WO2005075627A1 - Process for the preparation of l-amino acids using strains of the family enterobacteriaceae - Google Patents

Process for the preparation of l-amino acids using strains of the family enterobacteriaceae Download PDF

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WO2005075627A1
WO2005075627A1 PCT/EP2005/000767 EP2005000767W WO2005075627A1 WO 2005075627 A1 WO2005075627 A1 WO 2005075627A1 EP 2005000767 W EP2005000767 W EP 2005000767W WO 2005075627 A1 WO2005075627 A1 WO 2005075627A1
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gene coding
gene
coding
orf
microorganisms
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PCT/EP2005/000767
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French (fr)
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Mechthild Rieping
Nicole Dusch
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Degussa Ag
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Priority to AT05701199T priority Critical patent/ATE433483T1/en
Priority to EP05701199A priority patent/EP1711593B1/en
Priority to CN2005800040826A priority patent/CN1918283B/en
Priority to DE602005014846T priority patent/DE602005014846D1/en
Priority to DK05701199T priority patent/DK1711593T3/en
Publication of WO2005075627A1 publication Critical patent/WO2005075627A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to a process for the preparation of L-amino acids, especially L-threonine, using recombinant microorganisms of the family Enterobacteriaceae in which the open reading frame (ORF) denoted by yibD is enhanced or, in particular, overexpressed, and to said microorganisms .
  • ORF open reading frame
  • L-amino acids especially L-threonine
  • L-threonine are used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.
  • the productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains that are resistant to antimetabolites, e.g. the threonine analogue ⁇ -amino- ⁇ - hydroxyvaleric acid (AHV) , or auxotrophic for metabolites of regulatory significance, and produce L-amino acids, e.g. L-threonine.
  • antimetabolites e.g. the threonine analogue ⁇ -amino- ⁇ - hydroxyvaleric acid (AHV)
  • auxotrophic for metabolites of regulatory significance e.g. L-threonine.
  • the object which the inventors set themselves was to provide novel procedures for improving the preparation of L-amino acids, especially L-threonine, by fermentation.
  • the invention provides recombinant microorganisms of the family Enterobacteriaceae which contain an enhanced or overexpressed yibD open reading frame coding for a polypeptide called a putative glycosyl transferase, or nucleotide sequences coding for its gene product, and which produce L-amino acids, especially L-threonine, in an improved manner.
  • microorganisms that are not recombinant for the yibD ORF and contain no enhanced yibD ORF serve in each case as the starting point for the comparison.
  • These microorganisms include especially those of the family Enterobacteriaceae in which a polynucleotide is enhanced that codes for a polypeptide whose amino acid sequence is at least 90%, especially at least 95%, preferably at least 98% or at least 99%, particularly preferably 99.7% and very particularly preferably 100% identical to an amino acid sequence selected from the group comprising SEQ ID No. 4 and SEQ ID No . 6.
  • Amino acid sequences identical to those of SEQ ID No. 4 or SEQ ID No. 6 are preferred.
  • Said microorganisms contain enhanced or overexpressed polynucleotides selected from the group:
  • the invention also provides a fermentation process for the preparation of L-amino acids, especially L-threonine, using recombinant microorganisms of the family Enterobacteriaceae which, in particular, already produce L-amino acids and in which at least the open reading frame (ORF) called yibD, or nucleotide sequences coding for its gene product, is (are) enhanced.
  • ORF open reading frame
  • L-amino acids or “amino acids” mentioned hereafter is understood as meaning one or more amino acids, including their salts, selected from the group comprising 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-homoserine. L-threonine is particularly preferred.
  • the term "enhancement” describes the increase, in a microorganism, of the intracellular activity or concentration of one or more enzymes or proteins encoded by the appropriate DNA, for example by increasing the copy number of the gene(s) or ORF(s) by at least one (1) copy, functionally linking a strong promoter to the gene, or using a gene, allele or ORF coding for an appropriate enzyme/protein with a high activity, and optionally combining these measures.
  • Open reading frame is understood as meaning a segment of a nucleotide sequence that codes or can code for a protein/polypeptide or ribonucleic acid to which no function can be assigned according to the state of the art. After a function has been assigned to the segment of nucleotide sequence in question, it is generally referred to as a gene. Alleles are generally understood as meaning alternative forms of a given gene. The forms are distinguished by differences in the nucleotide sequence.
  • Gene product is generally understood as meaning the protein encoded by a nucleotide sequence, i.e. an ORF, a gene or an allele, or the encoded ribonucleic acid.
  • the activity or concentration of the appropriate protein is generally increased at least by 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, and at most by up to 1000% or 2000%, based on the activity or concentration of the wild-type protein or that of the protein in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein.
  • Non- recombinant microorganism or parent strain is understood as meaning the microorganism on which the measures according to the invention are performed.
  • the invention provides a process for the preparation of L-amino acids by the fermentation of recombinant microorganisms of the family Enterobacteriaceae, wherein
  • the microorganisms producing the desired L-amino acid in which the yibD open reading frame, or nucleotide sequences coding for the gene product, or alleles, is (are) enhanced or, in particular, overexpressed, are cultivated 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, constituents of the fermentation broth, and/or all or part (> 0 to 100%) of the biomass, optionally remaining in the isolated product or being completely removed.
  • the microorganisms can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, optionally starch or optionally cellulose, or from glycerol and ethanol .
  • Said microorganisms are representatives of the family Enterobacteriaceae selected from the genera Escherichia, Erwinia, Providencia and Serratia.
  • the genera Escherichia and Serratia are preferred.
  • the species Escherichia coli and Serratia marcescens may be mentioned in particular among the genera Escherichia and Serratia respectively.
  • Recombinant microorganisms are generally produced by transformation, transduction or conjugation, or a combination of these methods, with a vector containing the desired ORF, the desired gene, an allele of this ORF or gene or parts thereof, and/or a promoter that enhances the expression of the ORF or gene.
  • This promoter can be the promoter originating by enhancing mutation from the inherent regulatory sequence located upstream from the gene or ORF, or an efficient promoter has been fused with the gene or ORF.
  • suitable strains particularly L-threonine- producing strains, of the genus Escherichia, and especially of the species Escherichia coli, are:
  • L-threonine-producing strains of the genus Serratia and especially of the species Serratia marcescens, are:
  • L-threonine-producing strains of the family Enterobacteriaceae preferably possess, inter alia, one or more genetic or phenotypic characteristics selected from the group comprising resistance to ⁇ -amino- ⁇ -hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to ⁇ -methylserine, resistance to diaminosuccinic acid, resistance to ⁇ -aminobutyric acid, resistance to borrelidine, resistance to cyclopentanecarboxylic acid, resistance to rifampicin, resistance to valine analogues such as valine hydroxamate, resistance to purine analogues such as 6-dimethylaminopurine, need for L-methionine, optionally partial and compensatable need for L-isoleucine, need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to threonine
  • 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, defective threonine dehydrogenase, optionally capability for sucrose utilization, enhancement of the threonine operon, enhancement of homoserine dehydrogenase I-aspartate kinase I, preferably of the feedback-resistant form, enhancement of homoserine kinase, enhancement of threonine synthase, enhancement of aspartate kinase, optionally of the feedback-resistant form, enhancement of aspartate semialdehyde dehydrogenase, enhancement of phosphoenolpyruvate carboxylase, optionally of the feedback-resistant form, enhancement of phosphoenolpyruvate synthase, enhancement of transhydrogenase,
  • L-amino acids especially L-threonine
  • ORF open reading frame
  • the nucleotide sequences of the genes or open reading frames (ORFs) of Escherichia coli belong to the state of the art and can be taken from the genome sequence of Escherichia coli published by Blattner et al . (Science 277, 1453-1462 (1997)). It is known that the N-terminal amino acid methionine can be split off by host-specific enzymes ( ethionine aminopeptidase) .
  • the nucleotide sequence for the yibD ORF is likewise known from Salmonella typhimurium, which also belongs to the family Enterobacteriaceae.
