MXPA06007137A - Process for preparing l-amino acids using strains of the enterobacteriaceae family - Google Patents

Abstract

The invention relates to a process for preparing L-amino acids by fermenting recombinant microorganisms of the Enterobacteriaceae family, characterized in that a) the desired L-amino acid-producing microorganisms, in which the yaaU ORF, or nucleotide sequences or alleles encoding the gene products, is/are enhanced, in particular overexpressed, is cultured in a medium under conditions under which the desired L-amino acid is enriched in the medium or in the cells, and b) the desired L-amino acid is isolated, with, optionally, constituents of the fermentation broth, and/or the biomass remaining in its/their entirety or in portions (from=0 to 100%) in the isolated product or being removed completely.

Description

PROCESS FOR PREPARING L-AMINO ACIDS USING SCRAPS OF THE ENTEROBACTERIACEAE FAMILY Field of the Invention This invention relates to a process for preparing L-amino acids, in particular L-threonine, using recombinant strains of microorganisms of the family Enterobacteriaceae in which, the open reading structure (ORF) designated yaaU is improved, in particular, overexpressed, and said microorganisms.

Background of the Invention L-amino acids, in particular L-threonine, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry and, most particularly, in animal nutrition. It is known that L-amino acids can be prepared by fermentation strains of Enterobacteriaceae, in particular, Escherichia coli (E. coli) and Serratia marcescens. Due to the great importance, efforts are continually being made to improve the preparation methods. Methodological improvements can be related to measures that refer to fermentation technology, such as agitation or oxygen supply, or composition of the REF .: 173651 nutrient medium, such as the concentration of sugar during fermentation, or the raising to the product form, for example, by means of ion exchange chromatography, or the intrinsic operating properties of the microorganism itself. The methods of mutagenesis, selection and choice of mutant are used to improve the operating properties of these microorganisms. This results, in turn, in strains which are resistant to antimetabolites, such as the thiomine analog OI-amino-β-hydroxyvaleric acid (AHV), or auxotrophic, by metabolites of regulatory importance and produce L-amino acids such as L- threonine. For a number of years now, recombinant DNA methods have also been used for the improvement of strains that produce L-amino acid from the Enterobacteriaceae family by amplifying individual amino acid biosynthesis genes and investigating the effect on production. The information compiled on the cellular biology and molecular biology of Escherichia coli and Salmonella, can be found in Neidhardt (ed): Escherichia coli and Salmonella, Cellular and Molecular Biology, 2nd. Edition, ASM Press, Washington, D.C., USA, (1996).

Summary of the Invention The object of this invention is to provide new measures to improve the preparation of L-amino acids, in particular, L-threonine.

Detailed Description of the Invention The invention relates to recombinant microorganisms of the family Enterobacteriaceae, which contains an improved or overexpressed yaaU-ORF, which encodes a polypeptide that is annotated as a putative sugar transporter, and which produces L- amino acids, in particular L-threonine, in a better way. In such a case, the microorganisms which are not recombinant for the yaaU-ORF, and which do not contain any recombinant yaaU-ORF, and which do not contain any improved yaaU-ORF, are used as the starting point for the comparison. These recombinant microorganisms include, in particular, microorganisms of the Enterobacteriaceae family in which a polynucleotide encoding a polypeptide whose amino acid sequence is at least 90%, in particular at least 95%, preferably at least 98%, at least is improved. 99%, particularly preferably 99.7% and very particularly, preferably 100%, identical to one amino acid sequence selected from the group of SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8. Preference is given to amino acid sequences which are identical to those from SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8. Said microorganisms contain enhanced or overexpressed polynucleotides selected from the group: a) polynucleotide having the nucleotide sequence SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; b) polynucleotide having a nucleotide sequence which corresponds to SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7, within the limits of the degeneracy of the genetic code; c) polynucleotide sequence having a sequence which hybridizes, under stringent conditions, to the sequence which is complementary to SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; d) polynucleotide having a sequence of SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7, which contains functionally neutral sense mutants, with the polynucleotides encoding a putative sugar transporter. The invention also relates to a process for fermentatively preparing L-amino acids, in particular L-threonine, using recombinant microorganisms of the family Enterobacteriaceae which, in particular, already produces L-amino acids and in which, at least the open reading structure (ORF) having the designation yaaU, or nucleotide sequences that encode its gene product, is or are improved. Preference is given to the use of microorganisms which are described. When L-amino acids or amino acids are mentioned, which follow, by this means, they mean one or more amino acids, including their salts, selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L- glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-proline, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and L-homoserin. L-threonine is particularly preferred. In this context, the term "improved" describes the increase, in a microorganism, of the intracellular activity or concentration of one or more enzymes or proteins, which are encoded by the corresponding DNA, with for example, the number of copies of the gene or genes, or of the ORF or the ORFs, being increased by at least one (1) copy, making use of a strong promoter operatively linked to the gene or of a gel or allele or ORF which encodes a corresponding enzyme or protein that has a high activity, and where appropriate, these measures are combined. A segment of a nucleotide sequence which encodes, or may encode, a protein and / or polypeptide or ribonucleic acid to which the prior art is incapable of assigning any function, is designated an open reading structure (ORF). After a function has been assigned to the nucleotide sequence segment in question, this segment is generally referred to as a gene. Alleles are generally understood to be alternative forms of a given gene. The forms are distinguished by differences in the nucleotide sequence. In general, the protein or ribonucleic acid, encoded by a nucleotide sequence, i.e. an ORF, a gene or allele, is designated a gene product. Improved measures, particularly overexpression, generally increase the activity or concentration of the corresponding protein by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% , maximum up to 1000% or 2000%, based on the native type protein or on the activity or concentration of the protein in the original strain or microorganism, which is not recombinant for the corresponding enzyme or protein. The microorganism or non-recombinant original strain is understood to be the microorganism in which the measurements according to the invention are carried out.

