阅读说明：本技术 使用改良的肠杆菌科菌株制备l氨基酸的方法 (Method for producing L-amino acids using improved strains of the Enterobacteriaceae family ) 是由 M·里平 于 2019-08-09 设计创作，主要内容包括：本发明涉及一种重组的分泌L-氨基酸的肠杆菌科微生物,其包含功能性地连接编码膜蛋白的多核苷酸的具有启动子活性的DNA片段,特征在于所述具有启动子活性的DNA片段包含SEQ ID NO:8。(The present invention relates to a recombinant L-amino acid-secreting Enterobacteriaceae microorganism comprising a DNA fragment having promoter activity functionally linked to a polynucleotide encoding a membrane protein, characterized in that the DNA fragment having promoter activity comprises SEQ ID NO 8.)

1. A recombinant microorganism of the Enterobacteriaceae (Enterobacteriaceae) family which secretes L-amino acids, comprising a DNA fragment having promoter activity which is functionally linked to a polynucleotide encoding a membrane protein, characterized in that the DNA fragment having promoter activity comprises SEQ ID NO 8.

2. Microorganism according to claim 1, characterized in that the DNA fragment having promoter activity is linked at the 3' end to a second DNA fragment carrying a ribosome binding site.

3. The microorganism as claimed in claim 1, characterized in that the DNA fragment having promoter activity is linked at the 3' end to a second DNA fragment having the nucleotide sequence of positions 174 and 204 of SEQ ID NO 9.

4. The microorganism as claimed in claim 1, characterized in that the DNA fragment having promoter activity is linked at its 3 'end to a second DNA fragment having the nucleotide sequence of positions 174 and 204 of SEQ ID NO 9, which second DNA fragment is linked at its 3' end to a polynucleotide which codes for the membrane protein.

5. Microorganism according to any of the preceding claims, characterized in that the DNA fragment having promoter activity is linked at the 5' end to a DNA fragment having the nucleotide sequence in positions 1 to 138 of SEQ ID NO 9.

6. Microorganism according to any of the preceding claims, characterized in that the membrane protein is a protein with amino acid transporter activity.

7. Microorganism according to claim 6, characterized in that the protein with amino acid transporter activity is a protein with amino acid exporter activity.

8. Microorganism according to claim 7, characterized in that the protein having amino acid export protein activity has an amino acid sequence which is at least 90% identical to the sequence of SEQ ID NO. 2.

9. Microorganism according to claim 8, characterized in that the protein having amino acid export protein activity has an amino acid sequence which is at least 95% identical to the sequence of SEQ ID NO. 2.

10. Microorganism according to claim 9, characterized in that the protein with amino acid export protein activity comprises the amino acid sequence of SEQ ID No. 2 and/or in that the protein with amino acid export protein activity is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID No. 1.

11. Microorganism according to any one of claims 1 to 9, characterized in that the DNA fragment having promoter activity is present in the chromosome of the microorganism or, alternatively, in that the DNA fragment having promoter activity is located on an extrachromosomal replicating vector.

12. Microorganism according to any of claims 1 to 11, characterized in that it produces L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine.

13. A method of preparing an L-amino acid or a feed additive comprising an L-amino acid, the method comprising:

(i) fermenting the microorganism of any one of claims 1-12 in a culture medium;

(ii) enriching the L-amino acid in the fermentation broth and/or cells; and optionally

(iii) Isolating the L-amino acid.

14. A DNA fragment having promoter activity which is functionally linked to a polynucleotide encoding a membrane protein, characterized in that said DNA fragment having promoter activity comprises SEQ ID NO 8.

15. Use of a DNA fragment comprising SEQ ID NO 8 as a promoter for regulating the expression level of a gene encoding a membrane protein or an amino acid transporter.

Technical Field

The present invention relates to a method for the fermentative preparation of L-amino acids, such as L threonine, using recombinant microorganisms of the enterobacteriaceae family comprising specific DNA fragments having promoter activity functionally linked to polynucleotides encoding membrane proteins or amino acid transporters, and to various (respecitive) microorganisms.

Background

L-amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, are used in human medicine and in the pharmaceutical industry, in the food industry and in animal nutrition.

It is known that L-amino acids are prepared by fermentation of strains of the Enterobacteriaceae family, in particular of Escherichia coli (E.coli) and Serratia marcescens (Serratia marcescens). Due to the great importance, there are continuous attempts to improve the preparation process. Improvements in the methodology may relate to measures relating to the fermentation technology, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the choice of the sugars used or the sugar concentration during the fermentation, or the modification of the product form, for example by ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.

In wild-type strains, strict regulatory mechanisms prevent the production of metabolites, such as amino acids, in excess of those required by the strain and prevent release into the culture medium. Therefore, from the manufacturer's point of view, the construction of amino acid overproducing strains requires overcoming these metabolic regulations.

Strains are thus obtained which are resistant to antimetabolites, such as the threonine analogue α -amino- β -hydroxyvaleric Acid (AHV), or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as L-threonine.

For many years, recombinant DNA methods have likewise been used to improve L-amino acid-producing strains of the Enterobacteriaceae family in a specific manner by amplifying, for example, individual amino acid biosynthesis genes or changing the properties of particular genes and investigating the influence on the production. Comparative information on the cell and Molecular Biology of E.coli and Salmonella can be found in Neidhardt (ed) Escherichia coli and Salmonella, Cellular and Molecular Biology,2^ndedition, ASMPress, Washington, d.c., USA, (1996). A review of L-threonine metabolism and production is given by Debabov (Adva)Was published by sources in Biochemical Engineering Vol.79,113-136(2003)) and by ripping and Hermann (Microbiology monograms, Vol.5,71-92, ISSN 1862-5576 (print) and 1862-5584 (on-line), Springer Verlag Berlin/Heidelberg (2007)).

It has previously been found that proteins having amino acid export protein activity (gene products of the rhtC gene) catalyze the export of the amino acid L-threonine. Thus, overexpression of rhtC leads to the external accumulation of this metabolite by mediating resistance to threonine (Zakataeva et al, FEBS Letters 452(3):228-32(1999), Kruse et al, applied microbiological Biotechnology 59(2-3):205-10 (2002)). Amino acid exporters belong to the Rht family of amino acid exporters (Aleshin et al, Trends in Biochemical Science 24(4):133-5(1999)) and are assumed to function as threonine/proton antiporters.

The nucleotide sequences of the wild-type rhtC gene and the upstream region which code for the amino acid export protein of E.coli are generally available in the databases of the National Center for Biotechnology Information (NCBI) of the national library of medicine (Bethesda, MD, USA) under the accession number NC000913(region: 4005780-.

European patent application EP 1013765A 1 describes the beneficial effect of rhtC gene overexpression on the production and production of different amino acids such as L-threonine, L-homoserine, L-valine and L-leucine by Escherichia (Escherichia) strains, in which case the overexpression is achieved by increasing the copy number of the rhtC gene or by combining the rhtC gene with a promoter effective in Escherichia.