  • the yibD ORF of Escherichia coli K12 is described inter alia by the following data:
  • the yibD open reading frame codes for a 40.5 kDa protein; the isoelectric point is 9.4; the yibD ORF is located on a chromosome e.g. in the case of Escherichia coli K12 MG1655 in the intergenic region of the yibQ open reading frame coding for a hypothetical protein, and the tdh gene coding for threonine dehydrogenase Reference: Blattner et al., Science 277(5331), 1453- 1474 (1997) Accession no. : AE000439 Alternative gene name: b3615
  • the nucleic acid sequences can be taken from the data banks of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) , the nucleotide sequence data bank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany, or Cambridge, UK) or the DNA data bank of Japan (DDBJ, Mishima, Japan) .
  • NCBI National Center for Biotechnology Information
  • EMBL European Molecular Biologies Laboratories
  • EMBL European Molecular Biologies Laboratories
  • DDBJ Mishima, Japan
  • the known sequence of the yibD ORF of Escherichia coli is shown as SEQ ID No . 3 and the known sequence of the yibD ORF of Salmonella typhimurium is shown as SEQ ID No. 5.
  • the proteins encoded by these reading frames are shown as SEQ ID No. 4 and SEQ ID No. 6.
  • the open reading frames described in the cited literature references can be used according to the invention. It is also possible to use alleles of the genes, or open reading frames, which result from the degeneracy of the genetic code or from neutral sense mutations. The use of endogenous genes or endogenous open reading frames is preferred.
  • endogenous genes or “endogenous nucleotide sequences” is understood as meaning the genes, open reading frames or alleles, or nucleotide sequences, present in the population of a species.
  • Alleles of the yibD ORF that contain neutral sense mutations include, inter alia, those which result in at most 40, at most 30 or at most 20, preferably at most 10 or at most 5, very particularly preferably at most 3 or at most 2 or at least one conservative amino acid exchange in the protein encoded by them.
  • aromatic amino acids one refers to conservative exchanges when phenylalanine, tryptophan and tyrosine are exchanged for one another.
  • hydrophobic amino acids one refers to conservative exchanges when leucine, isoleucine and valine are exchanged for one another.
  • polar amino acids one refers to conservative exchanges when glutamine and asparagine are exchanged for one another.
  • basic amino acids one refers to conservative exchanges when arginine, lysine and histidine are exchanged for one another.
  • acidic amino acids one refers to conservative exchanges when aspartic acid and glutamic acid are exchanged for one another.
  • nucleotide sequences that code for variants of said proteins which additionally comprise a lengthening or shortening by at least one (1) amino acid at the N or C terminus . This lengthening or shortening amounts to no more than 50, 40, 30, 20, 10, 5, 3 or 2 amino acids or amino acid residues.
  • Suitable alleles also include those coding for proteins in which at least one (1) amino acid is inserted (insertion) or deleted (deletion).
  • the .maximum number of such changes, called indels, can affect 2, 3, 5, 10 or 20 amino acids, but under no circumstances more than 30 amino acids.
  • /Suitable alleles also include those obtainable by hybridization, especially under stringent conditions, using SEQ ID No. 3 or SEQ ID No . 5 or portions thereof, especially the coding regions or the sequences complementary thereto.
  • SEQ ID No. 3 or SEQ ID No . 5 or portions thereof especially the coding regions or the sequences complementary thereto.
  • Those skilled in the art will find instructions on the identification of DNA sequences by means of hybridization in, inter alia, the handbook "The DIG System User's Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al . (International Journal of Systematic Bacteriology 41, 255-260 (1991)).
  • the hybridization takes place under stringent conditions, i.e. 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, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is generally carried out at relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .
  • the hybridization reaction can be carried out using e.g. a buffer corresponding to 5x SSC buffer at a temperature of approx. 50°C - 68°C. It is also possible here to hybridize probes with polynucleotides that are less than 80% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved e.g. by lowering the salt concentration to 2x SSC and optionally 0.5x SSC thereafter (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) , the temperature being set to approx. 50°C - 68°c approx. 52°C - 68°C, approx.
  • polynucleotide fragments that are e.g. at least 80%, 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% identical to the sequence of the probe used or to the nucleotide sequences shown in SEQ ID No. 3 or SEQ ID No. 5. Further instructions on hybridization are commercially available in kit form (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, catalogue no. 1603558).
  • Enhancement can be achieved for example by increasing the expression of the genes, open reading frames or alleles or enhancing the catalytic properties of the protein. Both measures may optionally be combined.
  • Overexpression can be achieved for example by increasing the copy number of the appropriate genes or open reading frames or mutating the promoter and regulatory region or the ribosome binding site located upstream, from the structural gene.
  • Expression cassettes incorporated upstream from the structural gene work in the same way.
  • Inducible promoters additionally make it possible to increase expression in the course of L-threonine production by fermentation; the use of promoters for gene expression which allows another transitory gene expression can also be advantageous. Measures for prolonging the life of the mRNA also improve expression.
  • the enzyme activity is also enhanced by preventing degradation of the enzyme protein.
  • the ORFs, genes or gene constructs can either be located in plasmids of variable copy number or be integrated and amplified in the chromosome. Alternatively, it is also possible to achieve overexpression of the genes in question by changing the composition of the media and the culture technique.
  • 5,538,873 can be used as vectors.
  • Phages for example phage mu, as described in EP 0332448, or phage lambda ( ⁇ ) , can also be used as vectors .
  • the copy number can also be increased by incorporating another copy into another site of the chromosome, for example into the att site of phage ⁇ (Yu and Court, Gene 223, 77-81 (1998)).
  • US 5,939,307 describes that expression could be increased by incorporating expression cassettes or promoters such as the tac promoter, trp promoter, lpp promoter or P promoter and P R promoter of phage ⁇ , for example upstream from the chromosomal threonine operon.
  • the promoters of phage T7, the gearbox promoters or the nar promoter can likewise be used.
  • Such expression cassettes or promoters can also be used to overexpress plasmid-bound genes , as described in EP 0 593 792.
  • the expression of plasmid-bound genes can in turn be controlled by using the lacI Q allele (Glascock and Weickert, Gene 223, 221-231 (1998)). It is further possible to increase the activity of promoters by modification of their sequence through one or more nucleotide exchanges, by insertion (s) and/or by deletion (s) .
  • Another transitory gene expression can be achieved by using the growth phase-dependent fis promoter, for example as described in Walker et al.
  • Plasmid vectors replicatable in Enterobacteriaceae e.g. cloning vectors derived from pACYC184 (Bartolome et al . , Gene 102, 75-78 (1991)), pTrc99A (Amann et al . , Gene 69, 301-315 (1988)) or pSClOl derivatives (Vocke and Bastia, Proceedings of the National Academy of Sciences USA 80(21), 6557-6561 (1983)), can be used.
  • a strain transformed with a plasmid vector said plasmid vector carrying at least one nucleotide sequence coding for the yibD ORF or its gene product, or allele.
  • transformation is understood as meaning the uptake of an isolated nucleic acid by a host (microorganism) .
  • mutations which affect the expression of the appropriate genes or open reading frames can be transferred to different strains by sequence exchange (Hamilton et al . , Journal of Bacteriology 171, 4617-4622 (1989)), conjugation or transduction.
  • L-amino acids especially L-threonine
  • strains of the family Enterobacteriaceae it can be advantageous not only to enhance the yibD open reading frame, but also to enhance one or more enzymes of the known threonine biosynthetic pathway, or enzymes of the anaplerotic metabolism, or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate, or glycolytic enzymes, or PTS enzymes, or enzymes of sulfur metabolism.
  • endogenous genes is generally preferred.
  • sucB gene of the sucABCD operon coding for the dihydrolipoyl transsuccinase E2 subunit of 2-ketoglutarate dehydrogenase (WO 03/008614),
  • L-amino acids especially L-threonine
  • the term "attenuation” describes the decrease or switching-off of the intracellular activity or concentration, in a microorganism, of one or more enzymes/ proteins encoded by the appropriate DNA, for example by using a weaker promoter than in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein, or a gene or allele which codes for an appropriate enzyme/protein with a low activity, or inactivating the appropriate enzyme/protein, the open reading frame or the gene, and optionally combining these measures .
  • the attenuation measures generally reduce the activity or concentration of the appropriate protein to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein.
  • Non-recombinant microorganism or parent strain is understood as meaning the microorganism on which the measures according to the invention are performed.
  • Attenuation can be achieved for example by reducing or switching off the expression of the genes or open reading frames or the catalytic properties of the enzyme proteins . Both measures may optionally be combined.