The invention relates to a process for preparing L-amino acids by recombinant fermentation microorganisms of the Enterobacteriaceae family, characterized in that a) the microorganisms that produce desired L-amino acids, in which the open reading structure yaaU, or nucleotide or allele sequences which encode the products of the gene thereof, is / are improved, in particular overexpressed, are cultured in a medium under conditions in which the desired L-amino acid is enriched in medium or in cells, and b) the desired L-amino acid is isolated, with, optionally, the constituents of the fermentation broth and / or the biomass remaining in the whole or in portions (from _> g to 100%) in the isolated product or being completely removed. Microorganisms which have an improved or overexpressed open reading structure (ORF), designated yaaU, and which are in particular recombinants, are likewise part of the subject matter of the present invention, can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, where appropriate starch and where appropriate cellulose or glycerol and ethanol. The microorganisms are representative of the family Enterobacteriaceae and are selected from the genera Escherichia, Erwinia, .Providencia y Serratia. The genera Escherichia and Serratia are preed. The species of Escherichia coli can be mentioned, in particular, in the case of the genus Escherichia, while the species Serratia marcescens can be mentioned, in particular, in conjunction with the genus Serratia. In general, the recombinant organisms are generated by means of transformation, transduction or conjugation, or a combination of these methods, with a vector which contains the desired ORF, the desired gene, an allele of this ORF or gene, or parts of the themselves, and / or a promoter, which improves the expression of the ORF or gene. This promoter can be the promoter which has been produced by improving the mutation from the endogenous regulatory sequences located upstream of the gene or ORF; alternatively, an efficient promoter has been fused to the ORF gene. Examples of suitable strains which, in particular, produce L-threonine of the genus Escherichia, in particular of the species Escherichia coli are Escherichia coli H4581 (EP 0 301 572) -Escherichia coli KY10935 (Document Bioscience Biotechnology and Biochemistry 61 (11 ): 1877-1882 (1997) -Escherichia coli VNIIgenetics MG442 (US-A-4278,765) Escherichia coli VNIIgenetics MI (US-A-4,321,325) -Escherichia coli VNIIgenetics 472T23 (US-A-5,631,157) -Escherichia coli BKIIM B-3996 (US-A-5,175,107) -Escherichia coli cat 13 (WO 98 / 04715) -Escherichia coli KCCM-10132 (WO 00/09660) Examples of suitable L-threonine producing strains of the genus Serratia, in particular of the Serratia marcescens species, are Serratia marcescens HNr21 (Applied and Environmental Microbiology 38 Document ( 6): 1045-1051 (1979)). -Serratia marcescens TLrl56 (Document Gene 57 (2-3): 151-158 (1987)) -Serratia marcescens T-2000 (Applied Biochemistry and Biotechnology 37 (3): 255-265 (1992)) The strains that produce L -threonine from the family Enterobacteriaceae, preferably possesses, inter alia, one or more of the genetic or phenotypic characteristics selected from the group: resistance to a-amino-β-hydroxyvaleric acid, resistance to tialisin, resistance to methionine, resistance to a-methylserine, resistance to diaminosuccinic acid, resistance to a-aminobutyric acid, resistance to borrelidin, acid resistance cyclopentenecarboxylic, resistance to rifampicin, resistance to valine analogues such as valine hydroxamate, resistance to purine analogues, such as 6-dimethylaminopurine, requirement for L-methionine, possible compensatory and partial requirement for L-isoleucine, requirement for mesodiaminopimelic acid, auxotrophy with respect to dipeptides containing threonine, resistance to L-threonine, resistance to raphinate threonine, resistance to L-homoserin, resistance to L-lysine, resistance to L-methionine, resistance to L-glutamic acid, resistance to L- aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, resistance to L-valine, sensitivity to fluoropyruvate, threonine dehydrogenase defective, possible ability to use sucrose, improvement of the operon of threonine, improvement of homoserine dehydrogenase I-aspartate kinase, preferably of the refed-resistant form, improved kinase homoserin, improvement of threonine synthase, aspartate kinase improvement, possibly the resistant form of feedback, improvement of semialdehyde of aspartate dehydrogenase, improvement of phosphoenolpyruvate carboxylase, possibly in the form resistant to feedback, improvement of phosphoenolpyruvate synthase , improvement of transhydrogenase, improvement of the product of the RhtB gene, improvement of the product of the RhtC gene, improvement of the Yfik gene product, improvement of a pyruvate carboxylase and attenuation of the formation of acetic acid. It has been found that, after overexpression of the gene or the open reading structure (ORF) yaaU, or its alleles, microorganisms of the family Enterobacteriaceae produce L-amino acids, in particular L-threonine, in a better way. The nucleotide sequences of the genes Escherichia coli or open reading structures (ORF) belong to the prior art and can be obtained from the genome sequence of Escherichia coli published by Blattner et al. (Science 277: 1453-1462 (1997)). It is known that endogenous enzymes (methionine aminopeptidase), are capable of unfolding the methionine of the N-terminal amino acid. The nucleotide sequences for the yaaU-ORF of Shigella flexneri and Salmonella typhimirium, which similarly belong to the family Enterobacteriaceae, have also been described. The YaaU ORF of Escherichia coli K12, is described, inter alia, by the following data: Designation: open reading structure Function: annotated as a putative transport protein, a membrane transporter the MSF family (main guide superfamily). The transported substrates vary greatly; The MFS family contains monosaccharide, disaccharide and oligosaccharide transporters, potassium and amino acid transporters, transporters for tricarboxylic acid cycle intermediates and also transporters which pump antibiotics out of the cells. Description: the yaaU open reading structure encodes a 48.7 KDa protein; the isoelectric point is 8.8; when located in the chromosome, yaaU is present for example, in the case of Escherichia coli K12 MG1655, in the intergenic region of the carB genes, which encode the long chain of the ca.rbamoyl phosphate synthase, and kefC, which encodes a protein of the potassium efflux system regulated by (K (+) / H (+) lutathione; Reference: Blattner et al., Science 277 (5331): 1453-1474 (1997) Accesses No. AE000114 Alternative gene name : B0045 Nucleic acid sequences can be obtained from the database belonging to the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA), the nucleic acid sequence database from the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK) or the Japanese DNA database (DDBJ, Mishima, Japan). For reasons of greater clarity, the known nucleotide sequence of the yaaU-ORF of Escherichia coli is given in SEQ ID NO. 3 and the sequences known for the yaaU-ORF of Shigella flexneri (AE015041) and Salmonella typhirium (AE008697), are given under SEQ ID NO. 5, and respectively, SEQ ID NO. 7. The amino acid sequences of the proteins encoded by these reading structures are represented as SEQ ID NO. 4, SEQ ID NO. 6 and, respectively, SEQ ID NO. 8. The open reading structures described in the indicated passages can be used in accordance with the invention. In addition, it is possible to use alleles of genes or open reading structures, which results from the degeneracy of the genetic code, or as a consequence of functionally neutral sense mutations. Preference is given to the use of endogenous genes or endogenous open reading structures.

