# Patent

## Title
Method of production of L-amino acids

## Abstract

An isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, with the L-aspartic acid at position 5 of the amino acid sequence replaced by another proteinogenic amino acid, and possesses citrate synthase activity. In addition, a vector comprises the polynucleotide and a bacterium comprises the vector. An isolated polynucleotide comprises a nucleotide sequence comprising, from position 1 to 39, the nucleotide sequence corresponding to position 1 to 39 of SEQ ID NO: 11, from position 40 to 105, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12, with each proteinogenic amino acid except L-aspartic acid being present at position 5. A method of producing an L-amino acids is also described.

## Background
This application is a divisional application of U.S. application Ser. No. 11/777,423, filed Jul. 13, 2007, now U.S. Pat. No. 7,785,840 issued Aug. 31, 2010, which claims priority to U.S. Provisional Application Ser. No. 60/830,331, filed Jul. 13, 2006.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to novel polynucleotides coding for a polypeptide with citrate synthase activity, bacteria containing the polynucleotides and polypeptides and methods of production of amino acids using these bacteria.
2. Discussion of the Background
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
Amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and quite particularly in animal nutrition.
Amino acids may be produced by fermentation of strains of coryneform bacteria, preferably Corynebacterium glutamicum. Owing to their great importance, work is constantly in progress for improving the production processes. Process improvements may relate to the fermentation technology, for example, stirring and supply of oxygen, or to the composition of the nutrient media, for example, the sugar concentration during fermentation, or processing to the product form by, for example, ion-exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
Methods of mutagenesis, selection and mutant screening are employed for improving the performance characteristics of these microorganisms. In this way we obtain strains that are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance, and which produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology have also been used for some years now for strain improvement of L-amino acid-producing strains of the genus Corynebacterium, preferably Corynebacterium glutamicum, by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
Synoptic descriptions of the biology, genetics and biotechnology of Corynebacterium glutamicum are given in “Handbook of Corynebacterium glutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis, 2005), in the special issue of the Journal of Biotechnology (Chief Editor: A. Pühler) with the title “A new era in Corynebacterium glutamicum biotechnology” (Journal of Biotechnology 104/1-3, (2003)) and in the book by T. Scheper (Managing Editor) “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering/Biotechnology 79, Springer Verlag, Berlin, Germany, 2003).
The nucleotide sequence of the genome of Corynebacterium glutamicum is described in Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109 (2003)), in EP 1 108 790 and in Kalinowski et al. (Journal of Biotechnology 104/1-3, 2003)).
The nucleotide sequence of the genome of Corynebacterium efficiens is described in Nishio et al. (Genome Research, 13 (7), 1572-1579 (2003)).
The nucleotide sequences of the genome of Corynebacterium glutamicum and Corynebacterium efficiens are also available in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan (DDBJ, Mishima, Japan) or in the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).
The wild-type sequence of the coding region of the gltA gene of Corynebacterium glutamicum is presented in SEQ ID NO: 1 in the specification of the present application. In addition, the sequences located upstream and downstream of the coding region are shown in SEQ ID NO: 3 and 25. The amino acid sequence of the encoded GltA polypeptide (citrate synthase) is accordingly given in SEQ ID NOs: 2, 4 and 26.
SUMMARY OF THE INVENTION
It was an object of the present invention to provide novel measures for the improved production of L-amino acids, preferably L-lysine, L-valine and L-isoleucine, and more preferably L-lysine.
This and other objects have been achieved by the present invention the first embodiment of which includes an isolated polynucleotide, encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the L-aspartic acid at position 5 of the amino acid sequence is replaced by another proteinogenic amino acid and wherein the polypeptide possesses citrate synthase activity.
The invention further provides a vector comprising the isolated polynucleotide and a bacterium that has been transformed with the vector.
The invention also provides a method of production of an L-amino acid.



BRIEF DESCRIPTION OF THE DRAWING
The FIGURE represents a map of the plasmid pK18mobsacB_gltAD5V.



DETAILED DESCRIPTION OF THE INVENTION
The invention relates to an isolated polynucleotide that codes for a polypeptide which comprises the amino acid sequence of SEQ ID NO: 2, wherein the L-aspartic acid at position 5 of the amino acid sequence is replaced by another proteinogenic amino acid, preferably L-valine, L-leucine and L-isoleucine, and more preferably L-valine, and wherein the polypeptide possesses citrate synthase activity (EC No. 4.1.3.7). Optionally, the polypeptide comprises at least one conservative amino acid substitution, with the citrate synthase activity of the polypeptide being essentially unchanged by the conservative amino acid substitutions.
Proteinogenic amino acids are understood as meaning the amino acids that occur in natural proteins, i.e. in proteins of microorganisms, plants, animals and humans. These include in particular L-amino acids, selected from the group 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 terms polypeptide and protein are used as synonyms.
The invention further relates to vectors and bacteria, preferably of the genus Corynebacterium and Escherichia, and more preferably of the species Corynebacterium glutamicum and Escherichia coli, which contain the stated polynucleotide or were produced using the stated polynucleotide.
The invention also relates finally to bacteria preferably of the genus Corynebacterium and Escherichia, and more preferably of the species Corynebacterium glutamicum and Escherichia coli, which have been transformed with the stated vector.
The term transformation comprises all methods for transferring polynucleotides, preferably DNA, into a desired bacterium. Among other things these include the use of isolated DNA in transformation, electrotransformation or electroporation, transfer by cellular contact as in conjugation or the transfer of DNA by particle bombardment.
A further aspect of the invention relates to a bacterium, that may be a recombinant bacterium, of the genus Corynebacterium, which comprises a polynucleotide that codes for a polypeptide with citrate synthase activity, which comprises the amino acid sequence of SEQ ID NO: 2, wherein each proteinogenic amino acid except L-aspartic acid, preferably L-valine, L-leucine and L-isoleucine, and preferably L-valine, is contained at position 5 of the amino acid sequence. Optionally, the polypeptide may contain one or more conservative amino acid substitution(s), with the citrate synthase activity of the polypeptide being essentially unchanged by the conservative amino acid substitutions.
A further aspect of the invention relates to a method of production of L-amino acids, preferably L-lysine, L-valine and L-isoleucine, and more preferably L-lysine, comprising the following steps:
a) fermentation of the recombinant bacteria of the genus Corynebacterium according to the invention in a suitable nutrient medium, and
b) accumulation of the L-amino acid in the nutrient medium or in the cells of the bacteria.
“L-amino acids” means the proteinogenic amino acids.
If L-lysine or lysine is mentioned hereinafter, this is intended to mean not only the bases, but also the salts, for example L-lysine monohydrochloride or L-lysine sulfate.
With regard to the bacteria of the genus Corynebacterium, L-amino acid-excreting strains are preferred, based on the following species:
Corynebacterium efficiens, for example the strain DSM44549,
Corynebacterium glutamicum, for example the strain ATCC13032,
Corynebacterium thermoaminogenes, for example the strain FERM BP-1539, and
Corynebacterium ammoniagenes, for example the strain ATCC6871,
the species Corynebacterium glutamicum being preferred.
Some representatives of the species Corynebacterium glutamicum are also known under different designations. Examples include:
Corynebacterium acetoacidophilum ATCC 13870,
Corynebacterium lilium DSM20137,
Corynebacterium melassecola ATCC 17965,
Brevibacterium flavum ATCC14067,
Brevibacterium lactofermentum ATCC 13869, and
Brevibacterium divaricatum ATCC14020.
Known representatives of amino acid-excreting strains of the genus Corynebacterium are, for example, the L-lysine producing strains:
Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in EP 0 358 940,
Corynebacterium glutamicum MH20-22B (=DSM16835) described in Menkel et al. (Applied and Environmental Microbiology 55(3), 684-688 (1989)),
Corynebacterium glutamicum AHP-3 (=FERM BP-7382) described in EP 1 108 790,
Corynebacterium glutamicum DSM16834 described in (PCT/EP2005/012417),
Corynebacterium glutamicum DSM17119 described in (PCT/EP2006/060851),
Corynebacterium glutamicum DSM17223 described in (PCT/EP2006/062010),
Corynebacterium glutamicum DSM16937 described in (PCT/EP2005/057216), and
Corynebacterium thermoaminogenes AJ12521 (=FERM BP-3304) described in U.S. Pat. No. 5,250,423;
or the L-valine producing strains:


    
    
        Brevibacterium lactofermentum FERM BP-1763 described in U.S. Pat. No. 5,188,948,
        Brevibacterium lactofermentum FERM BP-3007 described in U.S. Pat. No. 5,521,074,
        Corynebacterium glutamicum FERM BP-3006 described in U.S. Pat. No. 5,521,074, and
        Corynebacterium glutamicum FERM BP-1764 described in U.S. Pat. No. 5,188,948;

or the L-isoleucine producing strains:

        Brevibacterium flavum FERM-BP 759 described in U.S. Pat. No. 4,656,135,
        Corynebacterium glutamicum FERM-BP 757 described in U.S. Pat. No. 4,656,135,
        Brevibacterium flavum FERM-BP 760 described in U.S. Pat. No. 4,656,135,
        Corynebacterium glutamicum FERM-BP 758 described in U.S. Pat. No. 4,656,135,
        Brevibacterium flavum FERM BP-2215 described in U.S. Pat. No. 5,705,370, and
        Brevibacterium flavum FERM BP-2433 described in U.S. Pat. No. 5,705,370.
    