  • Gene expression can be reduced by an appropriate culture technique, by genetic modification (mutation) of the signal structures of gene expression or else by an antisense RNA technique.
  • Signal structures of gene expression are e.g. represspr genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators .
  • missense mutations results in an exchange of a given amino acid in a protein for another amino acid, said amino acid exchange being in particular non-conservative. This impairs the functioning ability or activity of the protein and reduces it to a value. of 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5%.
  • a nonsense mutation results in a stop codon in the coding region of the gene and hence in a premature termination of translation.
  • Insertions or deletions of at least one base pair in a gene result in frame shift mutations, the outcome of which is that false amino acids are incorporated or translation is prematurely terminated. If a stop codon is produced in the coding region as a consequence of the mutation, this also results in a premature termination of translation. Deletions of at least one (1) or several codons typically also result in a complete loss of enzyme activity.
  • Suitable mutations in the genes can be incorporated into suitable strains by gene or allele exchange.
  • a common method is that of gene exchange with the aid of a conditionally replicating pSClOl derivative pMAK705, as described by Hamilton et al. (Journal of Bacteriology 171, 4617-4622 (1989)).
  • Other methods described in the state of the art for example that of Martinez-Morales et al . (Journal of Bacteriology 181, 7143-7148 (1999)) or that of Boyd et al. (Journal of Bacteriology 182, 842-847 (2000)), can likewise be used.
  • L-amino acids especially L-threonine
  • microorganisms prepared according to the invention can be cultivated by the batch process, the fed batch process, the repeated fed batch process or a continuous process (DE 102004028859.3 or US 5,763,230).
  • a summary of known cultivation methods is provided in the textbook by Chmiel (Bioreatechnik 1. Einf ⁇ hrung in die Biovons- technik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).
  • the culture medium to be used must appropriately meet the demands of the particular strains . Descriptions of culture media for various microorganisms are contained in "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington DC, USA, 1981) .
  • Carbon sources which can be used are sugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmitic acid, stearic acid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids, e.g. acetic acid. These substances can be used individually or as a mixture.
  • Nitrogen sources which can be used are organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen sources can be used individually or as a mixture.
  • Phosphorus sources which can be used are phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium salts .
  • the culture medium must also contain metal salts, e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
  • metal salts e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
  • essential growth-promoting substances 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 feed materials can be added to the culture medium all at once or fed in appropriately during cultivation.
  • the fermentation is generally carried out at a pH of 5.5 to 9.0, especially of 6.0 to 8.0.
  • the pH of the culture is controlled by the appropriate use of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid.
  • Foaming can be controlled using antifoams such as fatty acid polyglycol esters .
  • the stability of plasmids can be maintained by adding suitable selectively acting substances, e.g. antibiotics, to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gaseous mixtures, e.g. air, into the culture.
  • the temperature of the culture is normally 25°C to 45°C and preferably 30°C to 40°C.
  • the culture is continued until the formation of L-amino acids or L-threonine has reached a maximum. This objective is normally achieved within 10 hours to 160 hours.
  • L-amino acids can be analysed by means of anion exchange chromatography followed by ninhydrin derivatization, as described by Spackman et al. (Analytical Chemistry 30, 1190-1206 (1958)), or by reversed phase HPLC, as described by Lindroth et al . (Analytical Chemistry 51, 1167-1174 (1979) ) .
  • the process according to the invention is used to prepare L-amino acids, for example L-threonine, L-isoleucine, L-valine, L-methionine, L-homoserine, L-tryptophan and L- lysine, especially L-threonine, by fermentation.
  • L-amino acids for example L-threonine, L-isoleucine, L-valine, L-methionine, L-homoserine, L-tryptophan and L- lysine, especially L-threonine, by fermentation.
  • M9 and complete medium (LB) used for Escherichia coli are described by J.H. Miller (A Short Course in Bacterial Genetics (1992), Cold Spring Harbor Laboratory Press) .
  • the isolation of plasmid DNA from Escherichia coli and all the techniques for restriction, ligation, Klenow treatment and alkaline phosphatase treatment are carried out according to Sambrook et al . (Molecular Cloning - A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press) . Unless described otherwise, the transformation of Escherichia coli is carried out according to Chung et al . (Proceedings of the National Academy of Sciences of the United States of America 86, 2172-2175 (1989)).
  • the incubation temperature in the preparation of strains and transformants is 37°C.
  • the yibD gene from E. coli K12 is amplified using the poly erase chain reaction (PCR) and synthetic oligonucleotides.
  • PCR poly erase chain reaction
  • the nucleotide sequence of the yibD ORF in E. coli K12 MG1655 (Accession Number AE000439, Blattner et al. (Science 277, 1453-1474 (1997))) is used as the starting material to synthesize PCR primers (MWG Biotech, Ebersberg, Germany) .
  • the sequences of the primers are modified to provide recognition sites for restriction enzymes.
  • the recognition sequence for Sad is chosen for the yibD-exl primer and the recognition sequence for Hindlll is chosen for the yibD-ex2 primer, said sequences being underlined in the nucleotide sequences shown below:
  • the chromosomal E. coli K12 MG1655 DNA used for the PCR is isolated with "Qiagen Genomic-tips 100/G" (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions.
  • An approx. 1114 bp DNA fragment can be amplified with the specific primers under standard PCR conditions (Innis et al. (1990), PCR Protocols.' A Guide to Methods and Applications, Academic Press) using Vent DNA polymerase (New England Biolabs GmbH, Frankfurt, Germany) (SEQ ID No . 3 ) .
  • the amplified yibD fragment is ligated with vector pCR-Blunt II-TOPO (Zero TOPO TA Cloning Kit, Invitrogen, Groningen, The Netherlands) in accordance with the manufacturer's instructions and transformed into the E. coli strain TOP10. Plasmid-carrying cells are selected on
  • pCRBluntyibD pCRBluntyibD
  • Vector pCRBluntyibD is then cleaved with the enzymes Hindlll and Sad and, after separation in 0.8% agarose gel, the yibD fragment is isolated from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilden, Germany) and ligated with vector pTrc99A (Pharmacia Biotech, Uppsala, Sweden) which has been digested with the enzymes Hindlll and Sad.
  • the E. coli strain XLlBlue MRF' (Stratagene, La Jolla, USA) is transformed with the ligation mixture and plasmid-carrying cells are selected on LB agar supplemented with 50 ⁇ g/ml of ampicillin.
  • the success of the cloning can be demonstrated, after isolation of the plasmid DNA, by control cleavage with the enzyme Pvul .
  • the plasmid is called pTrc99AyibD ( Figure 1) .
  • the L-threonine-producing E. coli strain MG442 is described in patent US-A-4, 278, 765 and is deposited in the Russian National Collection of Industrial Microorganisms (VKPM,
  • the strain MG442 is transformed with expression plasmid pTrc99AyibD, described in Example 1, and with vector pTrc99A and plasmid-carrying cells are selected on LB agar supplemented with 50 ⁇ g/ml of ampicillin. This procedure yields the strains MG442/pTrc99AyibD and MG442/pTrc99A.
  • 250 ⁇ l of each of these precultures are transferred to 10 ml of production medium (25 g/1 of (NH 4 ) 2 S0 4 , 2 g/1 of KH 2 P0 , 1 g/1 of MgS0 4 -7H 2 0, 0.03 g/1 of FeS0 4 -7H 2 0, 0.018 g/1 of MnS0 4 -lH 2 0, 30 g/1 of CaC0 3 , 20 g/1 of glucose, 50 mg/1 of ampicillin) and incubated for 48 hours at 37°C.
  • the formation of L-threonine by the original strain MG442 is verified in the same way except that no ampicillin is added to the medium.
  • the optical density (OD) of the culture suspension is determined using an LP2W photometer from Dr. Lange (Dusseldorf, Germany) at a measurement wavelength of 660 nm.
  • the concentration of L-threonine formed is then determined in the sterile-filtered culture supernatant using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by means of ion exchange chromatography and postcolumn reaction with ninhydrin detection.
  • Table 1 shows the result of the experiment.