"Endogenous genes" or "nucleotide-endogenous sequence" are understood to be genes or open reading structures or alleles or nucleotide sequences which are present in a population of species. Suitable alleles of the yaaU-ORF, which contain functionally neutral sense mutations, include, inter alia, those which carry at most 50 or at most 40 or at most 30 or at most 20, preferably at most 10 or more. 5, very particularly preferably, at most 3 or at most 2, or at least one conservative amino acid substitution in the protein which they encode. In the case of aromatic amino acids, the substitutions are said to be conservative, when phenylalanine, tryptophan and tyrosine are substituted with each other. In the case of hydrophobic amino acids, the substitutions are said to be conservative when leucine, isoleucine and valine are substituted with each other. In the case of polar amino acids, the substitutions are said to be conservative when glutamine and asparagine are substituted with each other. In the case of basic amino acids, the substitutions are said to be conservative when arginine, lysine and histidine are substituted with each other. In the case of amino acids, substitutions are said to be conservative when aspartic acid and glutamic acid are substituted with each other. At In case of amino acids that contain hydroxyl group, substitutions are said to be conservative when serious and threonine are substituted with each other. In the same way, it is also possible to use nucleotide sequences which encode variants of said proteins, in which the variants additionally contain an extension or truncation by at least (1) an amino acid to the N-term or C-term. This extension or truncation amounts, not more than 50, 40, 30, 20, 10, 5, 3, or 2 amino acids or amino acid residues. Suitable alleles also include those which encode proteins in which at least (1) amino acid has been inserted or deleted. The maximum number of such changes, called indelos, can affect 2, 3, 5, 10, 20, but in no case more than 30 amino acids. Further suitable alleles include those which can be obtained by means of hybridization, in particular, under stringent conditions using SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7, or parts thereof, in particular the coding regions or the sequences which are complementary to these. The skilled person finds instructions to identify DNA sequences by means of hybridization in, inter alia, the "The DIG System Users Guide for Filter Hybridization" manual provided by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). Hybridization takes place under stringent conditions, that only the only hybrids formed are those in which the probe and the target sequence, i.e., the polynucleotides treated with the probe, are at least 80% identical. It is known that the stringency of the hybridization, which includes the washing steps, is influenced and / or determined by the variation of the buffer composition, the temperature and the salt concentration. In general, the hybridization reaction is carried out at a stringency which is relatively low compared to that of the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996). For example, a buffer corresponding to the 5x SSC buffer can be used for the hybridization reaction at a temperature of about 50-80 ° C. Under these conditions, the probes can also hybridize with polynucleotides which possess less than 70% identity with the sequence of the probe. These hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by decreasing the salt concentration down to 2x SSC and, where appropriate, subsequently to 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995), with the temperature being adjusted to approx. 50 ° C-68 ° C, approx. 52 ° C-68 ° C, approx.- 54 ° C-68 ° C, approx. 56 ° C-68 ° C, approx. 58 ° C- 68 ° C, approx. 60 ° C-68 ° C, approx. 62 ° C-68 ° C, approx 64 ° C-68 ° C, approx. 66 ° C-68 ° C. The temperature ranges of approx. 64 ° C-68 ° C or approx. 66 ° C-68 ° C, are preferred. It is possible, where appropriate, to lower the salt concentration by lowering it to a concentration corresponding to 0.2x SSC or 0. Ix SSC. By increasing the step hybridization temperature, in steps of about 1-2 ° C, from 50 ° C to 68 ° C, it is possible to isolate the polynucleotide fragments, for example, they possess at least 80%, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of the probe used or with the nucleotide sequences shown in SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7. Additional instructions for hybridization can be obtained, commercially in the form of those which are called kits (eg, DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558). The nucleotide sequences which are thus obtained encode polypeptides which have at least 90%, in particular at least 95%, preferably at least 98% or at least 99%, very particularly preferably 99.7% identity with the sequences of amino acid represented in SEC ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8. To achieve improvement, it is possible for example to increase the expression of genes or open reading structures or alleles or increase the catalytic properties of the protein. Both measurements can be combined, where appropriate. To achieve overexpression, the number of copies of the corresponding genes or open reading structures can be increased, for example, or the promoter region and the regulatory region or the ribosome binding site which is located upstream of the structural gene, can be mutated The expression cassettes which are incorporated upstream of the structural gene, act in the same way. It is also possible to increase the expression during the course of the fermentative production of L-threonine, incorporating inducible promoters; In addition, using promoters for gene expression which allow different chronological gene expression may also be advantageous. The expression is likewise, improved by means of measurements to extend the expected life of the mRNA. In addition, the enzymatic activity is also improved by preventing the enzymatic protein from. be divided. ORFs, genes or gene constructs, can either be present in plasmids that have different copy numbers or are integrated, and amplified, in the chromosome. Alternatively, the overexpression of Related genes can also be achieved by altering the composition of the culture medium and conduit. Methods for overexpression are suitably described in the prior art, for example, in Makrides et al. (Microbiological Reviews 60 (3), 512-538 (1996)). Using vectors increases the number of copies by at least one (1) copy. The vectors used can be plasmids as described, for example, in US 5,538,873. The vectors used can also be phages, for example, phage Mu, as described in EP 0332448, or lambda phage (?). The number of copies can also be increased by incorporating an additional copy elsewhere in the chromosome, for example, in the att site of the phage? (and Court, Gene 223.77-81 (1998)). US 5,939,307 reports that it was possible to increase the expression by incorporating cassettes or expression promoters, such as the tac promoter, the trp promoter, the lpp promoter, or the PL promoter or phage PR promoter, upstream for example, of the chromosomal threonine operon. In the same way, it is possible to