The present inventors have recently found that high expression levels of membrane proteins, such as amino acid transporters and in particular amino acid exporters, such as rhtC, can lead to reduced production levels of the desired amino acid, depending on the expression system. In particular and with respect to amino acid production, strong promoter systems are not suitable for expressing the above-mentioned membrane proteins in the corresponding amino acid production systems.

Thus, for membrane proteins, it is particularly important to regulate the correct level of (constitutive or inducible) expression during fermentation when high concentrations of amino acids accumulate in the fermentation broth. EP 1013765 describes a process for preparing L-threonine-resistant bacteria by amplifying the copy number of the rhtC gene, and the L-threonine titer is increased relative to strains in which the rhtC gene is not enhanced. However, the effect of overexpression in Escherichia strains that produce commercially relevant amounts of amino acids is not described.

Gene expression is controlled in particular by a promoter region in the 5' region of the gene. Promoters initiate transcription through the interaction of transcription factors and RNA polymerase. As a result, the promoter contains many conserved sequence motifs which can be determined based on its consensus sequence (Fournier et al, Antimicrobial Agents and Chemistry 39(6): 1365. sup. -. 1368 (1995); Chapon, EMBO Journal 1: 369. sup. -. 374 (1982); Smith et al, Journal of bacteriological Chemistry 257: 9043. sup. -. 9048 (1982)).

The following general bacterial promoter elements were classified on the basis of the consensus sequence of genes transcribed by means of the sigma-70 factor in the best studied bacterial model organism Escherichia coli (Rosenberg et al, Nature 272:414-423 (1978); Hawley and McClure, Nucleic Acids Research 11(8):2237-2255 (1983)):

35 region (35 base pair sequence upstream of the transcription start point), consensus sequence: 5'-TTGACA-3',

-10 region (this sequence can be found approximately 10 base pairs upstream of the transcription start point), also known as Pribnow box, consensus sequence: 5 '-TATAAT-3'.

The sigma factor of RNA polymerase binds to both regions, and the polymerase then induces transcription of downstream genes. "consensus sequences" for strong and weak promoters can be obtained by comparing the DNA sequences of the respective promoters. The position of the promoter elements with respect to each other and with respect to the transcription start site is also important. The distance from-10 to the transcription start site in the consensus sequence is 5-7 base pairs, with-10 and-35 regions separated by 16-18 base pairs. However, the similarity of the promoter to the consensus sequence does not necessarily provide high expression levels in every strain of Escherichia, and other regulatory mechanisms controlling the expression level may be more important than the optimized consensus sequence.

In view of the above findings regarding the expression level of membrane proteins, there is still a need to provide improved methods and tools for regulating the expression level of membrane proteins, in particular amino acid exporters such as rhtC, to further improve the fermentative production of L-amino acids, in particular L-threonine, L-homoserine, L-lysine, L-tryptophan, L-valine, L-leucine, L-isoleucine and L-histidine, by L-amino acid-producing microorganisms of the Enterobacteriaceae family.

Disclosure of Invention

The present invention relates to a recombinant L-amino acid-secreting enterobacteriaceae microorganism comprising a DNA fragment having promoter activity functionally linked to a polynucleotide encoding a membrane protein, characterized in that said DNA fragment having promoter activity comprises the amino acid sequence of SEQ ID NO: 8.

the invention further provides a novel method or process for preparing L-amino acid or feed additive containing L-amino acid by using the microorganism.

Furthermore, the present invention relates to a polypeptide comprising SEQ ID NO:8 and functionally linked to a DNA fragment having promoter activity of a polynucleotide encoding a membrane protein.

Finally, the invention relates to a polypeptide comprising SEQ ID NO:8 as a promoter for regulating the expression level of a gene encoding a membrane protein or an amino acid transporter.

Brief Description of Drawings

FIG. 1: map of expression plasmid pMW219_ P (allel) rhtC.

FIG. 2: map of the gene replacement vector pKO3 rhtC-Pmut.

The base pair numbers are approximations obtained in reproducibility measurements. Abbreviations and names used have the following meanings:

BssHII: cleavage site for restriction enzyme BssHII

HindIII: cleavage site for restriction enzyme HindIII

KpnI: cleavage site for restriction enzyme KpnI

NcoI: cleavage site for restriction enzyme NcoI

SpeI: cleavage site for the restriction enzyme SpeI

XbaI: cleavage site for restriction enzyme XbaI

Cm: chloramphenicol resistance gene

Km: kanamycin resistance gene

lacZ "5' portion of lacZ α Gene fragment

3 'part of the fragment of the' lacZ α Gene

oriC: origin of replication

rhtC: threonine export protein RhtC gene

And (5) sacB: sacB gene

repA: genes for replication protein RepA

Further details can be found in the examples.

The invention is illustrated below by means of non-limiting examples and exemplary embodiments.

Detailed Description

The present invention relates to recombinant L-amino acid-secreting microorganisms of the Enterobacteriaceae family which comprise a DNA fragment having promoter activity functionally linked to a polynucleotide encoding a membrane protein and which secrete (i.e.are produced and concentrated in cells or culture media) increased amounts of L-amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, wherein the DNA fragment having promoter activity comprises the amino acid sequence of SEQ ID NO: 8.

according to SEQ ID NO:8 is derived from a polynucleotide according to SEQ ID NO: 7(wt) by replacement of the nucleobase cytosine at position 24 with thymine.

The present inventors have surprisingly found that the use of the above-specified promoter sequences enables the regulation of the expression level of genes encoding membrane proteins, in particular amino acid transporters such as amino acid exporters such as rhtC, in such a way that the ability of the fermentative production of amino acids is improved when commercially significant concentrations are accumulated in the fermentation broth.

The term "modulation of the expression level" as used in the context of the present invention refers to setting a balanced expression level resulting in an optimal yield of the amino acid product. As described in detail below, the gene expression level can be tailored to the specific needs of transporters abundant in the cell membrane, by using the above-mentioned DNA fragments as promoter elements in higher production strains to regulate the expression level of genes encoding membrane proteins or amino acid transporters, in particular amino acid exporter genes such as rhtC, without affecting the adaptability and productivity of the cells.

The invention further provides a microorganism comprising a DNA fragment having promoter activity, characterized in that the DNA fragment is linked at the 3' end to a second DNA fragment carrying a ribosome binding site.

In the microorganism according to an embodiment of the present invention, the above-mentioned DNA fragment may be ligated at its 3' end with a DNA having the sequence of SEQ ID NO:9, which is the naturally occurring 3' -flanking region of said DNA segment. Has the sequence shown in SEQ ID NO:9 the second DNA fragment of the nucleotide sequence at position 174-204 may be ligated at its 3' end with a polynucleotide encoding a membrane protein.

Preferably, the membrane protein is a protein having amino acid transporter activity, in particular an amino acid export protein, such as RhtC.

The DNA fragment having promoter activity according to the present invention may be ligated at the 5' end with a DNA fragment having the sequence of SEQ ID NO:9, which is a naturally occurring 5' -flanking region of said DNA fragment.

The microorganisms according to the invention include in particular those of the Enterobacteriaceae family in which the promoter activity is present and which comprise the amino acid sequence of SEQ ID NO:8 is functionally linked at the 3' end to a polynucleotide whose amino acid sequence is at least 70% or at least 80%, or at least 90%, in particular at least 95%, preferably at least 98% or at least 99%, particularly preferably up to 99.6%, very particularly preferably up to 100% identical to the amino acid sequence of SEQ ID No. 2.