    


Information on the taxonomic classification of strains of this group of bacteria can be found inter alia in Seiler (Journal of General Microbiology 129, 1433-1477 (1983)), Kämpfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)), Liebl et al. (International Journal of Systematic Bacteriology 41, 255-260 (1991)) and in U.S. Pat. No. 5,250,434.
Strains with the designation “ATCC” can be obtained from the American Type Culture Collection (Manassas, Va., USA). Strains with the designation “DSM” can be obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). 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 Corynebacterium thermoaminogenes (FERM BP-1539) is described in U.S. Pat. No. 5,250,434.
For production of the polynucleotides, it is possible to use classical in-vivo mutagenesis techniques with cell populations of bacteria of the genus Corynebacterium using mutagenic substances such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or using ultraviolet light. Mutagenesis techniques are described for example in the Manual of Methods for General Bacteriology (Gerhard et al. (Eds.), American Society for Microbiology, Washington, D.C., USA, 1981) or in Tosaka et al. (Agricultural and Biological Chemistry 42(4), 745-752 (1978)) or in Konicek et al. (Folia Microbiologica 33, 337-343 (1988)).
From the mutagenized cell population, those mutants are taken and multiplied which require L-glutamic acid or citric acid in order to be able to grow on a minimal agar or whose growth on the minimal agar is improved by adding L-glutamic acid or citric acid. It is also possible, starting from mutants requiring L-glutamic acid or citric acid, to isolate so-called revertants, which do not require L-glutamic acid or citric acid for their growth. These L-glutamic acid-auxotrophic or citric acid-auxotrophic mutants or their respective revertants are then investigated. Technical details on the isolation of mutants with defective citrate synthase activity can be found for example in Shiio et al. (Agricultural and Biological Chemistry 46(1), 101-107 (1982)).
Next, DNA is prepared or isolated from the mutants and by means of, for example, the polymerase chain reaction (PCR) using primer pairs which allow the amplification of the gltA gene or gltA allele, the corresponding polynucleotide is synthesized and isolated.
For this, it is possible to select any primer pairs from the nucleotide sequence located upstream and downstream of the coding region and the nucleotide sequence that is complementary to it (see SEQ ID NOs: 3 and 25). A primer of a primer pair then comprises preferably at least 15, at least 18, at least 20, at least 21 or at least 24 consecutive nucleotides selected from the nucleotide sequence between position 1 and 1000 of SEQ ID NO: 25. The associated second primer of a primer pair comprises at least 15, at least 18, at least 20, at least 21 or at least 24 consecutive nucleotides selected from the complementary nucleotide sequence between position 3314 and 2312 of SEQ ID NO: 25.
A person skilled in the art will find instructions and information on PCR for example in the handbook “PCR Strategies” (Innis, Felfand and Sninsky, Academic Press, Inc., 1995), in the handbook by Diefenbach and Dveksler “PCR Prime—a laboratory manual” (Cold Spring Harbor Laboratory Press, 1995), in Gait's handbook “Oligonucleotide Synthesis: a Practical Approach” (IRL Press, Oxford, UK, 1984) and in Newton and Graham “PCR” (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
Further instructions on PCR can be found for example in WO 06/100177 on pages 15 to 17.
In a further step, the nucleotide sequence of the polynucleotide is then determined. This can for example be determined by the chain-terminating technique of Sanger et al. (Proceedings of the National Academies of Sciences, USA, 74, 5463-5467 (1977)) with the modifications stated by Zimmermann et al. (Nucleic Acids Research 18, 1067 pp (1990)).
The polypeptide encoded by this nucleotide sequence can then be analyzed with respect to the amino acid sequence. For this, the nucleotide sequence is input in a program for translating a DNA sequence into an amino acid sequence. Suitable programs are for example the “Patentin” program, which is obtainable from patent offices, for example the US Patent Office (USPTO), or the “Translate Tool”, which is available on the ExPASy Proteomics Server on the World Wide Web (Gasteiger et al., Nucleic Acids Research 31, 3784-3788 (2003)).
It is also possible for the polynucleotide, which is also designated hereinafter as gltA allele, to be produced by methods of in-vitro genetics.
Suitable methods for in-vitro mutagenesis including among others treatment with hydroxylamine according to Miller (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 the use of a polymerase chain reaction using a DNA polymerase, which has a high error rate. Such a DNA polymerase is for example the Mutazyme DNA Polymerase (GeneMorph PCR Mutagenesis Kit, No. 600550) of the company Stratagene (La Jolla, Calif., USA). It is also possible to use mutagenic oligonucleotides, as described by T. A. Brown (Gentechnologie für Einsteiger, Spektrum Akademischer Verlag, Heidelberg, 1993) and R. M. Horton (PCR-mediated recombination and mutagenesis, Molecular Biotechnology 3, 93-99 (1995)). The method using the “Quik Change Site-directed Mutagenesis Kit” of the company Stratagene (La Jolla, Calif., USA) described by Papworth et al. (Strategies 9 (3), 3-4 (1996)) can also be used.
Methods for the determination of citrate synthase activity can be found in Eikmanns et al. (Microbiology 140, 1817-1828 (1994)) and in Shiio et al. (Agricultural and Biological Chemistry 46 (1), 101-107 (1982)).
It is moreover possible to overexpress the citrate synthase allele according to the invention in Corynebacterium glutamicum or Escherichia coli, and it can then be prepared in purified or isolated form.
A polynucleotide with the nucleotide sequence shown in SEQ ID NO: 5 was isolated in this way. The polypeptide encoded by this polynucleotide is shown in SEQ ID NO: 6 and 8. It contains L-valine instead of L-aspartic acid at position 5 of the amino acid sequence.
It was found that when the strain ATCC 13032 is provided, instead of the wild-type gltA gene, with the gltA allele according to the invention, which codes for the citrate synthase according to SEQ ID NO: 6 (ATCC13032::gltA D5V), in comparison with the wild-type strain ATCC13032, which contains the citrate synthase according to SEQ ID NO: 2, with enzyme activity reduced by approx. 40% up to a maximum of approx. 90%, preferably with enzyme activity reduced by approx. 70% up to a maximum of approx. 90%.
It is known that conservative amino acid substitutions only change the enzyme activity insignificantly. Accordingly, the invention also relates to polynucleotides that code for polypeptides with citrate synthase activity, which in addition to the amino acid substitutions at position 5 of the amino acid sequence contain one (1) or more conservative amino acid substitution(s), which does not alter the enzyme activity substantially. That is, it remains essentially unchanged. The term “not altered substantially,” “essentially unchanged,” or “substantially unchanged” means in this context that the citrate synthase activity of the polypeptide is altered by at most 20%, preferably at most 10% and more preferably at most 5% to at most 2% in comparison with the citrate synthase activity of the polypeptide according to SEQ ID NO: 10 or SEQ ID NO: 6, preferably SEQ ID NO: 6.
For an experimental test, the gltA gene of strain ATCC13032 is substituted for the gltA allele, which codes for a polypeptide containing the amino acid substitution at position 5 and at least one conservative amino acid substitution. Then the strain is cultivated, a cellular extract is produced and the citrate synthase activity is determined. As a reference, the citrate synthase activity in strain ATCC13032::gltA D5V is determined.
Instead of strain ATCC13032, it is also possible to use L-lysine-excreting strains of Corynebacterium glutamicum which comprises the coding region of the gltA gene of the wild type including the nucleotide sequences located upstream, corresponding to the nucleotide sequence between position 1 and 2064 of SEQ ID NO: 3, preferably SEQ ID NO: 3. Suitable strains are, for example, DSM16833 described in PCT/EP2005/012417, DSM13994 described in EP 1 239 040 A2 or DSM17576 described in DE 102005045301. In these strains, the appropriate mutation(s) can be inserted in the coding region of the gltA gene by, for example, allelic substitution.
It is also possible to purify the polypeptides and to conduct the comparative tests on the purified polypeptides.
The enzyme citrate synthase (EC No. 4.1.3.7) catalyzes the condensation reaction of oxaloacetate and acetyl-CoA, with formation of citric acid and coenzyme A (CoA) as reaction products. The enzyme is assigned the number EC 2.3.3.1 in the Kyoto Encyclopedia of Genes and Genomes (KEGG, Kanehisa Laboratory, Bioinformatics Center, Institute for Chemical Research, Kyoto University, Japan).
In the case of aromatic L-amino acids, we talk of conservative substitutions when L-phenylalanine, L-tryptophan and L-tyrosine are substituted for one another. In the case of hydrophobic L-amino acids, we talk of conservative substitutions when L-leucine, L-isoleucine and L-valine are substituted for one another. In the case of polar L-amino acids, we talk of conservative substitutions when L-glutamine and L-asparagine are substituted for one another. In the case of basic L-amino acids, we talk of conservative substitutions when L-arginine, L-lysine and L-histidine are substituted for one another. In the case of acidic L-amino acids, we talk of conservative substitutions when L-aspartic acid and L-glutamic acid are substituted for one another. In the case of L-amino acids containing hydroxyl groups, we talk of conservative substitutions when L-serine and L-threonine are substituted for one another.
Preferably the polypeptide contains at most two (2), at most three (3), at most four (4) or at most five (5) conservative amino acid substitutions in addition to the substitution at position 5 of SEQ ID NO: 2.
It is known that the terminal methionine may be removed during protein synthesis by enzymes that are intrinsic to the host, so-called aminopeptidases.
The isolated polynucleotides, which code for the citrate synthase variant, or portions thereof, can be used for producing recombinant strains of the genus Corynebacterium, preferably Corynebacterium glutamicum, which comprises the amino acid substitution at position 5 of the amino acid sequence of the citrate synthase polypeptide and which provide improved release of L-amino acids into the surrounding medium or accumulation of them inside the cell, compared with the starting or parent strain.
The initial strains preferably used are those which already possess the capacity to excrete at least 1 g/l, preferably at least 5 g/l, and more preferably at least 10 g/l of the desired L-amino acid into the surrounding nutrient medium.
A widely used method for incorporating mutations in genes of bacteria of the genus Corynebacterium, preferably of the species Corynebacterium glutamicum, is allelic substitution, which is also known as “gene replacement”. In this technique, a DNA fragment that contains the mutation of interest is transferred into the desired strain and the mutation is incorporated in the chromosome of the desired strain by at least two recombination events or cross-over events or a gene sequence present in the strain in question is replaced by the mutated sequence.
In this method, the DNA fragment containing the mutation of interest may be located in a vector, preferably a plasmid, which preferably is not replicated by the strain that is to be provided with the mutation, or such replication is limited, i.e. occurs under selected culture conditions. A bacterium of the genus Escherichia, preferably of the species Escherichia coli, may be used as auxiliary or intermediate host, in which the vector can be replicated.
Examples of such plasmid vectors are the pK*mob and pK*mobsacB vectors, for example pK18mobsacB, described by Schäfer et al. (Gene 145, 69-73 (1994)), and the vectors described in WO 02/070685 and WO 03/014362. These vectors can replicate in Escherichia coli but not in Corynebacterium. Preferably, suitable vectors are those which contain a gene with conditionally negative dominant action for example the sacB gene (levansucrase gene) of for example Bacillus or the galK gene (galactose kinase gene) of for example Escherichia coli. “Gene with conditionally negative dominant action” means a gene which under certain conditions is disadvantageous, for example toxic to the host, but in other conditions does not have adverse effects on the host carrying the gene. These make it possible to select for recombination events in which the vector is eliminated from the chromosome.
Furthermore, Nakamura et al. (U.S. Pat. No. 6,303,383) described a temperature-sensitive plasmid for Corynebacterium, which can only replicate at temperatures below 31° C. It can also be used for the purposes of the invention.
The vector is then transferred into the Corynebacterium by conjugation, for example, by Schäfer's method (Journal of Bacteriology 172, 1663-1666 (1990)) or transformation, for example, by Dunican and Shivnan's method (Bio/Technology 7, 1067-1070 (1989)) or the method of Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)). Optionally, the transfer of the DNA can also be achieved by ballistic methods (e.g. particle bombardment).
After homologous recombination by means of a first cross-over event producing integration and a suitable second cross-over event causing an excision in the target gene or in the target sequence, incorporation of the mutation is achieved and a recombinant bacterium is obtained. “Target gene” means the gene in which the desired substitution is to take place.
The strains obtained can be identified and characterized using, among others, the methods of Southern blotting hybridization, polymerase chain reaction and sequencing, the method of fluorescence resonance energy transfer (FRET) (Lay et al. Clinical Chemistry 43, 2262-2267 (1997)) or methods of enzymology.
This method was used by Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)) for incorporating a lysA allele carrying a deletion, and a lysA allele carrying an insertion, into the chromosome of C. glutamicum instead of the wild-type gene.