use the phage T7 promoters, the transmission promoters or the nar promoter. Such expression cassettes or promoters can also be used as described in EP 0 593 792 to overexpress genes bound to plasmid. Using the lacIQ allele instead, it becomes possible to control the expression of genes linked to alleles (Glascock and Weickert, Gene 223, 221-231 (1998)). It is also possible, for the activity of the promoters to increase, to modify their sequence by means of one or more nucleotide substitutions, by means of (an) insertion (s) and / or by means of (a) suppression (s) . The expression of the different chronological gene can be achieved, for example, as described in Walker et al. (Journal of Bacteriology 181: 1269-80 (1999)), using the fis promoter dependent on the growth phase. The expert can find general instructions in this regard in, inter alia, Chang and Cohen (Journal of Bacteriology 134: 1141-1156 (1978)), Hartley and Gregori (Gene 13: 347-353 (1981)), Amann and Brosius (Gene 40: 183-190 (1985)), by Broer et al. (Proceedings of the National Academy of Sciences of the United States of America 80: 21-25 (1983)), LaVallie et al. (BIO / TECHNOLOGY 11: 187-193 (1993)), in PCT / US97 / 13359, Llosa et al. (Plasmid 26: 222-242 (1991)), Quandt and Klipp (Gene 80: 161-169 (1989)), Hamilton et al. (Journal of Bacteriology 171: 4617-4622 (1989)), Jensen and Hammer (Biotechnology and Bioengineering 58: 191-195 (1998)) and in known textbooks of genetics and molecular biology. Plasmid vectors which can be replicated in Enterobacteriaceae, such as cloning vectors derived from pACYC184, (Bartolomé et al., Gene 102: 75-78 (1991)), pTrc99A (Amann et al., Gene 69: 301) can be used. -315 (1988)) or derivatives of pSClOl (Vocke and Bastia; Proceedings of the National Academy of Sciences USA 80 (21): 6557-6561 (1983). In a process according to the invention, it is possible to use a strain which is transformed with a plasmid vector which carries at least one nucleotide or allele sequence, which encodes the yaaU ORF or its gene product. The term "transformation" is understood to mean the absorption of an isolated nucleic acid by a host (microorganism). It is also possible to use sequence exchange, (Hamilton et al, Journal of Bacteriology 171: 4617-4622 (1989)), conjugation or transduction to transfer mutations, which affect the expression of given genes or open reading structures, in different strains. More detailed explanations of the concepts of genetics and molecular biology can be found in well-known textbooks of genetics and molecular biology, such as the textbook by Birge (Bacterial and Bacteriophage Genetics, 4th ed., Springer Verlag, New York ( USA), 2000), or the textbook by Berg, Tymoczko and Stryer (Biochemistry, 5th ed., Freeman and Company, New York (USA), 2002) or the manual by Sambrook et al. (Molecular Cloning, A Laboratory Manual, (3 -Volume Set), Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). In addition, when family strains are used Enterobacteriaceae to produce L-amino acids, in particular L-threonine, it may be advantageous, in addition to improving the open reading structure and yaaU, to improve one or more enzymes from the path of threonine biosynthesis or enzymes of anaplerotic metabolism or enzymes to produce dinucleotide phosphate. Reduced nicotinamide adenine or glycosylation enzymes or PTS enzymes or sulfur metabolism enzymes. It is generally preferred to use endogenous genes. Thus, it is possible, for example, to simultaneously improve, in particular overexpression, one or more of the genes selected from the group • at least one gene of the thrABC operon encoding the kinase aspartate, homoserine dehydrogenase, homoserine kinase and threonine synthase (US Pat. A-4, 278, 765), • the pyc gene of Corynebacterium glutamicum encoding pyruvate carboxylase (WO 99/18228), • the pps gene encoding phosphoenolpyruvate synthase (Molecular and General Genetics 231 (2): 332). -336 (1992)), • the ppc gene encoding phosphoenolpyruvate carboxylase (WO 02/064808), • the pntA and pntB genes encoding the pyridine transhydrogenase subunits (European Journal of Biochemistry 158: 647-653 (1986 )), • the rhtB gene that encodes the protein that mediates homoserin resistance (EP-A-0 994 190), • the rhtC gene that encodes the protein that mediates threonine resistance (EP-A-1 013 765), • the thrE gene of Corynebacterium glutamicum that encodes the protein of the carrier that exports threonine (WO 01/92545), • the gdhA gene encoding glutamate dehydrogenase (Nucleic Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983)), • the pg gene encoding the phosphoglucomutase (WO 03/004598), • the fba gene encoding the fructose aldose biphosphate (WO 03/004664), • the ptsH gene of the ptsHIcrr operon encoding the phosphotransferase hexose of the phospho-histidine protein of the phosphotransferase system PTS ( WO 03/004674), • the ptsl gene of the pstHIcrr operon encoding enzyme I of the phosphotransferase system PTS (WO 03/004674), • the crr gene of the ptsHIcrr operon encoding the glucose specific component IIA of the system of phosphotransferase PTS (WO 03/004674), • the ptsG gene encoding the specific glucose IIBC component (WO 03/004670), • the lrp gene encoding the regiment of the gift of leucine (WO 03/004665), • the fadR gene encoding the fad regulor regulator (WO 03/038106), • the iclR gene encoding the central intermediary metabolism regulator (WO 03/038106), • the ahpC gene of the ahpCF operon encoding the small subunit of the alkyl hydroxy peroperoxide reductase (WO 03/004663), the ahpF gene of the ahpC operon encoding the large subunit of the alkyl hydroxyperoxide reductase (WO 03/004663), • the cysK gene encoding cysteine synthase-A (WO 03/006666), • the cysB gene coding for the cys regimen regulator (WO 03/006666), • the cysJ gene of the cysJIH operon encoding the NADPH sulfite reductase flavoprotein (document 03/06666), • the cysl gene of the cysJIH operon encoding the NADPH sulfite reductase hemoprotein (document 03/006666), • the cysH gene of the cysJIH operon encoding adenylyl sulfate reductase (WO 03/006666), • the rseA gene of the rseABC operon encoding a membrane protein, which possesses anti-sigmaE activity (WO 03/008612), • the rseC gene of the rseABC operon encoding a global regulator of the sigmaE factor (WO 03/008612), • the sucA gene of the sucABCD operon that encodes the decarboxylase subunit of 2-ketoglutarate dehydrogenase (WO 03/008614), • the sucB gene of the sucABCD operon encoding the E2 subunit of dihydrolipoyl transuccinase 2-ketoglutarate dehydrogenase (WO 03/008614), • the sucC gene of the sucABCD operon encoding the β-subunit of succinyl-CoA synthetase (WO 03/008615), • the sucD gene of the sucABCD operon encoding the succinyl-CoA synthetase a-subunit (WO 03/008615), • the aceE gene encoding the El component of the pyruvate dehydrogenase complex (WO 03/076635), • the aceF gene encoding the E2 component of the pyruvate dehydrogenase complex (WO 03/076635), • the rseB gene encoding the activity regulator of the SigmaE factor (Molecular Microbiology 24 (2): 355-371 (1997)), • the product of the open reading structure gene (ORF) yodA from Escherichia coli (Accession Number AE000288 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA, DE10361192.4)).