The microorganism comprises a polynucleotide selected from the group consisting of:

a) has a sequence selected from SEQ ID NO:1 and the nucleotide sequence complementary thereto;

b) having a sequence corresponding to SEQ ID NO: 1;

c) has a sequence that hybridizes under stringent conditions to the sequence of SEQ ID NO:1, said stringent conditions are preferably obtained by a washing step at a temperature in the range of 64 ℃ to 68 ℃ and a salt concentration of the buffer in the range of 2 x SSC to 0.1 x SSC;

d) has the sequence SEQ ID NO:1, comprising a functional neutral sense mutant,

preferably has the sequence of SEQ ID NO:1, which encodes an amino acid export protein.

Preferably, the amino acid export protein is RhtC.

The DNA fragment having promoter activity may be present in the chromosome of the microorganism. Alternatively, it may be located on an extrachromosomal replicating vector.

There are basically two possibilities for gene expression. In continuous expression, the gene is continuously expressed by a constitutive promoter, and the corresponding protein is accumulated in the cell.

Alternatively, inducible promoters may be used to induce gene expression. Expression of the target gene can be induced by an inducer. The method is used if (over) expression has a negative effect on the producing organism. The reason for this may be a high loading of metabolic sources during growth. The result is slower growth and therefore longer operating times of the bioreactor and the associated increased costs in the case of industrial production. The induced expression is also advantageous in the case of cytotoxic products. Here, autotoxicity and cell death occur after induction of expression. With regard to the economics of the production process, it is therefore attempted to subdivide the process into a growth phase and a production phase. As large a quantity of biomass as possible is produced during the growth phase, and then the target protein is produced by inducing the promoter during the production phase. In this way maximum yields can be obtained, and the process becomes significantly more economical.

The invention also relates to a process for the fermentative preparation of L-amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, using recombinant microorganisms of the Enterobacteriaceae family which secrete L-amino acids, in particular even before using the DNA fragments with promoter activity according to the invention, and in which at least one polynucleotide which encodes a membrane protein or a protein with the activity of an amino acid transporter, such as an amino acid exporter, e.g.rhtC, is functionally linked to the DNA fragments with promoter activity.

The method for producing an L-amino acid by fermentation of the recombinant microorganism of the present invention comprises the steps of:

(i) fermenting the microorganism of the invention in a culture medium;

(ii) enriching the fermentation broth and/or the microbial cells for L-amino acids; and optionally

(iii) Isolating the L-amino acid.

Furthermore, the present invention relates to a DNA fragment having promoter activity and comprising SEQ ID NO 8, said DNA fragment being functionally linked to a polynucleotide encoding a membrane protein.

Such a DNA fragment comprising SEQ ID NO 8 can be used as a promoter for regulating the expression level of a gene encoding a membrane protein or an amino acid transporter, particularly an amino acid exporter gene such as rhtC.

If mutations are made in the regulatory sequence upstream of the start codon, care must be taken as to the functionality of these elements as a function of the sequence and distance from the start codon. The expression "functionally linked" as used herein refers to a regulatory sequence such as a promoter controlling the expression of a gene.

Microorganisms which produce L-amino acids before the use of DNA fragments having promoter activity according to the invention are excluded here both in wild-type strains and in the usual laboratory strains, for example DH5 α, DH5 α mcr, W3110, MG1655, MC4100, Y1089, H560 and MM 152.

If L-amino acids or amino acids are mentioned below, this is intended to mean one or more amino acids selected from the group consisting of: l-asparagine, L-threonine, L-serine, L-glutamic acid, 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, L-glutamine, L-aspartic acid and L-homoserine. L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine are particularly preferred.

In this connection, the terms "use of a DNA fragment having promoter activity" or "use of a DNA fragment comprising SEQ ID NO:8 as a promoter" describe the incorporation of said DNA fragment upstream of the structural gene to regulate transcription.

Substitutions in the DNA fragments having promoter activity used in the microorganisms used in the method of the present invention can be generated by using, for example, the site-directed mutagenesis methods described in the prior art.

The mutagenesis generated may be determined and detected by DNA sequencing, for example by the method of Sanger et al (Proceedings of the National Academy of sciences USA 74(12): 5474: 5463 (547)) using the method of directed mutagenesis of the mutagenic Oligonucleotide (T.A. Brown: Gentechnologie f ü rEinstegiers [ Genetic engineering for probes ], Spektrum Akademischer Verlag, Heidelberg,1993) or the Polymerase Chain Reaction (PCR) as described by Gait: Oligonucleotides synthesis: APracial application (IRL Press, Oxford, UK,1984) or Newton and Graham (PCR, Spektrum ademischer Verlag, Heidelberg, 1994).

For the purpose of constructing base substitutions in the promoter region of a polynucleotide encoding a protein having amino acid exporter activity, for example, Q5 Site-Directed Mutagenesis kit of New England Biolabs GmbH (Frankfurt, Germany) can be used. In using these methods, a region of about 200 base pairs in the 5' region of the nucleotide sequence encoding a protein having amino acid export protein activity described in the prior art is cloned into an appropriate plasmid vector starting from the total DNA of the wild type strain by means of Polymerase Chain Reaction (PCR) amplification, and the DNA is then subjected to a mutagenesis process. Point mutations have been obtained by PCR using "GeneSOEing" (Gene spraying by overlapExtension, Horton, Molecular Biotechnology 3: 93-98 (1995)).

Furthermore, the toxicity caused by high concentrations of amino acids and/or related molecules enables the screening and selection of spontaneous mutations in the promoter region by increasing the level of resistance of the related strains.

The resulting promoter mutations can be incorporated into suitable strains, for example, by transformation and gene or allele replacement processes.

Hamilton et al (Journal of Bacteriology 174,4617-4622(1989)) describe the general approach by means of the conditional replication pSC101 derivative pMAK705 or the allele replacement method using pKO3 (Linket et al, Journal of Bacteriology 179: 6228-6237). Other methods described in the prior art, such as Martinez-Morales et al (Journal of Bacteriology 1999, 7143-.

The resulting promoter mutations can also be transferred to various strains by conjugation or transduction.

Therefore, the mutant DNA fragment having promoter activity can be stably integrated into the chromosome of a microorganism, thereby enabling constitutive expression of the downstream structural gene.

Mutant DNA fragments having promoter activity may also be present on extrachromosomal replicating vectors, thereby allowing overproduction of the resulting protein of the downstream structural gene on the expression plasmid.