  • FIG. 1 Map of plasmid pTrc99AyibD containing the yibD ORF

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Abstract

The invention relates to a process for the preparation of L-amino acids by the fermentation of recombinant microorganisms of the family Enterobacteriaceae, wherein a) the microorganisms producing the desired L-amino acid in which the yibD ORF, or nucleotide sequences coding for the gene product, or alleles, is (are) enhanced or, in particular, overexpressed are cultivated 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, consitutents of the fermentation broth, and/or all or part(≥ 0 to 100%) of the biomass, optionally remaining in the isolated product or being completely removed.

Description

Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae
Field of the invention
The present invention relates to a process for the preparation of L-amino acids, especially L-threonine, using recombinant microorganisms of the family Enterobacteriaceae in which the open reading frame (ORF) denoted by yibD is enhanced or, in particular, overexpressed, and to said microorganisms .
State of the art
L-amino acids, especially L-threonine, are used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.
It is known to prepare L-amino acids by the fermentation of strains of Enterobacteriaceae, especially Escherichia coli (E. coli) and Serratia marcescens. Because of their great importance, attempts are constantly being made to improve the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains that are resistant to antimetabolites, e.g. the threonine analogue α-amino-β- hydroxyvaleric acid (AHV) , or auxotrophic for metabolites of regulatory significance, and produce L-amino acids, e.g. L-threonine.
Methods of recombinant DNA technology have also been used for some years to improve L-amino acid-producing strains of the family Enterobacteriaceae by amplifying individual amino acid biosynthesis genes and studying the effect on production. A survey of the cellular 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 DC, USA (1996) .
Object of the invention
The object which the inventors set themselves was to provide novel procedures for improving the preparation of L-amino acids, especially L-threonine, by fermentation.
Summary of the invention
The invention provides recombinant microorganisms of the family Enterobacteriaceae which contain an enhanced or overexpressed yibD open reading frame coding for a polypeptide called a putative glycosyl transferase, or nucleotide sequences coding for its gene product, and which produce L-amino acids, especially L-threonine, in an improved manner.
The microorganisms that are not recombinant for the yibD ORF and contain no enhanced yibD ORF serve in each case as the starting point for the comparison. These microorganisms include especially those of the family Enterobacteriaceae in which a polynucleotide is enhanced that codes for a polypeptide whose amino acid sequence is at least 90%, especially at least 95%, preferably at least 98% or at least 99%, particularly preferably 99.7% and very particularly preferably 100% identical to an amino acid sequence selected from the group comprising SEQ ID No. 4 and SEQ ID No . 6.
Amino acid sequences identical to those of SEQ ID No. 4 or SEQ ID No. 6 are preferred.
Said microorganisms contain enhanced or overexpressed polynucleotides selected from the group:
a) polynucleotide with the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 5;
b) polynucleotide with a nucleotide sequence corresponding to SEQ ID No. 3 or SEQ ID No. 5 within the degeneracy of the genetic code;
c) polynucleotide with a sequence that hybridizes under stringent conditions with the sequence complementary to the sequence SEQ ID No. 3 or SEQ ID No. 5;
d) polynucleotide with a sequence SEQ ID No. 3 or SEQ ID No. 5 containing neutral sense mutants,
the polynucleotides coding for a putative glycosyl transferase. The invention also provides a fermentation process for the preparation of L-amino acids, especially L-threonine, using recombinant microorganisms of the family Enterobacteriaceae which, in particular, already produce L-amino acids and in which at least the open reading frame (ORF) called yibD, or nucleotide sequences coding for its gene product, is (are) enhanced.
It is preferable to use the microorganisms described.
The term "L-amino acids" or "amino acids" mentioned hereafter is understood as meaning one or more amino acids, including their salts, selected from the group comprising 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-homoserine. L-threonine is particularly preferred.
In this context the term "enhancement" describes the increase, in a microorganism, of the intracellular activity or concentration of one or more enzymes or proteins encoded by the appropriate DNA, for example by increasing the copy number of the gene(s) or ORF(s) by at least one (1) copy, functionally linking a strong promoter to the gene, or using a gene, allele or ORF coding for an appropriate enzyme/protein with a high activity, and optionally combining these measures.
Open reading frame (ORF) is understood as meaning a segment of a nucleotide sequence that codes or can code for a protein/polypeptide or ribonucleic acid to which no function can be assigned according to the state of the art. After a function has been assigned to the segment of nucleotide sequence in question, it is generally referred to as a gene. Alleles are generally understood as meaning alternative forms of a given gene. The forms are distinguished by differences in the nucleotide sequence.
Gene product is generally understood as meaning the protein encoded by a nucleotide sequence, i.e. an ORF, a gene or an allele, or the encoded ribonucleic acid.
Through the measures of enhancement, especially overexpression, the activity or concentration of the appropriate protein is generally increased at least by 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, and at most by up to 1000% or 2000%, based on the activity or concentration of the wild-type protein or that of the protein in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein. Non- recombinant microorganism or parent strain is understood as meaning the microorganism on which the measures according to the invention are performed.
The invention provides a process for the preparation of L-amino acids by the fermentation of recombinant microorganisms of the family Enterobacteriaceae, wherein
a) the microorganisms producing the desired L-amino acid, in which the yibD open reading frame, or nucleotide sequences coding for the gene product, or alleles, is (are) enhanced or, in particular, overexpressed, are cultivated 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, constituents of the fermentation broth, and/or all or part (> 0 to 100%) of the biomass, optionally remaining in the isolated product or being completely removed.
The microorganisms, especially recombinant microorganisms, with an enhanced or overexpressed open reading frame (ORF) called yibD, also provided by the present invention, can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, optionally starch or optionally cellulose, or from glycerol and ethanol . Said microorganisms are representatives of the family Enterobacteriaceae selected from the genera Escherichia, Erwinia, Providencia and Serratia. The genera Escherichia and Serratia are preferred. The species Escherichia coli and Serratia marcescens may be mentioned in particular among the genera Escherichia and Serratia respectively.
Recombinant microorganisms are generally produced by transformation, transduction or conjugation, or a combination of these methods, with a vector containing the desired ORF, the desired gene, an allele of this ORF or gene or parts thereof, and/or a promoter that enhances the expression of the ORF or gene. This promoter can be the promoter originating by enhancing mutation from the inherent regulatory sequence located upstream from the gene or ORF, or an efficient promoter has been fused with the gene or ORF.
Examples of suitable strains, particularly L-threonine- producing strains, of the genus Escherichia, and especially of the species Escherichia coli, are:
- Escherichia coli H4581 (EP 0 301 572) - Escherichia coli KY10935 (Bioscience, Biotechnology and Biochemistry 61(11), 1877-1882 (1997))
- Escherichia coli VNIIgenetika MG442 (US-A-4, 278, 765)
- Escherichia coli VNIIgenetika Ml (US-A-4, 321, 325)
- Escherichia coli VNIIgenetika 472T23 (US-A-5, 631, 157)
- Escherichia coli BKIIM B-3996 (US-A-5, 175, 107)
- Escherichia coli kat 13 (WO 98/04715)
- Escherichia coli KCCM-10132 (WO 00/09660)
Examples of suitable L-threonine-producing strains of the genus Serratia, and especially of the species Serratia marcescens, are:
- Serratia marcescens HNr21 (Applied and Environmental Microbiology 38(6) , 1045-1051 (1979)) - Serratia marcescens TLrl56 (Gene 57(2-3), 151-158 (1987))
- Serratia marcescens T-2000 (Applied Biochemistry and Biotechnology 37(3) , 255-265 (1992))
L-threonine-producing strains of the family Enterobacteriaceae preferably possess, inter alia, one or more genetic or phenotypic characteristics selected from the group comprising resistance to α-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to α-methylserine, resistance to diaminosuccinic acid, resistance to α-aminobutyric acid, resistance to borrelidine, resistance to cyclopentanecarboxylic acid, resistance to rifampicin, resistance to valine analogues such as valine hydroxamate, resistance to purine analogues such as 6-dimethylaminopurine, need for L-methionine, optionally partial and compensatable need for L-isoleucine, need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to threonine raffinate, resistance to L-homoserine, 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, defective threonine dehydrogenase, optionally capability for sucrose utilization, enhancement of the threonine operon, enhancement of homoserine dehydrogenase I-aspartate kinase I, preferably of the feedback-resistant form, enhancement of homoserine kinase, enhancement of threonine synthase, enhancement of aspartate kinase, optionally of the feedback-resistant form, enhancement of aspartate semialdehyde dehydrogenase, enhancement of phosphoenolpyruvate carboxylase, optionally of the feedback-resistant form, enhancement of phosphoenolpyruvate synthase, enhancement of transhydrogenase, enhancement of the RhtB gene product, enhancement of the RhtC gene product, enhancement of the YfiK gene product,' enhancement of a pyruvate carboxylase and attenuation of acetic acid formation.