Furthermore, for purposes of producing L-amino acids, in particular L-threonine, it may be advantageous in addition to improving the open reading structure and aaU, to attenuate, in particular, eliminate or reduce the expression of one or more of the genes selected from the group of • the tdh gene encoding threonine dehydrogenase (Journal of Bacteriology 169: 4716-4721 (1987)), • the mdh gene encoding malate dehydrogenase (E.C. 1.1.1.37) (Archives in Microbiology 149: 36-42 (1987)), • the gene product of the open reading structure (ORF) yj fA of Escherichia coli (Accession Number AAC77180 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA, (WO 02/29080)), • the gene product of the open reading structure (ORF) ytfP of Escherichia coli (Accession Number AAC77179 of the National Center for Biotechnology Information (NCBI, Bethesda , MD, USA, (WO 02/29080), • the pckA gene encoding the enzyme phosphoenolpyruvate carboxy kinase (WO 02/29080), • the poxB gene encoding pyruvate oxidase (WO 02/36797), • the dgsA gene (WO 02/081721), which is also known under the name of the mlc gene, which encodes the DgsA regulator of the phosphotransferase system, • the fruR gene (WO 02/081698), which is also known under the name of the gene was, which encodes the fructose repressor, • the rpoS gene (WO 01/05939), which is also known under the name of the katF gene, which codes for the sigma38 factor, and • the aspA gene that codes for the aspartate ammonium lyase (document 03/008603). In this context, the term "attenuation" describes the reduction or abolition, in a microorganism, of the intracellular activity or concentration of one or more enzymes or proteins, which are encoded by the corresponding DNA, by, for example, using a promoter weaker than in the original strain or non-recombinant microorganism for the corresponding enzyme or protein or a gene or allele which encodes a corresponding enzyme or protein having a lower activity, or inactivating the corresponding enzyme or protein, or the open reading structure or gene, and where appropriate, combine these measures. In general, attenuation measures decrease the activity or concentration of the corresponding protein down from 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10%, or from 0 to 5% of the activity or concentration of the native type protein or the activity or concentration of the protein for the original strain or microorganism, which is not recombinant for the corresponding enzyme or protein. The original strain or microorganism, which is not recombinant, is understood to be the microorganism in which tail compliance measurements are made. To achieve an attenuation, for example, the expression of genes or open reading structures, or the. catalytic properties of enzymatic proteins, can be reduced or abolished. Where appropriate, both measurements can be combined. The expression of the gene can be reduced by carrying out the cultivation in a suitable manner, by genetically altering (mutating) the signal structures for the expression of the gene, or by means of the antisense RNA technique. The signal structures for the expression of the gene are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the initiator codon and terminators. The skilled person can find information in this regard among other things, in and for example, Jensen and Hammer (Biotechnology and Bioengineering 58: 191-195 (1998)), in Carrier and Keasling (Biotechnology Progress 15: 58-64 (1999) ), in Franch and Gerdes (Current Opinion in Microbiology 3: 159-164 (2000)) and in fine textbooks Known genetics and molecular biology, such as textbooks by Kippers ("Molekulare Genetik [Molecular Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or those by Winnacker ("Gene und Klone [Genes and Clones] ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990). Mutations which lead to a change or reduction in the catalytic properties of the enzymatic proteins are known from the prior art. Examples which. can be mentioned are the articles by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Yano et al. (Proceedings of the National Academy of Sciences of the United States of America 95: 5511-5515 (1998)) and Wente and Schachmann (Journal of Biological Chemistry 266: 20833-20839 (1991)). The summaries can be found in well-known textbooks of genetics and molecular biology, such as that of Hagemann ("Allgemeine Genetik [General Genetics] ", Gustav Fischer Verlag, Stuttgart, 1986.) The mutations which come to be considered are transitions, transversions, insertions and deletions of at least one (1) base pair of nucleotides, depending on the effect of the substitution. of amino acid stimulated by mutation in enzymatic activity, reference is made to inverted nonsense mutations or nonsense mutations. it leads to the replacement of a given amino acid in a protein with a different amino acid, with the amino acid replacement in particular being non-conservative. This therapy impairs the capacity or functional activity of the protein and reduces down to a value from 0 to 75%, 0 to 50%, 0 to 25%, or 0 to 10%, or 0 to 5%. A nonsense mutation leads to a stop codon in the coding region of the gene and thus, to premature termination of the translation. Insertions or deletions of at least one base pair in a gene, lead to structural change mutations which, in turn, result in incorrect amino acids being incorporated or in the translation being prematurely terminated. If a stop codon is formed in the coding region as a consequence of the mutation, this also leads to the translation being terminated prematurely. Deletions of at least (1) or more codons typically also lead to complete loss of enzymatic activity. The directions for generating these mutations belong to the prior art and can be obtained from known textbooks of genetics and molecular biology, such as textbooks by Knippers ("Molekulare Genetik [Molecular Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), those by Winnacker "Gene und Klone, [Genes and Clones]", VHC Verlagsgesellschaft, Weinheim, Germany, 1990) or those by Hagemann ("Allgemeine Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart, 1986). Suitable mutations in the genes can be incorporated into suitable strains by means of gene or allele exchange. A common method is the method described by Ha ilton et al. (Journal of Bacteriology 171: 4617-4622 (1989)), gene exchange using a pMAK705 conditionally replicating pSClOl derivative. Other methods described in the prior art, such as those of Martinez-Morales et al. (Journal of Bacteriology 181: 7143-7148 (1999)) or those of Boyd et al (Journal of Bacteriology 182: 842-847 (2000), can also be used.) In the same way it is possible to transfer mutations in the relevant genes, or mutations which affect the expression of relevant genes or open reading structures, in different strains by means of conjugation or transduction.In addition, it may be advantageous for the purpose of producing L-amino acids, in