A more detailed explanation of the concepts of Genetics and molecular biology can be found in known textbooks of Genetics and molecular biology, such as Birge (Bacterial and bacteriophase Genetics, 4)^thed., Springer Verlag, New York (USA),2000) or Berg, Tymoczko and Stryer (Biochemistry, 5)^thed. textbook by Freeman Company, New York (USA),2002, or the Manual Sambrook et al (MolekularCloning, A Laboratory Manual, (3-Volume)Set),Cold Spring Harbor LaboratoryPress,Cold Spring Harbor(USA),2001)。

The concentration of the protein can be determined by one-and two-dimensional protein gel fractionation followed by optical identification of the protein concentration in the gel using appropriate evaluation software. A common method for preparing the protein gel and identifying the protein is that described by Hermann et al (Electrophoresis,22:1712-23 (2001)). Protein concentrations can likewise be determined by Western blot hybridization with antibodies specific for the protein to be detected (Sambrook et al, Molecular cloning: a laboratory Manual.2)^ndEd.cold Spring Harbor laboratory press, Cold Spring Harbor, n.y.,1989) and subsequent optical evaluation using appropriate concentration determination software (Lohaus and Meyer (1998) Biospektrum 5: 32-39; lottspeich, Angewandte Chemie38:2630-2647 (1999)).

Chemically, a gene or allele is a polynucleotide. Alternative terms herein are nucleic acids, in particular deoxyribonucleic acids.

In this context, a DNA fragment refers to a portion of a nucleotide sequence that does not encode a protein or polypeptide or a ribonucleic acid.

The terms polypeptide and protein are used interchangeably.

An Open Reading Frame (ORF) refers to a part of a nucleotide sequence which encodes or can encode a protein or polypeptide or ribonucleic acid whose function cannot be assigned according to the prior art. After assigning a function to a certain part of the nucleotide sequence in question, the latter is often referred to as a gene. Alleles generally mean alternative forms of a given gene. The forms are distinguished by differences in nucleotide sequence.

A gene product generally refers to a protein or ribonucleic acid, i.e., an ORF, gene, or allele, encoded by a nucleotide sequence.

Microorganisms, in particular recombinant microorganisms, which comprise a DNA fragment having promoter activity comprising SEQ ID NO 8, which DNA fragment is functionally linked to a polynucleotide which codes for a membrane protein or a protein having the activity of an amino acid transporter, for example an amino acid export protein such as rhtC, can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, suitable starch, suitable cellulose or from glycerol and ethanol and, where appropriate, also from mixtures, as subject of the invention.

The microorganism of the present invention is a representative microorganism of the family Enterobacteriaceae. For example, it may be selected from the genera Escherichia (Escherichia), Erwinia (Erwinia), Providencia (Providencia) and Serratia (Serratia). Preferred are the genera Escherichia and Serratia. Particularly, Escherichia coli belonging to the genus Escherichia and Serratia marcescens (Serratia marcescens) belonging to the genus Serratia are mentioned.

Recombinant microorganisms are usually produced by transformation, transduction or conjugation or a combination of these methods with a vector comprising the desired gene, the desired ORF, an allele of said gene or ORF or a part thereof and/or a promoter enhancing the expression of said gene or ORF.

For the preparation of L-amino acid-enriched strains of the Enterobacteriaceae family which comprise a DNA fragment having promoter activity and comprising SEQ ID NO:8, which is functionally linked to a polynucleotide encoding a protein having amino acid exporter activity, preference is given to using strains (starting strains or parent strains) which already have the ability to concentrate the desired L-amino acid in the cell and/or secrete it into the surrounding nutrient medium or accumulate it in the fermentation broth. The term "generating" may also be used herein. More particularly, the strains used in the measures according to the invention have the ability to concentrate or accumulate not less than (at least) 2.0g/L,. gtoreq.8.0 g/L,. gtoreq.10.0 g/L,. gtoreq.50 g/L,. gtoreq.100 g/L or not more than 150g/L of L-amino acids in cells and/or in nutrient media or fermentation broths within not more than (not more than) 120 hours,. ltoreq.96 hours,. ltoreq.48 hours,. ltoreq.36 hours,. ltoreq.24 hours or. ltoreq.12 hours. The strains may be prepared by mutagenesis and selection, by recombinant DNA techniques, or by a combination of both methods.

The L-amino acid-secreting strain produces one or more, preferably one or essentially one amino acid selected from the group consisting of: l-asparagine, L-threonine, L-serine, L-glutamic acid, 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, L-glutamine, L-aspartic acid and L-homoserine, preferably selected from L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine. The term L-amino acid or amino acid also includes salts thereof.

The term "one or substantially one amino acid" contemplates that one or more other amino acids (secondary amino acids) of the L-amino acid may be produced in addition to the desired L-amino acid. The proportion of these secondary amino acids is ≥ 0 to not more than 40%, preferably ≥ 0 to not more than 20%, particularly preferably ≥ 0 to not more than 10%, very particularly preferably ≥ 0 to not more than 5%, based on the desired amount of L-amino acid.

Examples which may be mentioned as suitable parent strains, in particular strains of the genus Escherichia, in particular of the species Escherichia coli, which produce or secrete L-threonine, are:

escherichia coli H4581(EP 0301572)

Escherichia coli KY10935(Bioscience Biotechnology and Biochemistry 61 (11): 1877-1882(1997))

EscherichiA coli VNIIgenetikA MG442(US-A-4278,765)

Coli VNIIgenetikA M1(US-A-4,321,325)

EscherichiA coli VNIIgenetikA 472T23 (U.S. Pat. No. 5,631,157)

Coli TH 14.97(WO 02/26993)

Coli TH 21.97(WO 02/26993)

Coli BKEIM B-3996(US-A-5,175,107)

Escherichia coli BKEIM B-3996 Δ tdh Δ pckA/pVIC40(WO 02/29080)

Escherichia coli kat 13(WO 98/04715)

Escherichia coli Kat 69.9(WO 02/26993)

Escherichia coli KCCM-10132(WO 00/09660)

Escherichia coli KCCM-10168(WO 01/14525)

Escherichia coli KCCM-10133(WO 00/09661)

Examples of suitable parent strains of the genus Serratia which produce or secrete L-threonine, in particular the species Serratia marcescens (Serratia marcescens), which may be mentioned are:

serratia marcescens HNr21(Applied and Environmental Microbiology 38(6):1045-1051(1979))

Serratia marcescens TLr156(Gene 57(2-3):151-158(1987))

Serratia marcescens T-2000(Applied Biochemistry and Biotechnology 37(3):255-265 (1992)).

L-threonine-producing or secreting Enterobacteriaceae strains preferably have one or more genetic or phenotypic characteristics, for example, selected from the group consisting of resistance to α -amino- β -hydroxypentanoic acid, resistance to thiolysine, resistance to ethionine, resistance to α -methylserine, resistance to diaminosuccinic acid, resistance to α -aminobuteric acid, resistance to borrelidine, resistance to cyclopentane-carboxylic acid, resistance to rifampicin, resistance to valine analogs such as valine hydroxamate (valine hydroxamate), resistance to purine analogs such as 6-dimethylaminopurine, requiring L-methionine, suitably partial or compensable L-isoleucine, requiring meso-diaminopimelic acid, resistance to threonine-containing dipeptides, resistance to L-threonine, resistance to threonine-extracts, resistance to L-homoserine, resistance to L-lysine, resistance to L-methionine, increased resistance to L-threonine-containing dipeptides, increased resistance to L-threonine-pyruvate dehydrogenase, increased resistance to L-pyruvate kinase, increased resistance to L-pyruvate kinase, increased feedback, increased resistance to L-pyruvate kinase, increased resistance to cysteine kinase, increased resistance to the enzyme, increased resistance to the form of the enzyme of the serine-pyruvate kinase, increased feedback, increased resistance to the serine-pyruvate kinase, increased resistance to the enzyme, increased.