It has been found that the production of L-amino acids, especially L-threonine, by microorganisms of the family Enterobacteriaceae is improved after overexpression of the yibD gene or open reading frame (ORF), or its alleles. The nucleotide sequences of the genes or open reading frames (ORFs) of Escherichia coli belong to the state of the art and can be taken from the genome sequence of Escherichia coli published by Blattner et al . (Science 277, 1453-1462 (1997)). It is known that the N-terminal amino acid methionine can be split off by host-specific enzymes ( ethionine aminopeptidase) .
The nucleotide sequence for the yibD ORF is likewise known from Salmonella typhimurium, which also belongs to the family Enterobacteriaceae.
The yibD ORF of Escherichia coli K12 is described inter alia by the following data:
Name: open reading frame
Function: putative glycosyl transferase Description: the yibD open reading frame codes for a 40.5 kDa protein; the isoelectric point is 9.4; the yibD ORF is located on a chromosome e.g. in the case of Escherichia coli K12 MG1655 in the intergenic region of the yibQ open reading frame coding for a hypothetical protein, and the tdh gene coding for threonine dehydrogenase Reference: Blattner et al., Science 277(5331), 1453- 1474 (1997) Accession no. : AE000439 Alternative gene name: b3615
The nucleic acid sequences can be taken from the data banks of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) , the nucleotide sequence data bank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany, or Cambridge, UK) or the DNA data bank of Japan (DDBJ, Mishima, Japan) .
For greater clarity, the known sequence of the yibD ORF of Escherichia coli is shown as SEQ ID No . 3 and the known sequence of the yibD ORF of Salmonella typhimurium is shown as SEQ ID No. 5. The proteins encoded by these reading frames are shown as SEQ ID No. 4 and SEQ ID No. 6.
The open reading frames described in the cited literature references can be used according to the invention. It is also possible to use alleles of the genes, or open reading frames, which result from the degeneracy of the genetic code or from neutral sense mutations. The use of endogenous genes or endogenous open reading frames is preferred.
The term "endogenous genes" or "endogenous nucleotide sequences" is understood as meaning the genes, open reading frames or alleles, or nucleotide sequences, present in the population of a species.
Alleles of the yibD ORF that contain neutral sense mutations include, inter alia, those which result in at most 40, at most 30 or at most 20, preferably at most 10 or at most 5, very particularly preferably at most 3 or at most 2 or at least one conservative amino acid exchange in the protein encoded by them.
In the case of aromatic amino acids , one refers to conservative exchanges when phenylalanine, tryptophan and tyrosine are exchanged for one another. In the case of hydrophobic amino acids, one refers to conservative exchanges when leucine, isoleucine and valine are exchanged for one another. In the case of polar amino acids, one refers to conservative exchanges when glutamine and asparagine are exchanged for one another. In the case of basic amino acids, one refers to conservative exchanges when arginine, lysine and histidine are exchanged for one another. In the case of acidic amino acids, one refers to conservative exchanges when aspartic acid and glutamic acid are exchanged for one another. In the case of amino acids containing hydroxyl groups, one refers to conservative exchanges when serine and threonine are exchanged for one another. Likewise, it is also possible to use nucleotide sequences that code for variants of said proteins which additionally comprise a lengthening or shortening by at least one (1) amino acid at the N or C terminus . This lengthening or shortening amounts to no more than 50, 40, 30, 20, 10, 5, 3 or 2 amino acids or amino acid residues.
Suitable alleles also include those coding for proteins in which at least one (1) amino acid is inserted (insertion) or deleted (deletion). The .maximum number of such changes, called indels, can affect 2, 3, 5, 10 or 20 amino acids, but under no circumstances more than 30 amino acids.
/Suitable alleles also include those obtainable by hybridization, especially under stringent conditions, using SEQ ID No. 3 or SEQ ID No . 5 or portions thereof, especially the coding regions or the sequences complementary thereto. Those skilled in the art will find instructions on the identification of DNA sequences by means of hybridization in, inter alia, the handbook "The DIG System User's Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al . (International Journal of Systematic Bacteriology 41, 255-260 (1991)). The hybridization takes place under stringent conditions, i.e. 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, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is generally carried out at relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .
The hybridization reaction can be carried out using e.g. a buffer corresponding to 5x SSC buffer at a temperature of approx. 50°C - 68°C. It is also possible here to hybridize probes with polynucleotides that are less than 80% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved e.g. by lowering the salt concentration to 2x SSC and optionally 0.5x SSC thereafter (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) , the temperature being set 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 or approx. 66°C - 68°C. Temperature ranges of approx. 64°C - 68°C or approx. 66°C - 68°C are preferred. It is optionally possible to lower the salt concentration to a value corresponding to 0.2x SSC or 0. Ix SSC. By gradually increasing the hybridization temperature from 50°C to 68°C in steps of approx. 1 - 2°C, it is possible to isolate polynucleotide fragments that are e.g. at least 80%, 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% identical to the sequence of the probe used or to the nucleotide sequences shown in SEQ ID No. 3 or SEQ ID No. 5. Further instructions on hybridization are commercially available in kit form (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, catalogue no. 1603558).
Enhancement can be achieved for example by increasing the expression of the genes, open reading frames or alleles or enhancing the catalytic properties of the protein. Both measures may optionally be combined.
Overexpression can be achieved for example by increasing the copy number of the appropriate genes or open reading frames or mutating the promoter and regulatory region or the ribosome binding site located upstream, from the structural gene. Expression cassettes incorporated upstream from the structural gene work in the same way. Inducible promoters additionally make it possible to increase expression in the course of L-threonine production by fermentation; the use of promoters for gene expression which allows another transitory gene expression can also be advantageous. Measures for prolonging the life of the mRNA also improve expression. Furthermore, the enzyme activity is also enhanced by preventing degradation of the enzyme protein. The ORFs, genes or gene constructs can either be located in plasmids of variable copy number or be integrated and amplified in the chromosome. Alternatively, it is also possible to achieve overexpression of the genes in question by changing the composition of the media and the culture technique.
Methods of overexpression are adequately described in the state of the art, for example in Makrides et al. (Microbiological Reviews 60 (3), 512-538 (1996)). The use of vectors increases the copy number by at least one (1) copy. Plasmids such as those described e.g. in US
5,538,873 can be used as vectors. Phages, for example phage mu, as described in EP 0332448, or phage lambda (λ) , can also be used as vectors . The copy number can also be increased by incorporating another copy into another site of the chromosome, for example into the att site of phage λ (Yu and Court, Gene 223, 77-81 (1998)). US 5,939,307 describes that expression could be increased by incorporating expression cassettes or promoters such as the tac promoter, trp promoter, lpp promoter or P promoter and PR promoter of phage λ, for example upstream from the chromosomal threonine operon. The promoters of phage T7, the gearbox promoters or the nar promoter can likewise be used. Such expression cassettes or promoters can also be used to overexpress plasmid-bound genes , as described in EP 0 593 792. The expression of plasmid-bound genes can in turn be controlled by using the lacIQ allele (Glascock and Weickert, Gene 223, 221-231 (1998)). It is further possible to increase the activity of promoters by modification of their sequence through one or more nucleotide exchanges, by insertion (s) and/or by deletion (s) . Another transitory gene expression can be achieved by using the growth phase-dependent fis promoter, for example as described in Walker et al. (Journal of Bacteriology 181, 1269-80 (1999)). Those skilled in the art will find relevant general instructions inter alia in 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)), Quandt and Klipp (Gene 80, 161-169 (1989)),
Hamilton et al. (Journal of Bacteriology 171, 4517-4622 (1989)), Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and well-known textbooks on genetics and molecular biology.