This method was used by Nakagawa et al. (EP 1108790) and Ohnishi et al. (Applied Microbiology and Biotechnology 58(2), 217-223 (2002)) for incorporating various mutations into the chromosome of C. glutamicum starting from the isolated alleles or polynucleotides. In this way, Nakagawa et al. succeeded in incorporating a mutation designated Val59Ala into the homoserine dehydrogenase gene (hom), a mutation designated Thr311Ile into the aspartate kinase gene (lysC or ask), a mutation designated Pro458Ser into the pyruvate carboxylase gene (pyc) and a mutation designated Ala213Thr into the glucose-6-phosphate-dehydrogenase gene (zwf) of C. glutamicum strains.
For inserting the mutation in the gltA gene into the chromosome by means of allelic substitution, it is possible to use a polynucleotide that codes for an amino acid sequence which has the amino acid substitution at position 5 of SEQ ID NO: 2, as shown in SEQ ID NO: 10, and possesses, upstream and downstream thereof, a nucleotide sequence with a length in each case of at least approx. 51 (cf. SEQ ID NO: 11 and 12) preferably in each case at least approx. 101 or 102 (cf. SEQ ID NO: 13 and 14), preferably in each case at least approx. 201 nucleobases (cf. SEQ ID NO: 15 and 16) and more preferably in each case at least approx. 500 or 498 nucleobases (cf. SEQ ID NO: 17 and 18) selected from SEQ ID NO: 9. The maximum length of the nucleotide sequence located upstream and downstream of the mutation is generally approx. 500, approx. 750, approx. 1000, approx. 1500, approx. 2000 to 2100 nucleobases. The nucleotide sequence located upstream of the mutation comprises, for example, the sequence between position 1 to 762 of SEQ ID NO: 9 or the sequence between position 1 to 1012 of SEQ ID NO: 25. The nucleotide sequence located downstream of the mutation comprises for example the sequence between position 766 to 2814 of SEQ ID NO:9 or the sequence between position 1016 to 3314 of SEQ ID NO:25. The total length of the polynucleotide used for the allelic substitution is accordingly at most approx. 1000, at most approx. 1500, at most approx. 2000, at most approx. 3000 or at most approx. 4000 to 4200 nucleobases.
Accordingly, the invention relates to a polynucleotide that comprises a nucleotide sequence which contains, from position 1 to 39, the nucleotide sequence corresponding to position 1 to 39 of SEQ ID NO: 11 and, from position 40 to 105, a nucleotide sequence that codes for the amino acid sequence according to SEQ ID NO: 12, having every proteinogenic amino acid except L-aspartic acid being contained at position 5.
In this context, the stated positions 1 to 39 of SEQ ID NO: 11 correspond to the stated positions 712 to 750 of SEQ ID NO: 9. The stated positions 40 to 105 of SEQ ID NO: 11 correspond to the stated positions 751 to 816 of SEQ ID NO: 9.
In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1, with the codon corresponding to position 52 to 54 coding for every proteinogenic amino acid except L-aspartic acid.
In another embodiment the polynucleotide comprises the nucleotide sequence from position 712 to 816 of SEQ ID NO: 7.
Accordingly, the invention also relates to a polynucleotide that comprises a nucleotide sequence which comprises, from position 1 to 89, the nucleotide sequence corresponding to position 1 to 89 of SEQ ID NO: 13 and, from position 90 to 206, a nucleotide sequence which codes for the amino acid sequence according to SEQ ID NO: 14, having every proteinogenic amino acid except L-aspartic acid being contained at position 5.
In this context, the stated positions 1 to 89 of SEQ ID NO: 13 correspond to the stated positions 662 to 750 of SEQ ID NO: 9. The stated positions 90 to 206 of SEQ ID NO: 13 correspond to the stated positions 751 to 867 of SEQ ID NO: 9.
In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13, with the codon corresponding to position 102 to 104 coding for every proteinogenic amino acid except L-aspartic acid.
In another embodiment, the polynucleotide comprises the nucleotide sequence from position 662 to 867 of SEQ ID NO: 7.
Accordingly, the invention also relates to a polynucleotide that comprises a nucleotide sequence which comprises, from position 1 to 189, the nucleotide sequence corresponding to position 1 to 189 of SEQ ID NO: 15 and, from position 190 to 405, a nucleotide sequence which codes for the amino acid sequence according to SEQ ID NO: 16, having every proteinogenic amino acid except L-aspartic acid being contained at position 5.
In this context, the stated positions 1 to 189 of SEQ ID NO: 15 correspond to the stated positions 562 to 750 of SEQ ID NO: 9. The stated positions 190 to 405 of SEQ ID NO: 15 correspond to the stated positions 751 to 966 of SEQ ID NO: 9.
In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15, with the codon corresponding to position 202 to 204 coding for every proteinogenic amino acid except L-aspartic acid.
In another embodiment, the polynucleotide comprises the nucleotide sequence from position 562 to 966 of SEQ ID NO: 7.
Accordingly, the invention also relates to a polynucleotide that comprises a nucleotide sequence which comprises, from position 1 to 488, the nucleotide sequence corresponding to position 1 to 488 of SEQ ID NO: 17 and, from position 489 to 1001, a nucleotide sequence which codes for the amino acid sequence according to SEQ ID NO: 18, with every proteinogenic amino acid except L-aspartic acid being contained at position 5.
In this context, the stated positions 1 to 488 of SEQ ID NO: 17 correspond to the stated positions 263 to 750 of SEQ ID NO: 9. The stated positions 489 to 1001 of SEQ ID NO: 15 correspond to the stated positions 751 to 1263 of SEQ ID NO: 9.
In one embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17, with the codon corresponding to position 501 to 503 coding for every proteinogenic amino acid except L-aspartic acid.
In another embodiment, the polynucleotide comprises the nucleotide sequence from position 263 to 1263 of SEQ ID NO: 7.
In another embodiment, the polynucleotide comprises the nucleotide sequence from position 9 to 1687 of SEQ ID NO: 31.
The invention also relates to vectors, preferably plasmids, comprising the stated polynucleotides.
The invention also relates to bacteria preferably of the genus Escherichia, more preferably of the species Escherichia coli, and Corynebacterium, more preferably of the species Corynebacterium glutamicum, comprising the stated vectors.
The invention also relates to strains of the genus Corynebacterium, preferably of the species Corynebacterium glutamicum, which have been produced using the polynucleotides or vectors comprising the polynucleotides.
It is also possible to insert the gltA allele at another site in the chromosome of Corynebacterium glutamicum. Possible examples are the sites or genes aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi and poxB, as described in WO 03/04037. Other possibilities are, for example, intergenic regions, DNA of prophages, defective phages and phage components, as described in WO 04/069996.
The obtained recombinant strains display, relative to the initial strain or parent strain used, increased excretion or production of the desired amino acid in a fermentation process.
The L-lysine-excreting starting strains that can be used for the purposes of the invention possess, in addition to other properties, in particular a lysine-insensitive aspartate kinase.
“Lysine-insensitive aspartate kinase” means a polypeptide or protein with aspartate kinase activity (EC No. 2.7.2.4), which in comparison with the wild form, have lower sensitivity to inhibition by mixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine) and threonine or lysine alone or AEC alone. Aspartate kinases of this kind are also called feedback-resistant or desensitized aspartate kinases. The nucleotide sequences coding for these desensitized aspartate kinases or aspartate kinase variants are also designated as lysCFBR alleles. Information on numerous lysCFBR alleles is available in public databases. The lysC-gene is also designated as the ask-gene by some authors.
The coding region of the wild-type lysC gene of Corynebacterium glutamicum corresponding to access number AX756575 of the NCBI database is shown in SEQ ID NO: 19 and the polypeptide encoded by this gene is shown in SEQ ID NO: 20. The nucleotide sequences located upstream of the 5′ end and downstream of the 3′ end of the coding region are also shown in SEQ ID NO: 21. SEQ ID NO: 20 corresponds to SEQ ID NO: 22.
The L-lysine-excreting bacteria preferably have a lysC allele, which codes for an aspartate kinase variant possessing the amino acid sequence of SEQ ID NO: 20, comprising one or more of the amino acid substitutions selected from the group:
a) LysC A279T (substitution of L-alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-threonine; see U.S. Pat. No. 5,688,671 and access numbers E06825, E06826, E08178 and 174588 to 174597),
b) LysC A279V (substitution of L-alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-valine, see JP 6-261766 and access number E08179),
c) LysC L297Q (substitution of L-leucine at position 297 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for another proteinogenic amino acid, preferably L-glutamine; see DE 102006026328),
d) LysC S301F (substitution of L-serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-phenylalanine; see U.S. Pat. No. 6,844,176 and access number E08180),
e) LysC S301Y (substitution of L-serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-tyrosine, see Kalinowski et al. (Molecular and General Genetics 224, 317-324 (1990)) and access number X57226),
f) LysC T308I (substitution of L-threonine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-isoleucine; see JP 6-261766 and access number E08181),
g) LysC T311I (substitution of L-threonine at position 311 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-isoleucine; see WO 00/63388 and U.S. Pat. No. 6,893,848),
h) LysC S317A (substitution of L-serine at position 317 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-alanine; see U.S. Pat. No. 5,688,671 and access number 174589),
i) LysC R320G (substitution of L-arginine at position 320 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for glycine; see Jetten et al. (Applied Microbiology and Biotechnology 43, 76-82 (995)) and access number L27125),
j) LysC G345D (substitution of glycine at position 345 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-aspartic acid; see Jetten et al. (Applied Microbiology and Biotechnology 43, 76-82 (995)) and access number L16848),
k) LysC T380I (substitution of L-threonine at position 380 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-isoleucine; see WO 01/49854 and access number AX192358), and
l) LysC S381F (substitution of L-serine at position 381 of the encoded aspartate kinase protein according to SEQ ID NO: 20 for L-phenylalanine; see EP 0435132).
Strains comprise aspartate kinase variants comprising the amino acid substitution LysC T311I or at least one amino acid substitution selected from the group LysC A279T, LysC L297Q, LysC S317A, LysC T380I and LysC S381F.
Naturally it is also possible for insertion of the mutation in the gltA gene of the chromosome of a bacterium of the genus Corynebacterium to be carried out first, followed by insertion of one or more of the desired mutation(s) in the lysC gene of the strain in question.
In one embodiment, the described aspartate kinases are overexpressed in the Corynebacterium which comprises the amino acid substitution according to the invention in the gltA gene.
Overexpression means an increase in the intracellular concentration or activity of a ribonucleic acid, a protein or an enzyme compared with the initial strain (parent strain) or wild-type strain. Initial strain (parent strain) means the strain on which the measure leading to overexpression was carried out.
The increase in concentration or activity can be achieved, for example, by increasing the copy number of the corresponding polynucleotides chromosomally or extrachromosomally by at least one copy.
A widely used method of increasing the copy number comprises inserting the corresponding polynucleotide in a vector, preferably a plasmid, which is replicated by a coryneform bacterium. Suitable plasmid vectors are, for example, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or the pSELF vectors described by Tauch et al. (Journal of Biotechnology 99, 79-91 (2002)). A review article on the subject of plasmids in Corynebacterium glutamicum can be found in Tauch et al. (Journal of Biotechnology 104, 27-40 (2003)).
Transposons, insertion elements (IS elements) or phages can also be used as vectors. Such genetic systems are stated for example in patent specifications U.S. Pat. No. 4,822,738, U.S. Pat. No. 5,804,414 and U.S. Pat. No. 5,804,414. Similarly, it is possible to use the IS element ISaB1 described in WO 92/02627 or the transposon Tn45 of plasmid pXZ10142 (cited in “Handbook of Corynebacterium glutamicum” (Publisher: L. Eggeling and M. Bott)).
Another widely used method for achieving overexpression is the technique of chromosomal gene amplification. In this method, at least one additional copy of the polynucleotide of interest is inserted in the chromosome of a coryneform bacterium. Such amplification techniques are described for example in WO 03/014330 or WO 03/040373.
Another method of achieving overexpression comprises operably linking the corresponding gene or allele with a promoter or an expression cassette. Suitable promoters for Corynebacterium glutamicum are described for example in FIG. 1 of the review article by Patek et al. (Journal of Biotechnology 104(1-3), 311-323 (2003)). The variants of the dapA promoter described by Vasicova et al. (Journal of Bacteriology 181, 6188-6191 (1999)), for example the promoter A25, can be used similarly. It is also possible to use the gap-promoter of Corynebacterium glutamicum (EP 06007373). Finally it is possible to use the sufficiently well-known promoters T3, T7, SP6, M13, lac, tac and trc described by Amann et al. (Gene 69(2), 301-315 (1988)) and Amann and Brosius (Gene 40(2-3), 183-190 (1985)). Such a promoter can for example be inserted upstream of the gene in question, typically at a distance of about 1-500 nucleobases from the start codon.
As a result of the measures for overexpression, the activity or concentration of the corresponding polypeptide is increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000% relative to the activity or concentration of the polypeptide in the strain prior to the measure leading to overexpression.