Examples of suitable Escherichia parent strains, in particular Escherichia coli species, which secrete or produce L-homoserine which may be mentioned are:

coli NZ10rhtA23/pAL 4(US 6,960,455).

The L-homoserine producing or secreting enterobacteriaceae strain preferably has one or more genetic or phenotypic characteristics, for example selected from the group consisting of: l-threonine, L-methionine, L-isoleucine, defective homoserine kinase, suitably sucrose utilization, increased homoserine dehydrogenase I/aspartokinase I, preferably in feedback resistant form, increased RhtA gene product.

Examples of suitable parent strains of Escherichia, in particular E.coli, which secrete or produce L-lysine which may be mentioned are:

escherichia coli VL613(VKPM B-3423) (EP1149911)

Escherichia coli AJ11442(FERM BP-1543) (U.S. Pat. No. 4,346,170)

The L-lysine-producing or secreting enterobacteriaceae strains preferably have one or more genetic or phenotypic characteristics, for example selected from the group consisting of: resistance to lysine analogs such as oxolysine, lysine hydroxamic acid, (S) -2-aminoethyl-L-cysteine (AEC), γ -methyllysine, chlorocaprolactam, and the like, desensitized aspartokinase, and desensitized phosphoenolpyruvate carboxylase.

Examples of suitable parent strains of Escherichia, in particular E.coli, which secrete or produce L-tryptophan, which may be mentioned are:

escherichia coli JP4735/pMU3028(DSM10122) (U.S. Pat. No. 5,756,345)

Escherichia coli JP6015/pMU91(DSM10123) (U.S. Pat. No. 5,756,345)

Escherichia coli AGX17(pGX44) (NRRL B-12263) (U.S. Pat. No. 4,371,614)

The L-tryptophan-producing or secreting enterobacteriaceae strain preferably has one or more genetic or phenotypic characteristics, for example selected from the group consisting of: the activity of at least one enzyme selected from the group consisting of: anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), 3-deoxy-D-arabinose-heptulosonate-7-phosphate synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolpyruvylshikimate-3-phosphate synthase (aroA), chorismate synthase (aroC), prephenate dehydratase, chorismate mutase, and tryptophan synthase (trpAB); the activity of chorismate mutase/prephenate dehydratase or chorismate mutase/prephenate dehydrogenase can be further attenuated in that one or more of anthranilate synthase and phosphoglycerate dehydrogenase is released from the feedback inhibition by L-tryptophan and L-serine.

Examples of suitable parent strains of Escherichia, in particular E.coli, which secrete or produce L-isoleucine, which may be mentioned are:

escherichia coli H-8670(FERM BP-4051) (US5,460,958)

Escherichia coli H-8683(FERM BP-4052) (US5,460,958)

Escherichia coli FERM BP-3628 (US5,362,637)

Escherichia coli FERM BP-3629 (US5,362,637)

Escherichia coli FERM BP-3630 (US5,362,637)

Escherichia coli H-9146(FERM BP-5055) (US5,695,972)

Escherichia coli H-9156(FERM BP-5056) (US5,695,972)

The strains of the family Enterobacteriaceae which produce or secrete L-isoleucine preferably have one or more genetic or phenotypic characteristics, for example selected from the group consisting of: resistance to isoleucine analogues such as thioisoleucine, resistance to ethionine, resistance to arginine hydroxamate, resistance to S (2 aminoethyl) -L-cysteine, and resistance to D-serine.

Examples of suitable parent strains of the genus Escherichia, in particular of the species Escherichia coli, which secrete or produce L-valine which may be mentioned are:

EscherichiA coli AJ11502(NRRL B-12288) (US-A-4391907)

Examples of suitable parent strains of the genus Escherichia, in particular of the species Escherichia coli, which secrete or produce L-leucine, which may be mentioned are:

escherichia coli H-9070(FERM BP-4704) (US5,744,331)

Escherichia coli H-9072(FERM BP-4706) (US5,744,331)

The L-leucine producing or secreting Enterobacteriaceae strain preferably has one or more genetic or phenotypic characteristics, for example selected from the group consisting of resistance to leucine analogues such as 4-azaleucine or 5,5, 5-trifluoroleucine, resistance to β -2-thienylalanine, suitably the ability to utilize sucrose, an enhanced leucine operon, an increased 2-isopropylmalate synthase, an increased 3-isopropylmalate dehydrogenase, an increased isopropylmalate isomerase, an increased leucine transaminase, an increased leucine aminotransferase, an increased leucine exporter.

Examples of suitable parent strains of Escherichia, in particular E.coli, which secrete or produce L-alanine which may be mentioned are:

escherichia coli strain K88(FERM BP-4121) (US5,559,016)

The L-alanine-producing or L-alanine-secreting Enterobacteriaceae strains preferably have, for example, a heterologous L-alanine dehydrogenase gene, preferably from the genus Arthrobacter (Arthrobacter) or Bacillus (Bacillus) or Actinomycetes (Actinomycetes), particularly preferably from the species Arthrobacter (Arthrobacter sp.) HAP 1.

Examples of suitable parent strains of Escherichia, in particular E.coli, which secrete or produce L-histidine that may be mentioned are:

escherichia coli AJ 11388 (FERM-P5048, NRRL B-12121) (US4,388,405)

The L-histidine producing or secreting enterobacteriaceae strain preferably has one or more genetic or phenotypic characteristics, for example selected from the group consisting of: resistance to histidine analogs such as 2-thiazolylalanine, 1,2, 4-triazolylalanine, 2-methylhistidine and histidine hydroxamate, suitably the ability to utilize sucrose, an enhanced histidine operon, an increased ATP phosphoribosyltransferase, an increased phosphoribosyl ATP pyrophosphohydrolase, an increased phosphoribosyl AMP cyclohydrolase, an increased cyclase HisF, an increased glutaminyltransferase HisH, an increased 1- (5-phosphoribosyl) -5- [ (5-phosphoribosylamino) methylene-amino ] imidazole-4-carboxamide isomerase, an increased imidazole phosphoglycerate dehydratase, an increased histidine phosphate transaminase, an increased histidinol phosphate phosphatase, an increased histidinol dehydrogenase.

In the studies on which the present invention was based, microorganisms of the Enterobacteriaceae family which comprise a DNA fragment having promoter activity which is functionally linked to a polynucleotide which encodes a membrane protein or a protein having the activity of an amino acid transporter, such as an amino acid exporter, e.g.rhtC, have been found to produce increased amounts of L-amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, and to concentrate them in cells or culture media.

The nucleotide sequence of the E.coli gene or Open Reading Frame (ORF) is part of the prior art and can be found in the E.coli genome sequence disclosed in Blattner et al (Science 277:1453-1462 (1997)). The endogenous enzyme (methionine aminopeptidase) of the host is known to remove the N-terminal amino acid methionine.