Plasmid vectors replicatable in Enterobacteriaceae, e.g. cloning vectors derived from pACYC184 (Bartolome et al . , Gene 102, 75-78 (1991)), pTrc99A (Amann et al . , Gene 69, 301-315 (1988)) or pSClOl derivatives (Vocke and Bastia, Proceedings of the National Academy of Sciences USA 80(21), 6557-6561 (1983)), can be used. In one process according to the invention, it is possible to use a strain transformed with a plasmid vector, said plasmid vector carrying at least one nucleotide sequence coding for the yibD ORF or its gene product, or allele.
The term "transformation" is understood as meaning the uptake of an isolated nucleic acid by a host (microorganism) .
Also, mutations which affect the expression of the appropriate genes or open reading frames can be transferred to different strains by sequence exchange (Hamilton et al . , Journal of Bacteriology 171, 4617-4622 (1989)), conjugation or transduction.
Further details on the concepts of genetics and molecular biology can be found in well-known textbooks on genetics and molecular biology, for example the textbook by Birge (Bacterial and Bacteriophage Genetics, 4th ed. , Springer Verlag, New York (USA) , 2000) , the textbook by Berg, Tymoczko and Stryer (Biochemistry, 5th ed., Freeman and Company, New York (USA), 2002) or the textbook by Sambrook et al. (Molecular Cloning, A Laboratory Manual (3-volume set) , Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) , 2001) .
Furthermore, for the production of L-amino acids, especially L-threonine, with strains of the family Enterobacteriaceae, it can be advantageous not only to enhance the yibD open reading frame, but also to enhance one or more enzymes of the known threonine biosynthetic pathway, or enzymes of the anaplerotic metabolism, or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate, or glycolytic enzymes, or PTS enzymes, or enzymes of sulfur metabolism. The use of endogenous genes is generally preferred.
Thus, for example, one or more genes selected from the group comprising:
• at least one gene of the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (US-A-4, 278, 765) ,
• the pyc gene of Corynebacterium glutamicum coding for pyruvate carboxylase (WO 99/18228), • the pps gene coding for phosphoenolpyruvate synthase (Molecular and General Genetics 231(2), 332-336 (1992)),
• the ppc gene coding for phosphoenolpyruvate carboxylase (WO 02/064808) ,
• the pntA and pntB genes coding for the subunits of transhydrogenase (European Journal of Biochemistry 158, 647-653 (1986)),
• the rhtB gene coding for the protein that confers homoserine resistance (EP-A-0 994 190) ,
• the rhtC gene coding for the protein that confers threonine resistance (EP-A-1 013 765) ,
• the thrE gene of Corynebacterium glutamicum coding for threonine export carrier protein (WO 01/92545) ,
• the gdhA gene coding for glutamate dehydrogenase (Nucleic Acids Research 11, 5257-5266 (1983); Gene 23, 199-209 (1983)),
• the pgm gene coding for phosphoglucomutase (WO 03/004598) ,
• the fba gene coding for fructose biphosphate aldolase (WO 03/004664) ,
• the ptsH gene of the ptsHIcrr operon coding for phosphohistidme protein hexose phosphotransferase of the phosphotransferase system PTS (WO 03/004674) ,
• the ptsl gene of the ptsHIcrr operon coding for enzyme I of the phosphotransferase system PTS (WO 03/004674) ,
• the err gene of the ptsHIcrr operon coding for the glucose-specific IIA component of the phosphotransferase system PTS (WO 03/004674), the ptsG gene coding for the glucose-specific IIBC component (WO 03/004670),
the Irp gene coding for the regulator of the leucine regulon (WO 03/004665),
the fadR gene coding for the regulator of the fad regulon (WO 03/038106) ,
the iclR gene coding for the regulator of the central intermediary metabolism (WO 03/038106) ,
the ahpC gene of the ahpCF operon coding for the small subunit of alkyl hydroperpxide reductase (WO 03/004663),
the ahpF gene of the ahpCF operon coding for the large subunit of alkyl hydroperoxide reductase (WO 03/004663),
the cysK gene coding for cysteine synthase A (WO 03/006666) ,
the cysB gene coding for the regulator of the cys regulon (WO 03/006666) ,
the cysJ gene of the cysJIH operon coding for the flavoprotein of NADPH sulfite reductase (WO 03/006666) ,
the cysl gene of the cysJIH operon coding for the haemoprotein of NADPH sulfite reductase (WO 03/006666) ,
the cysH gene of the cysJIH operon coding for adenylyl sulfate reductase (WO 03/006666),
the rseA gene of the rseABC operon coding for a membrane protein with anti-sigmaE activity (WO 03/008612),
the rseC gene of the rseABC operon coding for a global regulator of the sigmaE factor (WO 03/008612), • the sucA gene of the sucABCD operon coding for the decarboxylase subunit of 2-ketoglutarate dehydrogenase (WO 03/008614) ,
• the sucB gene of the sucABCD operon coding for the dihydrolipoyl transsuccinase E2 subunit of 2-ketoglutarate dehydrogenase (WO 03/008614),
• ' the sucC gene of the sucABCD operon coding for the β subunit of succinyl CoA synthetase (WO 03/008615) ,
• the sucD gene of the sucABCD operon coding for the α subunit of succinyl CoA synthetase (WO 03/008615) ,
• the aceE gene coding for the El component of the pyruvate dehydrogenase complex (WO 03/076635) ,
• the aceF gene coding for the E2 component of the pyruvate dehydrogenase complex (WO 03/076635) , • the rseB gene coding for the regulator of sigmaE factor activity (Molecular Microbiology 24 (2) , 355-371 (1997)),
• the gene product of the yodA open reading frame (ORF) of Escherichia coli (Accession Number AE000288 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), DE 10361192.4),
• the gene product of the yaaU open reading frame (ORF) of Escherichia coli (Accession Number AE005181 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), DE 10361268.8),
• the malT gene coding for the positive transcriptional activator of the maltose regulon (Gene 42, 201-208 (1986)) and
• the eno gene coding for enolase (The Journal of Biological Chemistry 246(22) , 6797-6802 (1971)) can be simultaneously enhanced or, in particular, overexpressed.
Furthermore, for the production of L-amino acids, especially L-threonine, it can be advantageous not only to enhance the yibD open reading frame, but also to attenuate or, in particular, switch off one or more genes selected from the group comprising:
• the tdh gene coding for threonine dehydrogenase (Journal of Bacteriology 169, 4716-4721 (1987)),
• the dh gene coding for malate dehydrogenase (E.C. 1.1.1.37) (Archives in Microbiology 149 , 36-42 (1987)),
• the gene product of the yjf open reading frame (ORF) 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 ytfP open reading frame (ORF) of Escherichia coli (Accession Number AAC77179 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), WO 02/29080),
• the pckA gene coding for the enzyme phosphoenolpyruvate carboxykinase (WO 02/29080),
• the poxB gene coding for pyruvate oxidase (WO 02/36797) ,
• the dgsA gene coding for the DgsA regulator of the phosphotransferase system (WO 02/081721) , which is also known as the mlc gene,
• the fruR gene coding for the fructose repressor (WO 02/081698) , which is also known as the era gene, • the rpoS gene coding for the sigma38 factor (WO 01/05939), which is also known as the katF gene, and
• the aspA gene coding for aspartate ammonium lyase (WO 03/008603) , or reduce the expression.
In this context the term "attenuation" describes the decrease or switching-off of the intracellular activity or concentration, in a microorganism, of one or more enzymes/ proteins encoded by the appropriate DNA, for example by using a weaker promoter than in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein, or a gene or allele which codes for an appropriate enzyme/protein with a low activity, or inactivating the appropriate enzyme/protein, the open reading frame or the gene, and optionally combining these measures .
The attenuation measures generally reduce the activity or concentration of the appropriate protein to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the microorganism or parent strain that is not recombinant for the appropriate enzyme/protein. Non-recombinant microorganism or parent strain is understood as meaning the microorganism on which the measures according to the invention are performed.
Attenuation can be achieved for example by reducing or switching off the expression of the genes or open reading frames or the catalytic properties of the enzyme proteins . Both measures may optionally be combined. Gene expression can be reduced by an appropriate culture technique, by genetic modification (mutation) of the signal structures of gene expression or else by an antisense RNA technique. Signal structures of gene expression are e.g. represspr genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators . Those skilled in the art will find relevant information inter alia in e.g. 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 Microbiology 3, 159-164 (2000)) and well-known textbooks on genetics and molecular biology, such as the textbook by Knippers ("Molekulare Genetik" , 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or the one by Winnacker (From Genes to Clones, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) .