In a further embodiment, the bacteria of the genus Corynebacterium, which preferably in addition comprises a polynucleotide that codes for a lysine-insensitive aspartate kinase variant, possess one or more of the characters selected from the group
a) overexpressed polynucleotide (dapA gene), which codes for a dihydrodipicolinate synthase (DapA, EC No. 4.2.1.52),
b) overexpressed polynucleotide (asd gene), which codes for an aspartate semialdehyde dehydrogenase (Asd, EC No. 1.2.1.11),
c) overexpressed polynucleotide (lysA gene), which codes for a diaminopimelate decarboxylase (LysA, EC No. 4.1.1.20),
d) overexpressed polynucleotide (aat gene), which codes for an aspartate aminotransferase (Aat, EC No. 2.6.1.1),
e) overexpressed polynucleotide (lysE gene), which codes for a polypeptide with L-lysine exporting activity (LysE, Lysin Efflux Permease),
f) switched-off or attenuated activity of malate dehydrogenase (Mdh, EC No. 1.1.1.37),
g) switched-off or attenuated activity of malate-quinone oxidoreductase (Mqo, EC No. 1.1.99.16),
h) overexpressed polynucleotide, which codes for a pyruvate carboxylase (Pyc, EC No. 6.4.1.1), and
i) switched-off or attenuated activity of the E1p subunit of the pyruvate dehydrogenase complex (AceE, EC No. 1.2.4.1).
Characters a) to g) are preferred.
The known genes, for example, the wild-type genes, of Escherichia coli (Blattner et al., Science 277(5), 1453-1462 (1997)), Bacillus subtilis (Kunst et al., Nature 390 (6657), 249-256 (1997)), Bacillus licheniformis (Veith et al., Journal of Molecular Microbiology and Biotechnology 7 (4), 204-211 (2004)), Mycobacterium tuberculosis (Fleischmann et al., Journal of Bacteriology 1841, 5479-5490 (2004)), Mycobacterium bovis (Gamier et al., Proceedings of the National Academy of Sciences USA 100 (13), 7877-7882 (2003)), Streptomyces coeliclor (Redenbach et al., Molecular Microbiology 21 (1), 77-96 (1996)), Lactobacillus acidophilus (Altermann et al., Proceedings of the National Academy of Sciences USA 102 (11), 3906-3912 (2005)), Lactobacillus johnsonii (Pridmore et al., Proceedings of National Academy of Sciences USA 101 (8), 2512-2517 (2004)), Bifidobacterium longum (Schell et al., Proceedings of National Academy of Sciences USA 99 (22), 14422-14427 (2002)), and Saccharomyces cerevisiae can be used for overexpression of the listed genes or polynucleotides. The genomes of the wild-type forms of these bacteria are available in sequenced or annotated form. Preferably the endogenous genes or polynucleotides of the genus Corynebacterium, more preferably of the species Corynebacterium glutamicum, are used.
“Endogenous genes or polynucleotides” means the open reading frames (ORF), genes or alleles or their polynucleotides present in the population of a species.
The dapA gene of Corynebacterium glutamicum strain ATCC13032 is described for example in EP 0 197 335. The MC20 and MA16 mutations of the dapA promoter, as described in U.S. Pat. No. 6,861,246, can also be used, among others, for overexpression of the dapA gene of Corynebacterium glutamicum. 
The asd gene of Corynebacterium glutamicum strain ATCC21529 is described for example in U.S. Pat. No. 6,927,046.
The lysA gene of Corynebacterium glutamicum ATCC13869 (Brevibacterium lactofermentum) is described for example in U.S. Pat. No. 6,090,597.
The aat gene of Corynebacterium glutamicum ATCC13032 is described for example in Kalinowski et al. (Journal of Biotechnology 104 (1-3), 5-25 (2003); see also access number NC—006958). There it is designated aspB gene. In U.S. Pat. No. 6,004,773 a gene coding for an aspartate aminotransferase is designated aspC. Marienhagen et al. (Journal of Bacteriology 187 (22), 7639-7646 (2005) denote the aat gene as aspT gene.
The lysE gene of Corynebacterium glutamicum R127 is described for example in U.S. Pat. No. 6,858,406. Strain R127 is a restriction-defective mutant of ATCC13032 (Liebl et al., FEMS Microbiology Letters 65, 299-304 (1989)). The lysE gene of strain ATCC13032 used in U.S. Pat. No. 6,861,246 can be used similarly.
The pyc gene of Corynebacterium glutamicum of strain ATCC13032 is described for example in WO 99/18228 and WO 00/39305. Furthermore, alleles of the pyc gene can be used, such as are described in U.S. Pat. No. 6,965,021. The pyruvate carboxylases described in this patent specification possess one or more of the amino acid substitutions selected from the group: Pyc E153D (substitution of L-glutamic acid at position 153 for L-aspartic acid), Pyc A182S (substitution of L-alanine at position 182 for L-serine), Pyc A206S (substitution of L-alanine at position 206 for L-serine), Pyc H227R (substitution of L-histidine at position 227 for L-arginine), Pyc A455G (substitution of L-alanine at position 455 for glycine), and Pyc D1120E (substitution of L-aspartic acid at position 1120 for L-glutamic acid). Similarly, it is possible to use the pyc allele described in EP 1 108 790, which codes for a pyruvate carboxylase containing the amino acid substitution Pyc P458S (substitution of L-proline at position 458 for L-serine).
“Switched-off or attenuated activity” means reduction or switching-off of the intracellular activity or concentration of one or more enzymes or proteins in a microorganism, which is encoded by the corresponding polynucleotide or DNA.
For production of a strain in which the intracellular activity of a desired polypeptide is switched off, a deletion or insertion of at least one (1) nucleobase, preferably of one (1) or of two (2) nucleobases, is inserted in the coding region of the corresponding gene. It is also possible to delete at least one (1) or more codon(s) within the coding region. These measures lead to a shift of the reading frame (frame shift mutations) and therefore typically to the synthesis of a nonfunctional polypeptide. The introduction of a nonsense mutation by transversion or transition of at least one (1) nucleobase within the coding region has a similar effect. Owing to the stop codon that forms, there is premature termination of translation. The stated measures are preferably carried out in the region between the start codon and the penultimate coding codon, more preferably in the 5′-terminal portion of the coding region, which codes for the N-terminus of the polypeptide. If the total length of a polypeptide (measured as the number of chemically bound L-amino acids) is designated as 100%, then the portion of the amino acid sequence which, reckoned from the start amino acid L-formyl methionine, contains 80% of the subsequent L-amino acids, belongs to the N-terminus of the polypeptide.
Genetic measures for switching off malate-quinone oxidoreductase (Mqo) or reducing its expression are described for example in U.S. Pat. No. 7,094,106. U.S. Pat. No. 7,094,106 describes switching off the mqo gene by incorporating deletions or insertions of at least one base pair or substitutions generating a stop codon into the mqo gene, wherein reduction of expression was achieved by placing the expression of the mqo gene under the control of the E. coli trc promoter/LacIq repressor system.
Genetic measures for switching off malate dehydrogenase (Mdh) are described for example in WO 02/02778 (equivalent to U.S. Pat. No. 6,995,002). In WO 02/02778, the mdh gene was switched off by the insertion of a plasmid unable to replicate in Corynebacterium glutamicum comprising a central part of the coding region of the mdh gene into the host mdh gene by homologous recombination.
Genetic measures for switching off the E1p subunit (AceE) of the pyruvate dehydrogenase complex are described for example in EP 06119615 and in Schreiner et al. (Journal of Bacteriology 187(17), 6005-6018 (2005)). EP 06119615 and Schreiner et al. describe switching off the aceE gene by deleting a central part of the coding region of the aceE gene.
It is also possible, by suitable amino acid substitutions, to lower the catalytic property of the polypeptide in question.
In the case of malate-quinone oxidoreductase (Mqo) this can be achieved, as described in WO 06/077004, by preparing or using alleles of the mqo gene of SEQ ID NO: 23, which code for an Mqo variant that possesses the amino acid sequence of SEQ ID NO: 24 and contains one or more amino acid substitutions selected from the group
a) substitution of the L-serine at position 111 for another proteinogenic amino acid, preferably L-phenylalanine or L-alanine, and
b) substitution of the L-alanine at position 201 for another proteinogenic amino acid, preferably L-serine.
WO 06/077004 (equivalent to U.S. Pat. No. 7,214,526) describes an isolated coryneform bacterium mutant which comprises a gene encoding a polypeptide possessing malate quinone oxidoreductase enzyme activity, wherein the polypeptide comprises an amino acid sequence in which any proteinogenic amino acid except L-serine is present at position 111 or a comparable position.
Strains that comprise an mqo allele that codes for an Mqo variant which comprises the amino acid sequence of SEQ ID NO: 24, and contains L-phenylalanine at position 111, are preferred.
By the measures of switching-off or attenuation, the activity or concentration of the corresponding protein is generally lowered 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 initial strain or parent strain.
The performance of the produced bacteria of the genus Corynebacterium or of the fermentation process using the produced bacteria with respect to one or more parameters selected from L-amino acid concentration (L-amino acid formed per volume), L-amino acid yield (L-amino acid formed per carbon source consumed), L-amino acid formation (L-amino acid formed per volume and time) and the specific L-amino acid formation (L-amino acid formed per cell dry mass or dry biomass and time or L-amino acid formed per cell protein and time) or other process variables and combinations thereof, is increased by at least 0.5%, at least 1%, at least 1.5% or at least 2% relative to the initial strain or parent strain or the fermentation process using them.
The produced bacteria of the genus Corynebacterium can be cultivated continuously, as described for example in PCT/EP2004/008882, or discontinuously in a batch process, a fed batch process or a repeated fed batch process, for the purpose of production of the desired L-amino acids. A summary of a general nature covering known culture methods is given in Chmiel's textbook (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)). PCT/EP2004/008882 (equivalent to WO 05/021772 and DE 10339847) describes a fermentation process comprising incubating and culturing in at least first nutrient medium a coryneform bacterium producing L-lysine, feeding continuously further nutrient media to the culture in one or several streams and removing at the same time culturing broth with a removal stream or streams corresponding to the feed streams, wherein over the entire period of time of feeding and removing concentration of the source of carbon is not more than 10 g/L and L-lysine is formed.
The culture medium or fermentation medium to be used matches the requirements of the particular strains. Descriptions of culture media for various microorganisms are given in “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are interchangeable.
Sugars and carbohydrates, for example glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from sugar beet or sugar cane production, starch, hydrolyzed starch and cellulose, oils and fats, for example soya oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example palmitic acid, stearic acid and linoleic acid, alcohols, for example glycerol, methanol and ethanol and organic acids, for example acetic acid or lactic acid can be used as the carbon source. These materials can be used individually or as a mixture.
Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used as the nitrogen source. The nitrogen sources 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 phosphorus source.
The culture medium in addition contains salts, for example, in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium and iron, for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances can be used, such as amino acids for example homoserine and vitamins for example thiamine, biotin or pantothenic acid, in addition to the aforementioned substances.
The aforementioned ingredients can be added to the culture as a single charge, or can be supplied in a suitable manner during cultivation.
Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner for pH control of the culture. The pH is generally adjusted to a value of 6.0 to 9.0, preferably 6.5 to 8. Antifoaming agents, for example polyglycol esters of fatty acids, can be used for controlling foaming. To maintain the stability of plasmids, suitable substances with selective action, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, for example air, are fed into the culture. The use of liquids enriched with hydrogen peroxide is also possible. Optionally, the fermentation is carried out at excess pressure, for example at a pressure of 0.03 to 0.2 MPa. The temperature of the culture is normally in the range from 20° C. to 45° C. and preferably from 25° C. to 40° C. In batch processes, cultivation is continued until a maximum of the desired L-amino acid has formed at given conditions. This goal is normally reached within 10 hours to 160 hours. Longer cultivation times are possible with continuous processes. The activity of the bacteria leads to enrichment (accumulation) of the L-amino acid in the fermentation medium and/or in the bacterial cells.
Examples of suitable fermentation media are given inter alia in patent specifications U.S. Pat. No. 5,770,409, U.S. Pat. No. 5,840,551 and U.S. Pat. No. 5,990,350 or U.S. Pat. No. 5,275,940.
Methods for the determination of L-amino acids are known. The analysis can be carried out for example as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion-exchange chromatography followed by ninhydrin derivatization, or it can be carried out by reversed phase HPLC, as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
Accordingly, the invention also relates to a method of production of an L-amino acid, wherein the following steps are carried out:
a) fermentation of the bacteria according to the invention in a suitable nutrient medium,
b) accumulation of the L-amino acid in the nutrient medium or in the cells of said bacteria.
These steps may be followed by collecting of the L-amino acid that accumulated in the nutrient medium, in the fermentation broth or in the cells of the bacteria, in order to obtain a solid or a liquid product.