Also known are the nucleotide sequences of polynucleotides encoding proteins having the activity of the amino acid export protein (rhtC gene) of Salmonella enterica (Salmonella enterica) and Erwinia carotovora (Erwinia carotovora) belonging to the family Enterobacteriaceae (accession No. NC-003198 (REGION: comparative (3454404 3455024) and accession No. NC-004547 (REGION: comparative (4661556) 4662179), further nucleotide sequences of the rhtC gene found in the family Enterobacteriaceae (Shigella flexneri) (accession No. CP000266, AE 005073), Shigella borealis (Shigella boydii) (accession No. CP000036), Shigella shigeldyseniae (Shigelia sonensis) (accession No. CP000034), Shigella sonneri (Shigella carotoviridae) (Salmonella typhi accession No. 0032), and Salmonella typhimurium (Sodania carotoviridae) (accession No. 008832).

The rhtC gene of E.coli K12 is described, for example, by the following information:

name: amino acid export proteins

The functions are as follows: catalysis of L-threonine amino acid export by threonine/proton antiporter function

Reference documents: zakataeva et al, FEBS Letters 452(3):228-32 (1999);

Kruse et al.,Applied Microbiological Biotechnology 59(2-3):205-10(2002)

registration number: NC000913(Region:4005780-4006400)

The encoded polypeptide is 206 amino acids in length.

Alternative gene names (from EcoCyc: Encyclopedia of Escherichia coli K-12 Genesand Metabolim, SRI International, Menlo Park, USA) b3823, yigJ

Nucleic acid sequences can be found in the databases of the National Center for Biotechnology Information (NCBI) of the national library of medicine (Bethesda, MD, USA), in the nucleotide sequence databases of the European molecular biology laboratories (EMBL, Heidelberg, Germany and Cambridge, UK), or in the DNA databases of Japan (DDBJ, Mishima, Japan).

For clarity, the sequence of the rhtC gene of E.coli is disclosed as described in SEQ ID NO 1. The protein encoded by this reading frame is described in SEQ ID NO 2. The sequence of the rhtC gene of E.coli, including the upstream and downstream nucleotide sequences, is described as SEQ ID NO:10, and the protein encoded by this reading frame is described as SEQ ID NO:11 (corresponding to SEQ ID NO: 2).

The genes or open reading frames described in the citations may be used according to the invention. Alleles of the genes or open reading frames which are the result of degeneracy of the genetic code or functionally neutral sense mutations can also be used. Preferably, endogenous genes or endogenous open reading frames are used.

"endogenous gene" or "endogenous nucleotide sequence" refers to a gene or open reading frame or allele or nucleotide sequence present in a population of matter.

Alleles of the rhtC gene which contain functional neutral sense mutations include, for example, those which lead to not more than 30 or not more than 20, preferably not more than 10 or not more than 5, particularly preferably not more than 3 or not more than 2 or at least 1 conservative amino acid substitutions in the protein which they encode. The present invention relates to conservative amino acid substitutions, wherein an amino acid is substituted with an amino acid having similar functionality, charge, polarity, or hydrophobicity.

In the case of aromatic amino acids, when phenylalanine, tryptophan, and tyrosine are substituted for each other, the substitutions are considered to be conservative. In the case of hydrophobic amino acids, when leucine, isoleucine and valine are substituted for one another, the substitutions are considered to be conservative. In the case of polar amino acids, when glutamine and asparagine are substituted for each other, the substitution is considered conservative. In the case of basic amino acids, when arginine, lysine and histidine are substituted for one another, the substitution is considered conservative. In the case of acidic amino acids, substitutions are considered to be conservative when aspartic acid and glutamic acid are substituted for one another. In the case of hydroxyl-containing amino acids, when serine and threonine are substituted for each other, the substitution is considered to be conservative.

In the same way, nucleotide sequences encoding variants of the proteins which additionally contain an extension or truncation of at least one (1) amino acid at the N-terminus or C-terminus can also be used. The extension or truncation is no more than 40, 30, 20, 10, 5,3 or 2 amino acids or amino acid residues.

Suitable alleles also include alleles encoding proteins in which at least one (1) amino acid has been inserted or deleted. The maximum number of such changes, called indels, can affect 2, 3, 5,10 amino acids, but in any case does not exceed 20 amino acids.

Suitable alleles also include those which can be obtained by hybridization, in particular under stringent conditions, using SEQ ID NO.1 or a part thereof or the sequence complementary thereto.

Guidance in The identification of DNA sequences by Hybridization is found by The skilled worker, for example, in The handbook "The DIG System Users' Guide for Filter Hybridization" supplied by Boehringer Mannheim GmbH (Mannheim, Germany,1993) and in Liebl et al (International Journal of Systematic Bacteriology 41:255-260 (1991)). Hybridization is carried out under stringent conditions, i.e., the only hybrids formed are those in which the probe and target sequence (i.e., the polynucleotide treated with the probe) are at least 70% identical. It is known that the stringency of hybridization, including washing steps, can be influenced and/or determined by varying the buffer composition, temperature and salt concentration. Typically, hybridization reactions are performed at relatively low stringency compared to washing steps (Hybaid hybridization Guide, Hybaid Limited, Teddington, UK, 1996).

For example, a buffer corresponding to a 5 XSSC buffer may be used for the hybridization reaction at a temperature of about 50 ℃ to 68 ℃. Under these conditions, the probe may also hybridize to a polynucleotide having less than 70% identity to the probe sequence. These hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by reducing The salt concentration to 2 XSSC and, where appropriate, subsequently to 0.5 XSSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany,1995) and adjusting The temperature to about 50 ℃ to 68 ℃, about 52 ℃ to 68 ℃, about 54 ℃ to 68 ℃, about 56 ℃ to 68 ℃, about 58 ℃ to 68 ℃, about 60 ℃ to 68 ℃, about 62 ℃ to 68 ℃, about 64 ℃ to 68 ℃, about 66 ℃ to 68 ℃. The preferred temperature range is about 64 deg.C-68 deg.C or about 66 deg.C-68 deg.C. The salt concentration can be reduced to a concentration corresponding to 0.2 XSSC or 0.1 XSSC, as appropriate. By increasing the hybridization temperature stepwise from 50 ℃ to 68 ℃ at about 1-2 ℃, polynucleotide fragments can be isolated which are, for example, at least 70%, or at least 80%, or at least 90%, or 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% identical to the sequence of the probe used or to the nucleotide sequence shown in SEQ ID No. 1. Additional guidance regarding hybridization can be obtained commercially in the form of so-called kits (e.g., DIG Easy Hyb from Roche diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

Furthermore, when L-amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, are produced using strains of the Enterobacteriaceae family, it may be advantageous, in addition to regulating the expression of genes which code for membrane proteins or for proteins having the activity of amino acid transporters, such as amino acid exporters, for example rhtC, to increase one or more enzymes of the known biosynthetic pathway or amino acid transport or enzymes of anaplerotic metabolism or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate or glycolytic enzymes or PTS enzymes or sulfur metabolizing enzymes in the promoter region of said genes by any of the mutagenesis measures mentioned above. It is generally preferred to use endogenous genes.