Mutations that result in a modification or reduction of the catalytic properties of enzyme proteins are known from the state of the art. Examples which may be mentioned are the papers 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)). Surveys can be found in well-known textbooks on genetics and molecular biology, for example the one by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .
Possible mutations are transitions, transversions, insertions and deletions of at least one (1) base pair/ nucleotide. Depending on the effect of the amino acid exchange caused by the mutation on the enzyme activity, the term "missense mutations" or "nonsense mutations" is used. A missense mutation results in an exchange of a given amino acid in a protein for another amino acid, said amino acid exchange being in particular non-conservative. This impairs the functioning ability or activity of the protein and reduces it to a value. of 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5%. A nonsense mutation results in a stop codon in the coding region of the gene and hence in a premature termination of translation. Insertions or deletions of at least one base pair in a gene result in frame shift mutations, the outcome of which is that false amino acids are incorporated or translation is prematurely terminated. If a stop codon is produced in the coding region as a consequence of the mutation, this also results in a premature termination of translation. Deletions of at least one (1) or several codons typically also result in a complete loss of enzyme activity.
Instructions on the production of such mutations belong to the state of the art and can be found in well-known textbooks on genetics and molecular biology, such as the textbook by Knippers ("Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) , the one by Winnacker (From Genes to Clones, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or the one by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .
Suitable mutations in the genes can be incorporated into suitable strains by gene or allele exchange.
A common method is that of gene exchange with the aid of a conditionally replicating pSClOl derivative pMAK705, as described by Hamilton et al. (Journal of Bacteriology 171, 4617-4622 (1989)). Other methods described in the state of the art, for example that of Martinez-Morales et al . (Journal of Bacteriology 181, 7143-7148 (1999)) or that of Boyd et al. (Journal of Bacteriology 182, 842-847 (2000)), can likewise be used.
It is also possible for mutations in the particular genes, or mutations affecting the expression of the particular genes or open reading frames, to be transferred to different strains by conjugation or transduction.
Furthermore, for the production of L-amino acids, especially L-threonine, it can be advantageous not only to enhance the yibD open reading frame, but also to switch off unwanted secondary reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .
The microorganisms prepared according to the invention can be cultivated by the batch process, the fed batch process, the repeated fed batch process or a continuous process (DE 102004028859.3 or US 5,763,230). A summary of known cultivation methods is provided in the textbook by Chmiel (Bioprozesstechnik 1. Einfύhrung in die Bioverfahrens- technik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).
The culture medium to be used must appropriately meet the demands of the particular strains . Descriptions of culture media for various microorganisms are contained in "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981) .
Carbon sources which can be used are sugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmitic acid, stearic acid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids, e.g. acetic acid. These substances can be used individually or as a mixture.
Nitrogen sources which can be used are organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.
Phosphorus sources which can be used are phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium salts . The culture medium must also contain metal salts, e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances 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 feed materials can be added to the culture medium all at once or fed in appropriately during cultivation.
The fermentation is generally carried out at a pH of 5.5 to 9.0, especially of 6.0 to 8.0. The pH of the culture is controlled by the appropriate use of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled using antifoams such as fatty acid polyglycol esters . The stability of plasmids can be maintained by adding suitable selectively acting substances, e.g. antibiotics, to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gaseous mixtures, e.g. air, into the culture. The temperature of the culture is normally 25°C to 45°C and preferably 30°C to 40°C. The culture is continued until the formation of L-amino acids or L-threonine has reached a maximum. This objective is normally achieved within 10 hours to 160 hours.
L-amino acids can be analysed by means of anion exchange chromatography followed by ninhydrin derivatization, as described by Spackman et al. (Analytical Chemistry 30, 1190-1206 (1958)), or by reversed phase HPLC, as described by Lindroth et al . (Analytical Chemistry 51, 1167-1174 (1979) ) .
The process according to the invention is used to prepare L-amino acids, for example L-threonine, L-isoleucine, L-valine, L-methionine, L-homoserine, L-tryptophan and L- lysine, especially L-threonine, by fermentation.
The following microorganism has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany) under the terms of the Budapest Convention:
• Escherichia coli strain E. coli MG442 as DSM 16574. The present invention is illustrated in greater detail below with the aid of Examples.
The minimum medium (M9) and complete medium (LB) used for Escherichia coli are described by J.H. Miller (A Short Course in Bacterial Genetics (1992), Cold Spring Harbor Laboratory Press) . The isolation of plasmid DNA from Escherichia coli and all the techniques for restriction, ligation, Klenow treatment and alkaline phosphatase treatment are carried out according to Sambrook et al . (Molecular Cloning - A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press) . Unless described otherwise, the transformation of Escherichia coli is carried out according to Chung et al . (Proceedings of the National Academy of Sciences of the United States of America 86, 2172-2175 (1989)).
The incubation temperature in the preparation of strains and transformants is 37°C.
Example 1
Construction of expression plasmid pTrc99AyibD
The yibD gene from E. coli K12 is amplified using the poly erase chain reaction (PCR) and synthetic oligonucleotides. The nucleotide sequence of the yibD ORF in E. coli K12 MG1655 (Accession Number AE000439, Blattner et al. (Science 277, 1453-1474 (1997))) is used as the starting material to synthesize PCR primers (MWG Biotech, Ebersberg, Germany) . The sequences of the primers are modified to provide recognition sites for restriction enzymes. The recognition sequence for Sad is chosen for the yibD-exl primer and the recognition sequence for Hindlll is chosen for the yibD-ex2 primer, said sequences being underlined in the nucleotide sequences shown below:
yibD-exl : 5'-GATCTAGAGCrcGTCAGGATAACTTCAGAGG-3' (SEQ ID No. 1)
yibD-ex2 :
5-GATCTAAGCTTAGCCCGAAGCGGCGAAGTTTA-3 ' (SEQ ID No . 2)
The chromosomal E. coli K12 MG1655 DNA used for the PCR is isolated with "Qiagen Genomic-tips 100/G" (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. An approx. 1114 bp DNA fragment can be amplified with the specific primers under standard PCR conditions (Innis et al. (1990), PCR Protocols.' A Guide to Methods and Applications, Academic Press) using Vent DNA polymerase (New England Biolabs GmbH, Frankfurt, Germany) (SEQ ID No . 3 ) .
The amplified yibD fragment is ligated with vector pCR-Blunt II-TOPO (Zero TOPO TA Cloning Kit, Invitrogen, Groningen, The Netherlands) in accordance with the manufacturer's instructions and transformed into the E. coli strain TOP10. Plasmid-carrying cells are selected on
LB agar supplemented with 50 μg/ml of kanamycin. After isolation of the plasmid DNA, the vector is cleaved with the enzymes Pvul and Hindlll/Sacl and, after the cleavage has been checked in 0.8% agarose gel, is called pCRBluntyibD.
Vector pCRBluntyibD is then cleaved with the enzymes Hindlll and Sad and, after separation in 0.8% agarose gel, the yibD fragment is isolated from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilden, Germany) and ligated with vector pTrc99A (Pharmacia Biotech, Uppsala, Sweden) which has been digested with the enzymes Hindlll and Sad. The E. coli strain XLlBlue MRF' (Stratagene, La Jolla, USA) is transformed with the ligation mixture and plasmid-carrying cells are selected on LB agar supplemented with 50 μg/ml of ampicillin.
The success of the cloning can be demonstrated, after isolation of the plasmid DNA, by control cleavage with the enzyme Pvul .
The plasmid is called pTrc99AyibD (Figure 1) .
Example 2
Preparation of L-threonine with the strain MG442/ pTrc99AyibD
The L-threonine-producing E. coli strain MG442 is described in patent US-A-4, 278, 765 and is deposited in the Russian National Collection of Industrial Microorganisms (VKPM,
Moscow, Russia) as CMIM B-1628 and in the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany) as DSM 16574 under the terms of the Budapest Convention.