A fermentation broth means a fermentation medium or nutrient medium in which a microorganism has been cultivated for a certain time and at a certain temperature. The fermentation medium or the media used during fermentation contain(s) all substances or components for ensuring multiplication of the microorganism and formation of the desired amino acid.
At the end of fermentation, the resulting fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism, formed as a result of multiplication of the cells of the microorganism, b) the desired L-amino acid that formed in the course of fermentation, such as L-lysine, L-valine or L-isoleucine, c) the organic by-products that formed in the course of fermentation, and d) the constituents of the fermentation medium or of the ingredients for example vitamins such as biotin, amino acids such as homoserine or salts such as magnesium sulfate, that were not consumed in the fermentation.
The organic by-products include substances which may be produced and may be excreted by the microorganisms used in the fermentation in addition to the particular desired organic compound. These also include sugars, for example trehalose.
In the case of the amino acids L-valine and L-isoleucine, isolation and purification, for example using one or more methods selected from the group comprising chromatographic techniques, crystallization techniques and the use of activated charcoal, is preferred, so that pure products are largely obtained, for example products with purity of ≧90 wt. % or ≧95 wt. %.
In the case of the amino acid L-lysine, essentially four different product forms are known.
One group of L-lysine-containing products comprises concentrated, aqueous, alkaline solutions of purified L-lysine (EP-B-0534865). Another group, as described for example in U.S. Pat. No. 6,340,486 and U.S. Pat. No. 6,465,025, comprises aqueous, acidic, biomass-containing concentrates of L-lysine-containing fermentation broths. Another group of solid products comprises powder or crystalline forms of purified or pure L-lysine, which may bey in the form of a salt, for example L-lysine monohydrochloride. Yet another group of solid product forms is described for example in EP-B-0533039. The product form described there contains, in addition to L-lysine, most of the ingredients employed during fermentation but not consumed, and possibly the biomass of the microorganism used at a proportion of >0%-100%.
In accordance with the various product forms, a great variety of methods is known for collecting, isolating or purifying the L-lysine from the fermentation broth, in order to produce an L-lysine-containing product or purified L-lysine.
Solid, pure L-lysine may be produced using methods of ion-exchange chromatography possibly with the use of activated charcoal and crystallization techniques. In this way we obtain the corresponding base or a corresponding salt, for example the monohydrochloride (Lys-HCl) or lysine sulfate (Lys2-H2SO4).
A method of production of aqueous, basic L-lysine-containing solutions from fermentation broths is described in EP-B-0534865. In the method described there, the biomass is separated from the fermentation broth and discarded. A pH between 9 and 11 is established by means of a base, for example, sodium, potassium or ammonium hydroxide. The mineral constituents (inorganic salts) are separated from the broth after concentration and cooling, and either used as fertilizers or discarded.
In the case of methods for production of lysine using the bacteria, methods are preferred in which products are obtained that contain the constituents of the fermentation broth. These are used in particular as animal feed additives.
Depending on what is required, the biomass can be removed from the fermentation broth completely or partially by separation techniques such as centrifugation, filtration, decanting or a combination thereof, or it can be left in it completely. Optionally, the biomass or the fermentation broth containing the biomass is inactivated during a suitable process step, for example, by thermal treatment (heating) or by adding acid.
In one embodiment, the biomass is removed completely or almost completely, so that the finished product has a biomass content of zero (0%) or max. 30%, max. 20%, max. 10%, max. 5%, max. 1% or max. 0.1%. In another embodiment, the biomass is not removed or only a small proportion is removed, so that the finished product contains all the biomass (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass. In a method according to the invention, the biomass is accordingly removed in proportions from ≧0% to ≦100%.
Finally, the fermentation broth obtained after the fermentation can be adjusted to an acid pH with an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid or organic acid such as propionic acid, before or after complete or partial removal of the biomass (GB 1,439,728 or EP 1 331 220). It is also possible to acidify the fermentation broth still containing all the biomass. Finally, the broth can also be stabilized by adding sodium bisulfite (NaHSO3, GB 1,439,728) or another salt for example ammonium, alkali or alkaline-earth salt of sulfurous acid.
In separating the biomass, any organic or inorganic solids contained in the fermentation broth are removed partially or completely. The organic by-products dissolved in the fermentation broth and the dissolved, unconsumed constituents of the fermentation medium (ingredients) remain in the product at least partially (>0%), preferably to at least 25%, preferably to at least 50% and more preferably to at least 75%. Optionally, these also remain in the product completely (100%) or almost completely, i.e. >95% or >98% or over 99%. If a product in this sense contains at least a proportion of the constituents of the fermentation broth, it is also described with the term “product based on fermentation broth”.
Then the broth is dewatered or thickened or concentrated using known methods, e.g. by means of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. This concentrated fermentation broth can then be processed by techniques of freeze drying, spray drying, spray granulation or by other methods, for example the circulating fluidized bed as described in PCT/EP2004/006655, to pourable products and preferably to a fine powder or preferably coarse granules. If required, a desired product can be isolated from the granules thus obtained by sieving or dust separation.
It is also possible for the fermentation broth to be dried directly, i.e. without previous concentration, by spray drying or spray granulation.
“Pourable” means powders which, from a series of glass discharge vessels with outlet openings of different sizes, are discharged freely from the vessel with the 5 mm (millimeter) opening (Klein: Seifen, Öle, Fette, Wachse 94, 12 1968)).
“Fine” means a powder having mainly (>50%) a grain size of 20 to 200 μm diameter. “Coarse” means a product having mainly (>50%) a grain size from 200 to 2000 μm diameter.
Grain size can be determined using methods of laser diffraction spectrometry. The relevant methods are described in the textbook on “Particle Size Measurement in Laboratory Practice” by R. H. Müller and R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in the textbook “Introduction to Particle Technology” by M. Rhodes, Publ. Wiley & Sons (1998).
The pourable, fine powder can be converted by suitable compaction or granulation techniques to a coarse, storable and largely dust-free product with good pourability.
The term “dust-free” means that the product only contains a small proportion (<5%) of particles with grain size under 100 μm diameter.
“Storable”, in the sense of this invention, means a product that can be stored in cool, dry conditions for at least one (1) year or longer, preferably at least 1.5 years or longer, more preferably two (2) years or longer, without any substantial loss (max. 5%) of the particular amino acid.
The invention further relates to a method of manufacturing an L-lysine comprising product, which is described in broad outline in DE 102006016158, and in which the fermentation broth obtained using the microorganisms according to the invention, from which the biomass has been optionally separated completely or partially, is further processed, by carrying out a process that comprises at least the following steps:
a) the pH is lowered to 4.0-5.2, preferably 4.9-5.1, by adding sulfuric acid, and a sulfate/L-lysine molar ratio of 0.85-1.2, preferably 0.9-1.0, more preferably >0.9 to <0.95, is established in the broth, if necessary by adding one or more additional sulfate-containing compound(s) and
b) the mixture thus obtained is concentrated by dewatering, and optionally granulated,
optionally with one or both of the following measures being carried out before step a):
c) measurement of the sulfate/L-lysine molar ratio for determining the required amount of sulfate-containing compound(s)
d) addition of a sulfate-containing compound selected from the group comprising ammonium sulfate, ammonium hydrogensulfate and sulfuric acid in suitable proportions.
Optionally, also prior to step b), a salt of sulfurous acid, preferably an alkali metal hydrogensulfite, and more preferably sodium hydrogensulfite, at a concentration of 0.01-0.5 wt. %, preferably 0.1-0.3 wt. %, more preferably 0.1-0.2 wt. % relative to the fermentation broth is used.
DE 102006016158 (equivalent to US2007082031 and WO 07/042363) describes relatively light and thermally stable granulated fermentation-broth-based animal feed additives having a high content of L-lysine and low-loss methods for production the additives from broths obtained by fermentation.
As preferred sulfate-containing compounds in the sense of the aforementioned process steps we may mention in particular ammonium sulfate and/or ammonium hydrogensulfate or corresponding mixtures of ammonia and sulfuric acid and sulfuric acid itself.
The sulfate/L-lysine molar ratio V is calculated from the formula: V=2×[SO42−]/[L-lysine]. This formula takes account of the fact that the SO42− anion, or sulfuric acid, is divalent. A ratio V=1 means that the Lys2-H2SO4 is of stoichiometric composition, whereas at a ratio of V=0.9 there is a 10% sulfate deficit and at a ratio of V=1.1 there is a 10% sulfate excess.
During granulating or compacting the usual organic or inorganic auxiliaries, or carriers such as starch, gelatin, cellulose derivatives or similar substances, as are usually employed in the processing of foodstuffs or animal feed as binders, gelling agents or thickeners, or other substances for example silicic acids, silicates (EP0743016A) or stearates may be used.
Treatment the surface of the obtained granules with oils, as described in WO 04/054381 may be used. The oils used can be mineral oils, vegetable oils or mixtures of vegetable oils. Examples of such oils are soya oil, olive oil, soya oil/lecithin mixtures. Similarly, silicone oils, polyethylene glycols or hydroxyethyl cellulose are also suitable. Treatment of the surfaces with the aforesaid oils gives increased abrasion resistance of the product and a reduction in the proportion of dust. The content of oil in the product is 0.02-2.0 wt. %, preferably 0.02-1.0 wt. %, and more preferably 0.2-1.0 wt. % relative to the total amount of the feed additive.
Products are preferred having a proportion of ≧97 wt. % of a grain size from 100 to 1800 μm or a proportion of ≧95 wt. % of a grain size from 300 to 1800 μm diameter. The proportion of dust, i.e. particles with a grain size <100 μm, is preferably >0 to 1 wt. %, max. 0.5 wt. % being more preferred.
Alternatively, the product can also be coated with an organic or inorganic carrier that is known and usual in animal feed processing, for example silicic acids, silicates, grits, bran, meal or flour, starch, sugars or other substances and/or mixed and stabilized with usual thickeners or binders. Examples of applications and the methods employed are described in the literature (Die Mühle+Mischfuttertechnik 132 (1995) 49, page 817).
Finally, using coating processes with film-forming agents such as metal carbonates, silicic acids, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920, the product can be brought to a state in which it is stable against digestion in animal stomachs preferably the stomach of ruminants. DE4100920 (equivalent to U.S. Pat. No. 5,279,832 and EP 0495349) describes an active-substance preparation for oral administration, preferably for ruminants, comprising an active-substance core comprising at least one biologically active substance and a coating around this core which delays the release of the core after oral administration due to its geometrical shape as well as a method of preparing an accordingly shaped core pellet by coating.
For establishing a desired L-lysine concentration in the product, depending on requirements, the L-lysine can be added during the process in the form of a concentrate or optionally a substantially pure substance or salt thereof in liquid or solid form. These can be added individually or as mixtures to the fermentation broth obtained or concentrated, or alternatively during the drying or granulation process.
The invention relates further to a method of production of a solid lysine-containing product, as described in broad outline in US 20050220933, and which comprises the processing of the fermentation broth obtained using the microorganisms according to the invention, in the following steps:
a) filtration of the fermentation broth, preferably with a membrane filter, so that a biomass-containing sludge and a filtrate are obtained,
b) concentration of the filtrate, preferably so that a solids content of 48-52 wt. % is obtained,
c) granulation of the concentrate obtained in step b), preferably at a temperature from 50° C. to 62° C., and
d) coating of the granules obtained in c) with one or more coating agent(s)
For the coating in step d), it is preferable to use coating agents that are selected from the group comprising
d1) the biomass obtained in step a)
d2) a compound containing L-lysine, preferably selected from the group comprising L-lysine hydrochloride or L-lysine sulfate,
d3) an essentially L-lysine-free material with L-lysine content <1 wt. %, preferably <0.5 wt. %, preferably selected from the group consisting of starch, carrageenan, agar, silicic acids, silicates, grits, bran and meal, and
d4) a water-repellent substance, preferably selected from the group consisting of oils, polyethylene glycols and liquid paraffins.
The content of L-lysine is adjusted to a desired value by the measures corresponding to steps d1) to d4), preferably d1) to d3).
In the production of L-lysine-containing products, the ratio of the ions is preferably adjusted so that the molar ionic ratio according to the following formula