particular L-threonine, in addition to improving the structure open reader and aU, eliminate undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms which are prepared according to the invention, can be grown in a batch process, in a batch feed process, in a batch feed process repeated or in a continuous process (documents DE102004028859.3 or US 5,763,230 ). Known cultivation methods are summarized in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas ( Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral installations] (Vieweg Verlag, Brunswick / Wiesbaden, 1994)). The culture medium to be used must satisfy the demands of the given strains in an appropriate manner. The American Society for Bacteriology manual, "Manual of Methods for General Bacteriology" (Washington D. C, USA, 1981), contains descriptions of the medium for cultivating a variety of microroganisms. Sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and where appropriate, cellulose, oils and fats, such as soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids, such as palmitic acid, stearic acid and linoleic acid, alcohols, such as glycerol and Ethanol and organic acids, such as acetic acid, can be used as the carbon source. These substances can be used individually or as a mixture. Compounds containing organic nitrogen, such as peptones, yeast extract, meat extract, mat extract, fermented corn liquor, soybean meal and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, phosphate Ammonium, ammonium carbonate and ammonium nitrate can be used as the nitrogen source. 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 source of phosphorus. In addition, the culture medium must contain metal salts, such as magnesium sulfate or iron sulfate, which are required for growth. Finally, essential growth promoters, such as amino acids and vitamins, can be used in addition to the substances mentioned above. Suitable precursors can also be added to the culture medium. Said ingredients can be added to the culture in the form of a mixture or suitably fed during cultivation. The fermentation is carried out in a general way, at a pH from 5.5 to 9.0, in particular, from 6.0 to 8. 0. Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used in a suitable manner to control the pH of the culture. Antifoams, such as polyglycol fatty acid esters, can be used to control the foam. Selectively acting substances, eg, antibiotics, can be added to the medium to maintain the stability of the plasmids Oxygen or oxygen-containing gas mixtures, such as air, are passed into the culture to maintain aerobic conditions. The culture temperature is usually from 25 ° C to 45 ° C and preferably from 30 ° C to 40 ° C. The culture is continued up to a maximum of L-amino acids or that L-threonine has been formed.This objective is normally achieved within 10 to 160 hours The L-amino acids can be analyzed by means of anion exchange chromatography, followed by derivatization with ninhydrin, as described in Spackman et al (Analytical Chemistry 30: 1190-1206 (1958)), or by reverse phase HPLC, as described in Lindroth et al (Analytical Chemistry 51: 1167-1174 (1979).) The process according to the invention can be used to prepare fermentatively, L-amino acids, such as L-threonine, L-isoleucine, L-valine, L-methionine,L-homoserin, L-tryptophan and L-lysine, in particular, L-threonine. The following microorganisms were deposited in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] (DSMZ, Brunswick, Germany) in accordance with the Budapest Treaty: • strain MG442 of Escherichia coli as DSM 16574). The present invention is explained in more detail below with the help of implementation examples. Complete medium (LB) and minimal (M)) used for Escherichia coli, is described by J. H. Miller (A short course in bacterial genetics (1992), Cold Spring Harbor Laboratory Press). Isolation of Escherichia coli plasmid DNA, and also all techniques for restricting, binding and treating with Klenow phosphatase and alkaline phosphatase, are carried out as described in Sambrook et al. (Molecular Cloning-A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press). Unless indicated otherwise, Escherichia coli are transformed as described in Chung et al (Proceedings of the National Academy of Sciences of the United States of America 86: 2172-2175 (1989)). The incubation temperature when the strains and transformants are prepared is 37 ° C.

Example 1 Construction of the expression plasmid pTrc99AyaaU The YaaU ORF of E. coli K12 is amplified using the poly-eraser chain reaction (PCR) and synthetic oligonucleotides. The PCR polymers are synthesized (MWG Biotech, Ebersberg, Germany), based on the nucleotide sequence of the yaaU ORF in E. coli K12 MG1655 (accession number AE000114), Blattner et al. (Science 277: 1453-1474 (1997)). The sequences of the primers are modified to form recognition sites for restriction enzymes. The EcoRI recognition sequence is selected by the yaaU-exl primer and the BamHI recognition sequence is selected for the yaaU-ex2 primer, with this sequence being underlined in the nucleotide sequences shown below: yaaU-exl: 5 '-GATCTGAATTCTAAGGAATAACCATGCAACCGTC- 3 '(SEQ ID No. 1) yaaU-ex2: 5' -GATCTAGGATCCCAATTTACCCCATTCTCTGC-3 '(SEQ ID No. 2) Chromosomal DNA of E. coli K12 MG1655 used for PCR, is isolated using "Genomic Quiagen 100 tips. / G "(QIAGEN, Hilden, Germany), in accordance with the manufacturer's instructions. A DNA fragment of approximately 1371 bp in size (SEQ ID NO: 3) can be amplified under standard CPR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) using Vent DNA polymerase (New England Biolaps GmbH, Frankfurt, Germany), and the specific primers. The amplified yaaU fragment is ligated to the pCR-Blunt II-TOPO vector (Zero TOPO TA Cloning Kit, Invitrogen, Groningen, Netherlands), in accordance with the manufacturer's instructions and transformed into the TOP10 strain of E. coli. Cells harboring plasmids are selected in LB agar containing 50 μg kanamycin / ml. After the plasmid DNA has been isolated, the vector is cleaved with the enzymes EcoRV and EcoRI, and, after the cleavage has been verified in 0.8% agarose gel, it is designated pCRBluntyaaU. The vector pCRBluntyaaU is then unfolded with the enzymes EcoRI and BamHI and the yaaU fragment is separated on a 0.8% agarose gel; then it is isolated from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilden, Germany) and ligated with the vector pTrc99A (Pharmacia Biotech, Uppsala, Sweden), which has been digested with the enzymes BamHI and EcoRI. The XLIBlue MRF 'strain from E. coli (Stratagene, La Jolla, USA), is transformed with the ligation mixture and the cells harboring plasmid are selected on LB agar containing 50 μg ampicillin / ml. The fact that the cloning has been successful may to be demonstrated, after the plasmid DNA has been isolated, performing a control splitting using the EcoRl / BamHI and EcoRV enzymes. The plasmid is designated pTrc99AyaaU (Figure 1).