In this connection, the term "enhancement" describes the increase in the intracellular activity or concentration of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, by increasing the copy number of, for example, one or more genes or one or more ORFs by at least one copy, functionally linking a strong promoter to said gene or using genes or alleles or ORFs which encode corresponding enzymes or proteins with high activity, and where appropriate combining these measures.

In appropriate cases, it may be advantageous to modulate the expression or production level of one or more enzymes/proteins in a microorganism, for example by only moderately increasing the intracellular concentration or activity of the respective enzyme/protein, since an increase in enzyme/protein concentration which is too high may lead, for example, to defective cell division or altered cell morphology and even toxicity (Guthrie and wicker, Journal of Bacteriology 172(10): 5555-.

The term "moderately increased" describes an increase of the intracellular activity or concentration of the corresponding protein of not more than 10-fold, 8-fold, 6-fold, 4-fold, 3-fold, 2-fold or 1.5-fold based on the wild-type protein or based on the activity or concentration of the protein in a non-recombinant microorganism or parent strain of the corresponding enzyme or protein. A non-recombinant microorganism or parent strain refers to a microorganism that is to undergo enhancement or overexpression according to the invention.

To achieve enhancement, for example, the expression of a gene or open reading frame or allele or the catalytic properties of a protein may be increased. Both measures may be combined, where appropriate.

To achieve overexpression, for example, the copy number of the corresponding gene or open reading frame can be increased, or the promoter region and the regulatory region or the ribosome binding site located upstream of the structural gene can be mutated. Expression cassettes incorporated upstream of the structural gene function in the same way. Can also increase expression in the process of amino acid fermentation production through an inducible promoter; furthermore, it is also advantageous to use gene expression promoters which allow for gene expression in different time sequences. At the level of translational regulation of gene expression, the initiation frequency (binding of ribosomes to mRNA) or the elongation (elongation phase) can be increased. Expression is likewise improved by measures which extend the life of the mRNA. In addition, the enzyme activity is also enhanced by preventing enzyme protein decomposition. The ORFs, genes or gene constructs may be present in plasmids with different copy numbers or integrated and amplified in the chromosome. Alternatively, overexpression of the relevant gene can also be achieved by changing the composition of the medium and the culture mode.

Methods of overexpression are well described in the prior art, for example Makrides et al (Microbiological Reviews 60(3), 512-. The copy number is increased by at least one (1) copy using a vector. The vector used may be a plasmid, as described in US5,538,873. The vector used may also be a bacteriophage, for example bacteriophage Mu, as described in EP 0332448, or bacteriophage lambda (. lamda.). The copy number can also be increased by incorporating an additional copy into another site in the chromosome, for example the att site of bacteriophage lambda (Yu and Coart, Gene 223,77-81 (1998)).

Furthermore, replacement of the start codon with ATG, the most common (77%) codon in E.coli, can significantly improve translation because AUG codons are 2-3 fold more efficient than e.g. the codons GUG and UUG (Khudyakov et al, FEBS Letters232(2):369-71 (1988); Reddy et al, Proceedings of the National Academy of sciences of the USA 82(17):5656-60 (1985)). The sequence around the start codon can also be optimized, since the combined effect of the start codon and flanking regions has been described (Stenstrom et al, Gene 273(2):259-65 (2001); Hui et al, EMBO Journal 3(3):623-9 (1984)).

The skilled worker can find general guidance in this respect, for example in the following documents: 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)), de 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)), PCT/US97/13359, Llosa et al (Plasmid 26:222-224(1991)), Quandedt and Klipp (Gene 80:161-169(1989)), Journal of Bacteriology 171:4617 (19817)), and molecular biology (Hamming 191: 4617 and Biotechnology (1998)).

Plasmid vectors which are replicable in the Enterobacteriaceae family, such as pACYC 184-derived cloning vectors (Bartolom et al; Gene 102:75-78(1991)), pTrc99A (Amann et al; Gene 69:301-315(1988)), or pSC 101-derived vectors (Vocke and basic; Proceedings of the National academy of Sciences USA 80(21):6557-6561(1983)) may be used. In the method according to the invention, strains transformed with plasmid vectors carrying at least the rhtC gene or the nucleotide sequence or allele encoding the gene product thereof can be used.

The term "transformation" is understood to mean the uptake of a nucleic acid by a host (microorganism).

Alleles in the microorganisms used in the process of the invention can be generated using, for example, site-directed mutagenesis methods as described in the prior art.

Thus, for example, for the production of L-threonine, it is possible to simultaneously enhance, in particular overexpress, one or more genes selected from the group consisting of:

at least one gene of the thrABC operon coding for aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (U.S. Pat. No. 4,278,765),

the pyc gene of Corynebacterium glutamicum coding for pyruvate carboxylase (WO 99/18228),

the pps gene which codes for phosphoenolpyruvate synthase (Molecular and General Genetics 231 (2): 332-336 (1992); WO 97/08333),

the ppc gene which codes for phosphoenolpyruvate carboxylase (WO 02/064808),

the pntA and pntB genes encoding subunits of pyridine transhydrogenase (European Journal of Biochemistry 158: 647-653 (1986); WO 95/11985),

the thrE gene of Corynebacterium glutamicum which codes for a threonine export carrier protein (WO 01/92545),

the gdhA Gene encoding glutamate dehydrogenase (Nucleic Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983); DE19907347),

the ptsHIcrr operon ptsH gene encoding phosphohistidine protein hexose phosphotransferase of the PTS phosphotransferase system (WO 03/004674),

the ptsHIcrr operon ptsI gene encoding enzyme I of the PTS phosphotransferase system (WO 03/004674),

the ptsHIcrr operon crr gene encoding the glucose-specific IIA component of the PTS phosphotransferase system (WO 03/004674),

the ptsG gene which codes for a glucose-specific IIBC component (WO 03/004670),

the cysK gene which codes for cysteine synthase A (WO 03/006666),

the cysB gene which codes for a regulatory protein of the cys regulon (WO 03/006666),

the cysJIH operon cysJ gene which codes for the NADPH sulfite reductase flavoprotein (WO 03/006666),

the cysJIH operon cysI gene which codes for the NADPH sulfite reductase hemoprotein (WO 03/006666),

the cysJIH operon cysH gene coding for adenylate sulfate reductase (WO 03/006666),

the sucABCD operon sucA gene encoding the decarboxylase subunit of 2-ketoglutarate dehydrogenase (WO 03/008614),

the sucABCD operon sucB gene of the subunit E2 of dihydrolipoic acid-succinyltransferase, which codes for 2-ketoglutarate dehydrogenase (WO 03/008614),

the sucABCD operon sucC gene encoding the β -subunit of succinyl-CoA synthase (WO 03/008615),

the sucABCD operon sucD gene encoding the α -subunit of succinyl-CoA synthase (WO 03/008615),

the gene product of the E.coli Open Reading Frame (ORF) yjcG from the national center for Biotechnology information (NCBI, Bethesda, Md., USA) (accession number NC000913(region 4281276-,

the gene product of the E.coli Open Reading Frame (ORF) yibD of the national center for Biotechnology information (NCBI, Bethesda, Md., USA) (accession number NC000913(region 3787070) -3788104),

the gene product of the Open Reading Frame (ORF) of E.coli yaaU (accession number NC000913(region 45807-,

the rhtA gene encoding L-threonine and L-homoserine export proteins (Astauroviva et al, applied Biochemical Microbiology 21:611-616 (1985); RU Patent No.974817),

the rhtB gene which encodes L-homoserine and homoserine-lactone exporters (Zakataeva et al, FEBS Letters 452(3): 228-.