2x[SO42−]+[Cl−]−[NH4+]−[Na+]−[K+]−2x[Mg2+]−2x[Ca2+]/[L-Lys] is 0.68-0.95,

preferably 0.68-0.90, as described by Kushiki et al. in US 20030152633.

In the case of lysine, the solid product based on fermentation broth produced in this way has a lysine content (lysine base) from 10 wt. % to 70 wt. % or 20 wt. % to 70 wt. %, preferably 30 wt. % to 70 wt. % and more preferably from 40 wt. % to 70 wt. % based on the dry mass of the product. Maximum contents of lysine base of 71 wt. %, 72 wt. %, 73 wt. % are also possible. The water content of the L-lysine-containing, solid product is up to 5 wt. %, preferably up to 4 wt. %, and more preferably less than 3 wt. %. Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
EXAMPLES
Example 1
Sequencing of the gltA Gene of the Strain DM678
The strain Corynebacterium glutamicum DM678 (U.S. Pat. No. 6,861,246) is a lysine-producing strain developed by mutagenesis and screening. It is auxotrophic for L-threonine and L-methionine-sensitive. The strain was deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as DSM12866.
The method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) was used to isolate chromosomal DNA from the strain DM678. The polymerase chain reaction was used to amplify a DNA segment which harbors the gltA gene. The following oligonucleotides were used as primers for this:









gltA_XL-A1 (SEQ ID NO: 27):



5′ tgagttctattggcgtgacc 3′



 



gltA_XL-E1 (SEQ ID NO: 28):