Example 2 Preparation of L-threonine using the strain MG442 / pTrc99AyaaU The E. coli strain MG442 producing L-threonine is described in the US-A-4,278,765 patent specification and is deposited in the Russian national collection of industrial microorganisms ( VKPM, Moscow, Russia), as CMIM B-1628 and in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] (DSMZ, Brunswick, Germany), in accordance with the Budapest Treaty, as DSM 16574). Strain MG442 is transformed with the expression plasmid pTrc99AyaaU described in Example 1, and with the vector pTrc99A, and the cells harboring the plasmid, are selected on LB agar containing 50 μg of ampicillin / ml. This results in strains MG442 / pTrc99AyaaU and MG442 / pTrc99A. The selected individual colonies are then further propagated in minimal medium having the following composition: 3.5 g of Na2HPO4 * 2H20 / l, 1.5 g of KH2P04 / 1, 1 g of NH4C1 / 1, 0.1 g of MgSO4 * 7H20 / l, 2 glucose g / 1, 20 g of agar / l, 50 mg ampicillin / 1. The formation of L-threonine is verified in cultures of lots of 10 ml, which are contained in Erlenmeyer flasks of 100 ml. For this, a pre-culture medium of 10 ml of the following composition: 2 g of yeast extract / 1, 10 g of (NH4) 2S04 / 1, 1 g of KH2P04 / 1, 0.5 g of MgSO4 * 7H20 / l, 15 g of CaCO3 / l, 20 g of glucose, 50 mg of ampicillin / 1, are inoculated and incubated at 37 ° C and 180 rpm for 16 hours, in a Kuhner AG ESR incubator (Birsfelden, Switzerland). In each case, 250 μl of this preliminary culture are inoculated on 10 ml of production medium (25 g of (NH4) 2S04 / 1, 2 g of KH2P04 / 1, 1 g of MgSO4 * 7H20 / l, 0.03 g of FeS04 * 7H20 / l, 0.018 g of MnS04 * lH20 / l, 30 g of CaCO3 / l, 20 g of glucose / 1, 50 mg of ampicillin / 1) and incubated at 37 ° C for 48 hours. The formation of L-threonine by the starting strain MG442 is verified in the same way, however, if adding ampicillin to the medium. After incubation, the optical density (OD) of the culture suspension is determined at a wavelength measurement of 660 nm using a photometer from Dr. Lange LP2W (Dusseldorf, Germany). An amino acid analyzer Eppendorf-BioTronik (Hamburg, Germany) is then used to determine, by means of ion exchange chromatography and post-column reaction involving the detection of ninhydrin, the concentration of the resulting L-threonine in the supernatant of culture, which has been sterilized by filtration. The result of the experiment is shown in Table 1.

Brief Description of the Figure: Figure 1 is a map of plasmid pTrc99Ayaaü containing the yaaU gene. The long specifications are considered to be approximate. The abbreviations and designations used have the following meanings: • bla: gene which codes for resistance to picilin. • lac Iq: gene for the repressor protein of the trc promoter. • trc: region of the trc promoter, inducible by IPTG. • yaaU: coding region of the yaaU gene. • 5S: 5S rRNA region. • rrnBT: rRNA terminator region Abbreviations for restriction enzymes have the following meanings: • Ba HI: Bacillus restriction endonuclease amyloliquefaciens H. • EcoRI: restriction endonuclease of Escherichia coli RY13. • EcoRV: restriction endonuclease of Escherichia coli 'B946.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (4)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. 1. Recombinant microorganism of the family Enterobacteriaceae, characterized in that it contains an improved or overexpressed yaaU ORF, which encodes a polypeptide which is annotated as a putative sugar transporter, and which produces L-amino acids in an improved manner.
2. 2. Microorganism according to claim 1, characterized in that a polynucleotide encoding a polypeptide whose amino acid sequence is at least 90% identical to an amino acid sequence selected from the group of SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8,. 3. Microorganism according to claim 2, characterized in that it contains an overexpressed or improved polynucleotide, which corresponds to the yaaU ORF and which is selected from the group: a) polynucleotide having the nucleotide sequence SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; b) polynucleotide having a nucleotide sequence which corresponds to SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7, within the limits of the degeneracy of the genetic code; c) polynucleotide sequence having a sequence which hybridizes, under stringent conditions, to the sequence which is complementary to SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 7; d) polynucleotide having a sequence of SEQ ID NO.
3. 3, SEQ ID NO. 5 or SEQ ID NO. 7, which contains functionally neutral sense mutants, 4. Microorganism according to claim 2, characterized in that the polypeptide possesses an amino acid sequence which is at least 95% identical to one of the sequences selected from the group of SEQ. DO NOT. 4, SEQ ID NO. 6 and SEQ ID NO. 8. The microorganism according to claim 2, characterized in that the polypeptide possesses the amino acid sequence which is 100% identical to that of SEQ ID NO.
