polynucleotide sequence of claim 1.