5′ ttcgccaacgatgatgtcag 3′






The depicted primers were synthesized by MWG Biotech (Ebersberg, Germany). They make it possible to amplify a DNA segment which was about 1.8 kb long and harbors the gltA gene. The primer gltA_XL-A1 binds to the region corresponding to position 490 to 509 of the strand complementary to SEQ ID NO: 3 (and SEQ ID NO: 7). The primer gltA_XL-E1 binds to the region corresponding to position 2266 to 2247 of the strand shown in SEQ ID NO: 3 (and SEQ ID NO: 7).
The PCR reaction was carried out using the Phusion high fidelity DNA polymerase (New England Biolabs, Frankfurt, Germany). The reaction mixture was made up as specified by the manufacturer and contained 10 μl of the 5× Phusion HF buffer supplied, deoxynucleoside triphosphates each in a concentration of 200 μM, primers in a concentration of 0.5 μM, approximately 50 ng of template DNA and 2 units of Phusion polymerase in a total volume of 50 μl. The volume was adjusted to 50 μl by adding H2O.
The PCR mixture was first subjected to an initial denaturation at 98° C. for 30 seconds. This was followed by a denaturation step at 98° C. for 20 seconds, repeated 35×, a step for binding the primers to the introduced DNA at 60° C. for 20 seconds and the extension step to extend the primers at 72° C. for 60 seconds. After the final extension step at 72° C. for 5 minutes, the PCR mixture was subjected to an agarose gel electrophoresis (0.85% agarose). A DNA fragment about 1.8 kb long was identified, isolated from the gel and purified using the QIAquick gel extraction kit from Qiagen (Hilden, Germany).
The nucleotide sequence of the amplified DNA fragment or PCR product was determined by Agowa (Berlin, Germany).
The nucleotide sequence of the coding region of the gltA allele from the strain DM678 contains thymine as nucleobase at position 14. The wild-type gene (see SEQ ID NO: 1) contains adenine as nucleobase at this position. This adenine-thymine transversion leads to an amino acid exchange from aspartate to valine at position 5 of the resulting amino acid sequence. This mutation is referred to hereinafter as gltAD5V. The allele gltAD5V is depicted in SEQ ID NO: 5, and the amino acid sequence of the protein which was revealed with the aid of the Patentin program is depicted in SEQ ID NO: 6.
Example 2
Construction of the Exchange Vector pK18mobsacB_gltAD5V
The polymerase chain reaction was used to amplify a DNA fragment which harbors part of the upstream region of the gltA gene and part of the coding region which contains the gltAD5V mutation. The chromosomal DNA obtained in example 1 from DM678 was used as template. The following oligonucleotides were selected as primers for the PCR:









gltA_1.p (SEQ ID NO: 29):



5′ CCGTCGACAATAGCCTGAA 3′



 



gltA_2.p (SEQ ID NO: 30):



5′ CC-GAATTC-TTCGAGCATCTCCAGAAC 3′






They were synthesized by MWG Biotech (Ebersberg, Germany) and make it possible to amplify a DNA segment about 1.7 kb long comprising 832 bp of the upstream region and nucleotides 1-855 bp of the coding region of the gltA gene from DM678.
The primer gltA—1.p binds to the region corresponding to position 169 to 187 of the strand complementary to SEQ ID NO: 25. Nucleotides 9 to 26 of the primer gltA—2.p bind to the region corresponding to position 1855 to 1838 of the strand shown in SEQ ID NO: 25. In addition, the primer gltA—1.p contains the native cleavage site of the restriction enzyme SalI, and the primer gltA—2.p contains the sequence of the cleavage site of the restriction enzyme EcoRI, which are each marked by underlining in the nucleotide sequence depicted above.
The PCR reaction was carried out using the Phusion high fidelity DNA polymerase (New England Biolabs, Frankfurt, Germany). The reaction mixture had the composition described above. The PCR was carried out as described above. The nucleotide sequence of the amplicon about 1.7 kb long is depicted in SEQ ID NO: 31.
The amplicon was treated with the restriction endonucleases SalI and EcoRI and identified by electrophoresis in a 0.8% agarose gel. It was subsequently isolated from the gel and purified using the QIAquick gel extraction kit from Qiagen.
The DNA fragment purified in this way contains the described gltAD5V mutation and has ends compatible with DNA cut with SalI and EcoRI (respectively gltAD5V fragment and gltA′ in the FIGURE). It was subsequently cloned into the mobilizable vector pK18mobsacB described by Schäfer et al. (Gene, 145, 69-73 (1994)) in order to make an allelic or mutation substitution possible. For this purpose, pK18mobsacB was digested with the restriction enzymes EcoRI and SalI, and the ends were dephosphorylated with alkaline phosphatase (alkaline phosphatase, Boehringer Mannheim, Germany). The vector prepared in this way was mixed with the gltAD5V fragment, and the mixture was treated with the ready-to-go T4 DNA ligase kit (Amersham-Pharmacia, Freiburg, Germany).
Subsequently, the E. coli strain S17-1 (Simon et al., Bio/Technologie 1: 784-791, 1993) was transformed with the ligation mixture (Hanahan, In. DNA cloning. A practical approach. Vol. 1. ILR-Press, Cold Spring Harbor, N.Y., 1989). Selection of plasmid-harboring cells took place by plating out the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, N.Y., 1989) which had been supplemented with 25 mg/l kanamycin.
Plasmid DNA was isolated from a transformant using the QIAprep spin miniprep kit from Qiagen and checked by restriction cleavage with the enzymes SalI and EcoRI and subsequent agarose gel electrophoresis. The plasmid was called pK18mobsacB_gltAD5V and is depicted in the FIGURE. The abbreviations and designations used have the following meaning. The stated numbers of base pairs are approximations obtained within the scope of the reproducibility of measurements.













Kan:
kanamycin-resistance gene


SalI:
cleavage site of the restriction enzyme SalI


EcoRI:
cleavage site of the restriction enzyme EcoRI


gltA’:
cloned DNA fragment containing the gltAD5V mutation


sacB:
sacB gene


RP4-mob:
mob region with the origin of replication for transfer (oriT)


oriV:
origin of replication V









Example 3
Incorporation of the gltAD5V Mutation into the Strain Corynebacterium glutamicum DM1797
The intention was to introduce the gltAD5V mutation into the strain Corynebacterium glutamicum DM1797. The strain DM1797 was an aminoethylcysteine-resistant mutant of Corynebacterium glutamicum ATCC13032 and described in PCT/EP/2005/012417. It was deposited under the number DSM16833 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany).
The vector pK18mobsacB_gltAD5V described in example 2 was transferred by conjugation according to the protocol of Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the C. glutamicum strain DM1797. The vector was incapable of independent replication in DM 1797 and was retained in the cell only if it was integrated into the chromosome as the result of a recombination event. Selection of transconjugants, i.e. of clones having integrated pK18mobsacB_gltAD5V, took place by plating out the conjugation mixture on LB agar which had been supplemented with 25 mg/l kanamycin and 50 mg/l of nalidixic acid. Kanamycin-resistant transconjugants were subsequently streaked onto LB agar plates supplemented with kanamycin (25 mg/l) and incubated at 33° C. for 24 hours. Mutants in which, as a result of a second recombination event, excision of the plasmid had taken place were selected by culturing the clones nonselectively in LB liquid medium for 30 hours, then streaking onto LB agar, which had been supplemented with 10% sucrose, and incubating at 33° C. for 24 hours.
The plasmid pK18mobsacB_gltAD5V contains, just like the initial plasmid pK18mobsacB, besides the kanamycin-resistance gene a copy of the sacB gene which codes for the levan sucrase from Bacillus subtilis. The sucrose-inducible expression of the sacB gene leads to the formation of levan sucrase which catalyzes the synthesis of the product levan which is toxic for C. glutamicum. Thus, the only clones to grow on sucrose-supplemented LB agar were those in which the integrated pK18mobsacB_gltAD5V has been excised as the result of a second recombination event. Depending on the position of the second recombination event in relation to the site of mutation, the excision is associated with allelic substitution or incorporation of the mutation instead, or the original copy remains in the host's chromosome.
A clone in which the desired exchange, i.e. the incorporation of the gltAD5V mutation, had taken place was then sought. For this purpose, the sequence of the gltA gene was determined for 10 clones with the phenotype “growth in the presence of sucrose” and “non-growth in the presence of kanamycin”. In this way, a clone harboring the gltAD5V mutation was identified. This strain was designated C. glutamicum DM1797_gltAD5V.
Example 4
Production of L-lysine
The strain DM1797_gltAD5V obtained in example 3 and the initial strain DM1797 were cultured in a nutrient medium suitable for producing lysine, and the lysine content in the culture supernatant was determined.
For this purpose, the clones were initially grown on brain-heart agar plates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. These agar plate cultures were each used for inoculation of a preculture (10 ml of medium in a 100 ml Erlenmeyer flask). The medium MM was used as medium for the precultures. The precultures were incubated at 33° C. and 240 rpm on a shaker for 24 hours. Each of these precultures was used to inoculate a main culture, so that the initial OD (660 nm) of the main culture was 0.1 OD. The medium MM was likewise used for the main cultures.
















Medium
MM











CSL
5
g/l



MOPS
20
g/l



Glucose (autoclaved separately)
50
g/l



Salts:





(NH4)2SO4)
25
g/l



KH2PO4
0.1
g/l



MgSO4 * 7 H2O
1.0
g/l



CaCl2 * 2 H2O
10
mg/l



FeSO4 * 7 H2O
10
mg/l



MnSO4 * H2O
5.0
mg/l



Biotin (sterilized by filtration)
0.3
mg/l



Thiamine * HCl (sterilized by filtration)
0.2
mg/l



CaCO3 
25
g/l










CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and the salt solution were adjusted to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions, and the dry autoclaved CaCO3, were then added.
Culturing took place in volumes of 10 ml which were present in 100 ml Erlenmeyer flasks with baffles. The temperature was at 33° C., the number of revolutions was 250 rpm and the humidity was 80%.
After 48 hours, the optical density (OD) was determined at a measurement wavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed was determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion-exchange chromatography and post-column derivatization with ninhydrin detection.