L-threonine producing microorganisms of the Enterobacteriaceae family generally have feedback-resistant or desensitized aspartate kinase I/homoserine dehydrogenase feedback-resistant aspartate kinase/homoserine dehydrogenase means that the aspartate kinase/homoserine dehydrogenase (encoded by thrA, EC:2.7.2.4/EC:1.1.1.3) is less sensitive to inhibition by threonine or a mixture of threonine and the threonine analog α -amino- β -hydroxypentanoic Acid (AHV) or AHV alone than the wild-type form, genes or alleles encoding these desensitized enzymes are also referred to as thrA^FBRAn allele. The prior art describes thrA encoding aspartokinase/homoserine dehydrogenase variants having amino acid substitutions compared to the wild-type protein^FBRAn allele. The coding region of the thrA wild-type gene of E.coli corresponding to accession number U00096.2 of the NCBI database (Bethesda, Md., USA) is shown in SEQ ID NO 3, and the polypeptide encoded by this gene is shown in SEQ ID NO 4.

Also disclosed is the nucleotide sequence of the thrA gene of Serratia marcescens, available at NCBI under accession number X60821. The coding region of the serratia marcescens thrA wild type gene is shown as SEQ ID NO. 5, and the polypeptide coded by the gene is shown as SEQ ID NO. 6.

The L-threonine-producing microorganisms of the Enterobacteriaceae family which have been used in the process of the invention preferably have a thrA allele which codes for an aspartokinase/homoserine dehydrogenase variant having the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 6 which comprises one or more amino acid substitutions selected from the group consisting of:

ThrA E253K (L-lysine replaces the L-glutamic acid at position 253 of the aspartokinase/homoserine dehydrogenase encoded by SEQ ID NO:4 or SEQ ID NO: 6; see Research Disclosure 505,537(2006)),

THRA G330D (substitution of L-aspartic acid for glycine at position 330 of the aspartokinase/homoserine dehydrogenase enzyme encoded by SEQ ID NO:4 or SEQ ID NO: 6; see Omori et al (Journal of Bacteriology175(3),785-794(1993)),

ThrA S345F (L-phenylalanine replaces the L-serine at position 345 of the aspartokinase/homoserine dehydrogenase encoded by SEQ ID NO:4 or SEQ ID NO: 6; see Lee et al, Journal of Bacteriology185(18):5442-5451(2003)),

ThrA S352, SEQ ID NO 4 or SEQ ID NO 6 encodes an aspartokinase/homoserine dehydrogenase in which the L-serine at position 352 is replaced by L-phenylalanine, L-tyrosine, L-asparagine, L-alanine, L-arginine, L-glutamine, L-glutamic acid, L-histidine, L-leucine, L-methionine, L-tryptophan or L-valine, preferably by L-phenylalanine; see, Omori et al (Journal of Bacteriology175 (3)), 785-,

ThrA A479T (L-threonine replaces the L-alanine at position 479 of the aspartokinase/homoserine dehydrogenase enzyme encoded by SEQ ID NO:4 or SEQ ID NO: 6; see Omori et al (Journal of Bacteriology175(3),785-794 (1993)).

4 according to SEQ ID NO, preferably thrA^FBRAllele, thrA E253K (replacement of the encoded aspartate kinase with L-lysine ™ in `L-glutamic acid at position 253 of homoserine dehydrogenase) or S345F (replacement of L-serine at position 345 of encoded aspartokinase/homoserine dehydrogenase by L-phenylalanine).

The above measures can be used to overexpress the thrA described herein encoding an aspartokinase/homoserine dehydrogenase^FBRAn allele.

Furthermore, for the production of L-Amino acids, in particular L-threonine, L-homoserine, L-histidine, L-lysine, L-tryptophan, L-valine, L-leucine and L-isoleucine, it may be advantageous, in addition to regulating the expression of genes coding for membrane proteins or proteins having the activity of Amino Acid transporters, such as Amino Acid exporters, e.g.rhtC, to eliminate undesired secondary reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: over production of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), academic Press, London, UK, 1982).

Thus, for the production of L-threonine, for example, the expression of one or more genes selected from the group consisting of:

the tdh gene encoding threonine dehydrogenase (Journal of Bacteriology 169:4716-4721 (1987)),

the mdh gene (Archives in Microbiology 149: 36-42(1987)) which codes for malate dehydrogenase (E.C.1.1.1.37),

the pckA gene which codes for phosphoenolpyruvate carboxykinase (WO 02/29080),

the poxB gene encoding pyruvate oxidase (WO 02/36797),

the dgsA gene which codes for the DgsA regulatory protein of the phosphotransferase system (WO 02/081721), also known as the mlc gene,

the fruR gene which codes for the fructose repressor (WO 02/081698), also known as the cra gene,

sigma of coding³⁸The rpoS gene of factor (WO 01/05939), also known as the katF gene, and

the aspA gene which codes for ammonium aspartate lyase (WO 03/008603).

These measures are carried out, where appropriate, in addition to or in appropriate combination with the specific measures for enhancing the gene in order to increase threonine production.

In this context, the term "attenuation" describes the reduction or elimination of the intracellular activity or concentration of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weaker promoter than in the parent strain or in the microorganism without recombination of the corresponding enzyme or protein, or a gene or allele which encodes the corresponding enzyme or protein with a lower activity, or inactivates the corresponding enzyme or protein or open reading frame or gene, and, where appropriate, combining these measures.

Typically, the attenuation reduces the activity or concentration of the corresponding protein to 0-75%, 0-50%, 0-25%, 0-10% or 0-5% of the activity or concentration of the wild-type protein or of the parent strain or of the microorganism in which the corresponding enzyme or protein is not recombinant. A parent strain or non-recombinant microorganism is understood as meaning a microorganism on which the attenuation or elimination measures according to the invention are carried out.

To achieve attenuation, for example, the expression of genes or open reading frames or the catalytic properties of enzyme proteins can be reduced or eliminated. Both measures may be combined, where appropriate.

Gene expression can be reduced by culturing in a suitable manner or by genetically altering (mutating) the signal structure of gene expression or by antisense RNA technology. Signal structures for gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. Information on this can be found by the person skilled in the art, for example, in Jensen and Hammer (Biotechnology and Bioengineering 58:191-195(1998)), Carrier and Keasling (Biotechnology progress 15:58-64(1999)), Franch and Gerdes (Current Opinion in Biotechnology 3:159-164(2000)), Kawano et al (Nucleic Acids Research 33(19),6268-6276(2005)) and also well-known textbooks of genetics and molecular biology such as Knippers ("molecular genetics ]", 6th edition, Georg Thie Verlag, Stuttgart, Germany,1995) or Wincar (winter uns [ Genes ], "clone, VClaim, 1990).