4. 4, SEQ ID NO. 6 or SEQ ID NO. 8. Microorganism according to claims 1 to 5, characterized in that it is produced by transformation, transduction or conjugation, or a combination of these methods, with a vector which contains the yaaU ORF, an allele of this ORF, or parts of it, and / or a promoter. 7. Microorganism according to claim 1 or 6, characterized in that the number of copies of the yaaU ORF or the alleles, has been increased by at least 1. 8. Microorganism according to claim 7, characterized in that the increase in the number of copies of the yaaU ORF by at least 1, is achieved by integrating the ORF or the alleles in the chromosome of the microorganism. 9. Microorganism according to claim 7, characterized in that the increase in the number of copies of the yaGU ORG by at least 1 is achieved by means of a vector which is extrachromosomally replicated. 10. -Microorganism according to claim 1 or 2, characterized in that to achieve the improvement, a) the promoter and the regulatory region or the ribosomal binding site upstream of the yaaU ORF are mutated, or b) the expression cassettes or promoters are incorporated upstream of the yaaU ORF. 11. Microorganism according to claim 1 or 2, characterized in that the yaaU ORF is under the control of a promoter that improves the expression of the gene. 12. Microorganism according to claims 1 to 11, characterized in that improving the yaaU ORF increases the concentration or activity of the yaaU (protein) gene product by at least 10%, based on the activity or concentration of the gene product in the original strain or non-recombinant microorganism for the yaaU ORF. 13. Microorganism according to claims 1 to 12, characterized in that others, genes of the metabolic path for the biosynthesis of the desired L-amino acid are also present in a particularly overexpressed form., improved. 14. Microorganism according to claim 13, characterized in that the microorganism is selected from the genera Escherichia, Erwinia, Providencia and Serratia. 15. Microorganism according to claims 1 to 14, characterized in that it produces L-threonine. 16. Process for preparing L-amino acids by fermenting recombinant microorganisms of the family Enterobacteriaceae, characterized because, a) the microorganisms that produce desired L-amino acids, in which the open reading structure yaaU, or nucleotide sequences or alleles encoding the products of the gene thereof, is / are improved, particularly overexpressed, are cultured in a medium under conditions in which the desired L-amino acid is enriched in the medium or in the cells, and b) the desired L-amino acid is isolated, with the constituents of the fermentation broth and / or the biomass remaining in the whole or in portions (from _> 0 to 100%) in the product isolated or being completely removed. 17. Process according to claim 16, characterized in that the microorganisms are used as claimed in claims 1 to 15. 18. Process according to claim 16 or 17, characterized in that, for purposes of preparing L-threonine , microorganisms of the family Enterobacteriaceae are fermented in which one or more genes are selected from the group: 18.1) at least one gene of the thrABC operon encoding the kinase aspartate, homoserine dehydrogenase, homoserine kinase and threonine synthase, 18.2) the pyc gene of Corynebacterium glutamicum encoding pyruvate carboxylase, 18.3) the pps gene encoding the phosphoenolpyruvate synthase, 18.4) the ppc gene encoding phosphoenolpyruvate carboxylase, 18.5) the pntA and pntB genes encoding the pyridine transhydrogenase subunits, 18. 6) the rhtB gene that encodes the protein that mediates homoserin resistance, 18.7) the rhtC gene that encodes the protein that mediates threonine resistance, 18.8) the thrE gene of Corynebacterium glutamicum that encodes the carrier protein that exports the threonine , 18.9) the gdhA gene encoding glutamate dehydrogenase, 18.10) the pgm gene encoding the phosphoglucomutase, 18.11) the fba gene encoding the aldose fructose biphosphate, 18.12) the ptsH gene encoding the phosphotransferase hexose of the phospho- histidine, 18.13) the ptsl gene encoding enzyme I of the phosphotransferase system, 18.14) the crr gene encoding the specific IXA component of glucose, 18.15) the ptsG gene encoding the specific IIBC component of glucose, 18.16) gene lrp coding for the regulator of the leucine regution, 18.17) the fadR gene coding for the regulator of the fad regimen, 18.18) the iclR gene that codes for the regulator of the central intermediate metabolism, 18. 19) the ahpC gene encoding the small subunit of the alkyl hydroxyperoxide reductase, 18.20) the ahpF gene encoding the large subunit of the alkyl hydroxyperoxide reductase, 18.21) the cysK gene encoding the cysteine synthase-A, 18.22) the cysB gene coding regulator cys, 18.23) the cysJ gene encoding the sulfite reductase flavoprotein NADPH, 18.24) the cysl gene encoding the sulfite reductase hemoprotein NADPH, 18.25) the cysH gene encoding adenylyl sulfate reductase, 18.26) the rseA gene that encodes a membrane protein, which has anti-sigmaE activity, 18.27) the rseC gene that encodes a global regulator of the sigmaE factor, 18.28) the sucA gene that encodes the decarboxylase subunit of 2-ketoglutarate dehydrogenase, 18.29 ) the sucB gene encoding the E2 subunit of dihydrolipoyl transuccinase 2-ketoglutarate dehydrogenase, 18.30) the sucC gene encoding the β-subunit of succinyl-CoA synthetase, 18. 31) the sucD gene encoding the a-subunit of succinyl-CoA synthetase, 18.32) the aceE gene encoding the El component of the pyruvate dehydrogenase complex, 18.33) the aceF gene encoding the? 2 component of the pyruvate dehydrogenase complex, 18.34 ) the rseB gene encoding the activity regulator of the SigmaE factor, 18.35) the product of the open reading structure gene (ORF) yodA of Escherichia coli. is / are additionally at the same time, improved, in particular, overexpressed. 19. Process according to claims 16 to 18, characterized in that use is made of microorganisms in which, the metabolic trajectories which reduce the formation of the desired L-amino acids, are at least partially attenuated. Process according to claim 19, characterized in that, for purposes of preparing L-threonine, the microorganisms of the family Enterobacteriaceae are fermented with one or more of the genes selected from the group of: 20.1) the tdh gene encoding threonine dehydrogenase, 20.2) the mdh gene encoding the alato dehydrogenase, 20.3) the gene product of the open reading frame (ORF) yjf A of Escherichia coli, 20.4) the gene product of the open reading structure (ORF) ytfP of Escherichia coli, 20.5) the pckA gene encoding the enzyme phosphoenolpyruvate carboxykinase, 20.6) the poxB gene encoding pyruvate oxidase, 20. 7) the dgsA gene, which encodes the DgsA regulator of the phosphotransferase system, 20.8) the fruR gene, which encodes the fructose repressor, 20.9) the rpoS gene, which codes for the a38 sig factor, and 20.10) the aspA gene that encodes aspartate ammonium lyase. It is / are additionally at the same time, attenuated, in particular eliminated, or its expression is reduced. 21. Process according to claims 16 to 20, characterized in that the L-amino acids selected from the group of L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L- are prepared. cysteine, L-valine, L-methionine, L-proline, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and L-homoserine. 22. Process according to claim 21, characterized in that the L-amino acids selected from the group of L-isoleucine, L-valine, L-methionine, L-homoserin, L-tryptophan and L-lysine are prepared. *