TABLE 1







Strain
OD (660)
Lysine HCl (g/l)









DM1797
11.9
4.8



DM1797_gltAD5V
11.2
5.3










Example 5
Incorporation of the gltAD5V Mutation into the Strain Brevibacterium lactofermentum FERM BP-1763
It was intended to introduce the gltAD5V mutation into the strain Brevibacterium lactofermentum FERM BP-1763. The strain FERM BP-1763 is a mycophenolic acid-resistant valine producer (U.S. Pat. No. 5,188,948). It is auxotrophic for L-isoleucine and L-methionine.
The vector pK18mobsacB_gltAD5V described in example 2 was transferred by conjugation according to the protocol of Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the strain FERM-BP-1763. The vector was incapable of independent replication in FERM BP-1763 and was retained in the cell only if it was integrated into the chromosome as the result of a recombination event. Selection of transconjugants, i.e. of clones having integrated pK18mobsacB_gltAD5V, took place by plating out the conjugation mixture on LB agar which had been supplemented with 25 mg/l kanamycin and 50 mg/l of nalidixic acid. Kanamycin-resistant transconjugants were subsequently streaked onto LB agar plates supplemented with kanamycin (25 mg/l) and incubated at 33° C. for 24 hours. Mutants in which, as a result of a second recombination event, excision of the plasmid had taken place were selected by culturing the clones nonselectively in LB liquid medium for 30 hours, then streaking onto LB agar, which had been supplemented with 10% sucrose, and incubating at 33° C. for 24 hours.
The plasmid pK18mobsacB_gltAD5V contained, just like the initial plasmid pK18mobsacB, besides the kanamycin-resistance gene a copy of the sacB gene which coded for the levan sucrase from Bacillus subtilis. The sucrose-inducible expression of the sacB gene led to the formation of levan sucrase which catalyzes the synthesis of the product levan which was toxic for C. glutamicum. Thus, the only clones to grow on sucrose-supplemented LB agar were those in which the integrated pK18mobsacB_gltAD5V has been excised as the result of a second recombination event. Depending on the position of the second recombination event in relation to the site of mutation, the excision was associated with allelic substitution or incorporation of the mutation instead, or the original copy remains in the host's chromosome.
A clone in which the desired exchange, i.e. the incorporation of the gltAD5V mutation, had taken place was then sought. For this purpose, the sequence of the gltA gene was determined for 10 clones with the phenotype “growth in the presence of sucrose” and “non-growth in the presence of kanamycin”. In this way, a clone harboring the gltAD5V mutation was identified. This strain was designated C. glutamicum FERM BP-1763_gltAD5V.
Example 6
Production of L-valine
The strain FERM BP-1763_gltAD5V obtained in example 5 and the initial strain FERM BP-1763 were cultured in a nutrient medium suitable for producing valine, and the valine content in the culture supernatant was determined.
For this purpose, the clones were initially grown on brain-heart agar plates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. These agar plate cultures were each used for inoculation of a preculture (10 ml of medium in a 100 ml Erlenmeyer flask).
The medium CgIII (2.5 g/l NaCl, 10 g/l Bacto peptone, 10 g/l Bacto yeast extract, pH 7.4, 20 g/l glucose (autoclaved separately) was used for the precultures. The precultures were incubated at 33° C. and 240 rpm on a shaker for 24 hours. Each of these precultures was used to inoculate a main culture, so that the initial OD (660 nm) of the main culture was 0.1 OD. The medium MM was likewise used for the main cultures.
















Medium
MM











CSL
5
g/l



MOPS
20
g/l



Glucose (autoclaved separately)
50
g/l



Salts:





(NH4)2SO4)
25
g/l



KH2PO4
0.1
g/l



MgSO4 * 7 H2O
1.0
g/l



CaCl2 * 2 H2O
10
mg/l



FeSO4 * 7 H2O
10
mg/l



MnSO4 * H2O
5.0
mg/l



Isoleucine (sterilized by filtration)
0.1
g/l



Methionine (sterilized by filtration)
0.1
g/l



Leucine (sterilized by filtration)
0.1
g/l



Thiamine * HCl (sterilized by filtration)
0.2
mg/l



CaCO3
25
g/l










CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and the salt solution were adjusted to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions, and the dry autoclaved CaCO3, were then added.
Culturing took place in volumes of 10 ml which were present in 100 ml Erlenmeyer flasks with baffles. The temperature was at 33° C., the number of revolutions was 250 rpm and the humidity was 80%.
After 48 hours, the optical density (OD) was determined at a measurement wavelength of 660 nm using the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of valine formed was determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion-exchange chromatography and post-column derivatization with ninhydrin detection.











TABLE 2







Strain
OD (660)
Valine (g/l)









FERM BP-1763
8.4
11.9



FERM BP-1763_dltAD5V
7.8
12.6










German patent application 102006032634.2, filed Jul. 13, 2006, and U.S. provisional application 60/830,331, filed Jul. 13, 2006, are incorporated herein by reference.
Numerous modification and variations on the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

## Claims

1. A method for producing L-lysine comprising:
a) fermenting a recombinant bacterium in a suitable nutrient medium to produce a fermentation broth containing biomass, wherein said recombinant bacterium comprises a polynucleotide that encodes a polypeptide possessing a citrate synthase activity, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, and (i) wherein L-aspartic acid at position 5 of the amino acid sequence is replaced by L-valine, and
b) accumulating L-lysine in the nutrient medium, fermentation broth or in the cells of said bacterium,
wherein the bacterium is Corynebacterium glutamicum.

2. The method as claimed in claim 1, wherein L-lysine is collected.

3. The method as claimed in claim 1, wherein L-lysine is isolated and purified.

4. The method as claimed in claim 1, wherein at least one member selected from the group consisting of a fermentation broth and biomass remains in the end product in their entirety or in proportions.

5. The method as claimed in claim 1, wherein the method is a batch process.

6. The method as claimed in claim 1, wherein the method is a fed batch process.

7. The method as claimed in claim 1, wherein the method is a continuous process.

8. The method as claimed in claim 1, wherein water is extracted from an L-lysine-containing fermentation broth and a product with a maximum water content of 5 wt. % is obtained, wherein the product is the L-amino acid.

9. The method as claimed in claim 1, wherein an L-lysine-containing fermentation broth is first concentrated and then spray-dried or spray-granulated.

10. The method as claimed in claim 1, further comprising;
a) lowering the pH value to 4.0 to 5.2 by adding sulfuric acid, and establishing a sulfate/L-lysine molar ratio of 0.85 to 1.2 in the broth, if necessary by adding at least one sulfate-containing compound, and
b) concentrating the obtained mixture by dewatering and, optionally, granulating the mixture.

11. The method as claimed in claim 10, wherein ammonium sulfate and/or ammonium hydrogensulfate or sulfuric acid and ammonia are added as sulfate-containing compound.

12. The method as claimed in claim 10, wherein prior to step a), the sulfate/L-lysine ratio is measured to determine the required amount of sulfate-containing compounds.

13. The method as claimed in claim 9, wherein the surface of the granules obtained is treated with an oil.

14. The method as claimed in claim 1, further comprising:
c) filtering the fermentation broth and obtaining a biomass-containing sludge and a filtrate,
d) concentrating the filtrate and obtaining a solids content of 48-52 wt. %,
e) granulating the concentrate obtained in step d), and
f) coating the granules obtained in step e) with at least one coating agent.

15. The method as claimed in claim 14, wherein at least one coating agent is used selected from the group consisting of
f1) the biomass containing sludge obtained in step c)
f2) a compound containing L-lysine, selected from L-lysine hydrochloride or L-lysine sulfate,
f3) an essentially L-lysine-free material with L-lysine content <1 wt. %, selected from starch, carrageenan, agar, silicic acids, silicates, grits, bran and meal,
f4) a water-repellent substance, selected from oils, polyethylene glycols and liquid paraffins, and
f5) mixtures thereof.

16. The method as claimed in claim 14, wherein step e) is conducted at a temperature from 50° C. to 62° C.

17. The method as claimed in claim 15, wherein the content of L-lysine in (f3) is <0.5 wt. %.

18. The method as claimed in claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5.

19. The method as claimed in claim 1, wherein the polypeptide further comprises (ii) at least one conservative amino acid substitution, and wherein the citrate synthase activity of the polypeptide having the substitutions (i) and (ii) is altered by at most 2% compared to the citrate synthase activity of the polypeptide having the substitution (i).

20. The method as claimed in claim 1, wherein the bacterium further comprises a polynucleotide encoding a polypeptide possessing an aspartate kinase activity, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 20, and at least one amino acid substitution selected from the group consisting of:
a) substitution of L-alanine at position 279 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-threonine (LysC A279T),
b) substitution of L-alanine at position 279 of the encoded aspartate kinase protein SEQ ID NO: 20 for L-valine (LysC A279V),
c) substitution of L-leucine at position 297 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-glutamine (LysC L297Q),
d) substitution of L-serine at position 301 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-phenylalanine (LysC 5301F),
e) substitution of L-serine at position 301 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-tyrosine (LysC 5301Y),
f) substitution of L-threonine at position 308 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-isoleucine (LysC T308I),
g) substitution of L-threonine at position 311 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-isoleucine (LysC T311I),
h) substitution of L-serine at position 317 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-alanine (LysC S317A),
i) substitution of L-arginine at position 320 of the encoded aspartate kinase protein of SEQ ID NO: 20 for glycine (LysC R320G),
j) substitution of glycine at position 345 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-aspartic acid (LysC G345D),
k) substitution of L-threonine at position 380 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-isoleucine (LysC T380I), and
l) substitution of L-serine at position 381 of the encoded aspartate kinase protein of SEQ ID NO: 20 for L-phenylalanine (LysC S381F).

21. The method as claimed in claim 20, wherein the polynucleotide encoding the polypeptide with the aspartate kinase activity is overexpressed.

22. The method as claimed in claim 1, wherein the bacterium additionally possesses at least one character selected from the group consisting of:
a) an overexpressed polynucleotide, encoding a dihydrodipicolinate synthase (DapA),
b) an overexpressed polynucleotide, encoding an aspartate semialdehyde dehydrogenase (Asd),
c) an overexpressed polynucleotide, encoding a diaminopimelate decarboxylase (LysA),
d) an overexpressed polynucleotide, encoding an aspartate aminotransferase (Aat),
e) an overexpressed polynucleotide, encoding a polypeptide with L-lysine exporting activity (LysE),
f) a switched-off or attenuated activity of malate dehydrogenase (Mdh),
g) a switched-off or attenuated activity of malate-quinone oxidoreductase (Mqo),
h) an overexpressed polynucleotide, encoding a pyruvate carboxylase (Pyc), and
i) a switched-off or attenuated activity of the E1p subunit of the pyruvate dehydrogenase complex (AceE).

23. The method as claimed in claim 22, wherein the bacterium comprises all of the characters a) to g).