The strain MG442 is transformed with expression plasmid pTrc99AyibD, described in Example 1, and with vector pTrc99A and plasmid-carrying cells are selected on LB agar supplemented with 50 μg/ml of ampicillin. This procedure yields the strains MG442/pTrc99AyibD and MG442/pTrc99A. Chosen individual colonies are then multiplied further on minimum medium of the following composition: 3.5 g/1 of Na2HP04-2H20, 1.5 g/1 of KH2P04, 1 g/1 of NH4Cl , 0.1 g/1 of MgS04-7H20, 2 g/1 of glucose, 20 g/1 of agar, 50 mg/1 of ampicillin. The formation of L-threonine is verified in 10 ml batch cultures contained in 100 ml conical flasks. This is done by inoculating 10 ml of preculture medium of the following composition: 2 g/1 of yeast extract, 10 g/1 of (NH4) S04, 1 g/1 of KH2P0 , 0.5 g/1 of MgS04-7H20, 15 g/1 of CaC03, 20 g/1 of glucose, 50 mg/1 of ampicillin, and incubating for 16 hours at 37°C and 180 rpm on an ESR incubator from Kύhner AG (Birsfelden, Switzerland) . 250 μl of each of these precultures are transferred to 10 ml of production medium (25 g/1 of (NH4)2S04, 2 g/1 of KH2P0 , 1 g/1 of MgS04-7H20, 0.03 g/1 of FeS04-7H20, 0.018 g/1 of MnS04-lH20, 30 g/1 of CaC03, 20 g/1 of glucose, 50 mg/1 of ampicillin) and incubated for 48 hours at 37°C. The formation of L-threonine by the original strain MG442 is verified in the same way except that no ampicillin is added to the medium. After incubation the optical density (OD) of the culture suspension is determined using an LP2W photometer from Dr. Lange (Dusseldorf, Germany) at a measurement wavelength of 660 nm.
The concentration of L-threonine formed is then determined in the sterile-filtered culture supernatant using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by means of ion exchange chromatography and postcolumn reaction with ninhydrin detection.
Table 1 shows the result of the experiment. Table 1
Figure imgf000032_0001
Brief description of the Figure:
Figure 1: Map of plasmid pTrc99AyibD containing the yibD ORF
The indicated lengths are to be understood as approximate. The abbreviations and symbols used are defined as follows :
• bla: gene coding for ampicillin resistance
• lac Iq: gene for the repressor protein of the trc promoter
• trc: trc promoter region, IPTG-inducible
• yibD: coding region of the yibD gene
5S: 5S rRNA region
• rrnBT: rRNA terminator region
The abbreviations for the restriction enzymes are defined as follows :
• Pvul: restriction endonuclease from Proteus vulgaris

Claims

What is claimed is:
1. Recombinant microorganisms of the family Enterobacteriaceae which contain an enhanced or overexpressed yibD ORF, called a putative glycosyl transferase, and which produce L-amino acids in an improved manner .
2. Microorganisms according to Claim 1 in which a polynucleotide is enhanced that codes for a polypeptide whose amino acid sequence is at least 90% identical to an amino acid sequence selected from the group comprising SEQ ID No. 4 and SEQ ID No. 6.
3. Microorganisms according to Claim 2, wherein they /' contain an overexpressed or enhanced polynucleotide corresponding to the yibD ORF and selected from the group : a) polynucleotide with the nucleotide sequence SEQ ID No. 3 or SEQ ID No. 5; b) polynucleotide with a nucleotide sequence corresponding to SEQ ID No. 3 or SEQ ID No. 5 within the degeneracy of the genetic code; c) polynucleotide with a nucleotide sequence that hybridizes under stringent conditions with the sequence complementary to the sequence SEQ ID No. 3 or SEQ ID No. 5; d) polynucleotide with a sequence SEQ ID No. 3 or SEQ ID No. 5 containing neutral sense mutants.
4. Microorganisms according to Claim 2 , wherein the polypeptide has an amino acid sequence that is at least 95% identical to a sequence selected from the group comprising SEQ ID No. 4 and SEQ ID No. 6.
5. Microorganisms according to Claim 2, wherein the polypeptide has the amino acid sequence that is 100% identical to that of SEQ ID No. 4 or SEQ ID No. 6.
6. Microorganisms according to Claims 1 to 5, wherein they are produced by transformation, transduction or conjugation, or a combination of these methods, with a vector containing the yibD ORF, an allele of this ORF or parts thereof, and/or a promoter.
7. Microorganisms according to Claim 1 or 6 in which the copy number of the yibD ORF or the alleles is increased by at least 1.
8. Microorganisms according to Claim 7, wherein the increase in the copy number of the yibD ORF by at least 1 is achieved by integration of the ORF or the alleles into the chromosome of the microorganisms.
9. Microorganisms' according to Claim 7, wherein the increase in the copy number of the yibD ORF by at least 1 is achieved by an extrachromosomally replicating vector.
10. Microorganisms according to Claim 1 or 2 , wherein the enhancement is achieved by a) mutating the promoter and regulatory region or the ribosome binding site upstream from the yibD ORF, or b) incorporating expression cassettes or promoters upstream from the yibD ORF.
11. Microorganisms according to Claim 1 or 2, wherein the yibD ORF is under the control of a promoter that enhances the expression of the gene.
12. Microorganisms according to Claims 1 to 11, wherein enhancement of the yibD ORF increases the concentration or activity of the yibD gene product (protein) by at least 10%, based on the activity or concentration of the gene product in the microorganism or parent strain that is not recombinant for the yibD ORF.
13. Microorganisms according to Claims 1 to 12, wherein they are selected from the genera Escherichia, Erwinia, Providencia and Serratia.
14. Microorganisms according to Claim 13, wherein other genes of the biosynthetic pathway of the desired L- amino acid are additionally enhanced or, in particular, overexpressed.
15. Microorganisms according to Claims 1 to 14, wherein they produce L-threonine.
16. Process for the preparation of L-amino acids by the fermentation of recombinant microorganisms of the family Enterobacteriaceae, wherein a) the microorganisms producing the desired L-amino acid, in which the yibD ORF, or nucleotide sequences coding for its gene product, or alleles, is (are) enhanced or, in particular, overexpressed, are cultivated 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, constituents of the fermentation broth, and/or all or part (> 0 to 100%) of the biomass, remaining in the isolated product or being completely removed.
17. Process according to Claim 16, wherein the microorganisms according to Claims 1 to 15 are used.
18. Process according to Claim 16 or 17, wherein, to prepare L-threonine, microorganisms of the family Enterobacteriaceae are fermented in which additionally one or more genes selected from the group comprising:
18.1 at least one gene of the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase,
18.2 the pyc gene of Corynebacterium glutamicum coding for pyruvate carboxylase,
18.3 the pps gene coding for phosphoenolpyruvate synthase,
18.4 the ppc gene coding for phosphoenolpyruvate carboxylase,
18.5 the pntA and pntB genes coding for the subunits of pyridine transhydrogenase,
18.6 the rhtB gene coding for the protein that confers homoserine resistance, 18.7 the rhtC gene coding for the protein that confers threonine resistance,
18.8 the thrE gene of Corynebacterium glutamicum coding for threonine export carrier protein,
18.9 the gdhA gene coding for glutamate dehydrogenase,
18.10 the pg gene coding for phosphoglucomutase,
18.11 the fba gene coding for fructose biphosphate aldolase,
18.12 the ptsH gene coding for phosphohistidme protein hexose phosphotransferase,
18.13 the ptsl gene coding for enzyme I of the phosphotransferase system,
18.14 the err gene coding for the glucose-specific IIA component,
, 18.15 the ptsG gene coding for the glucose-specific IIBC component,
18.16 the lrp gene coding for the regulator of the leucine regulon,
18.17 the fadR gene coding for the regulator of the fad regulon,
18.18 the iclR gene coding for the regulator of the central intermediary metabolism,
18.19 the ahpC gene coding for the small subunit of alkyl hydroperoxide reductase,
18.20 the ahpF gene coding for the large subunit of alkyl hydroperoxide reductase,
18.21 the cysK gene coding for cysteine synthase A,
18.22 the cysB gene coding for the regulator of the cys regulon,
18.23 the cysJ gene coding for the flavoprotein of NADPH sulfite reductase,
18.24 the cysl gene coding for the haemoprotein of NADPH sulfite reductase,
18.25 the cysH gene coding for adenylyl sulfate reductase,
18.26 the rseA gene coding for a membrane protein with anti-sigmaE activity,
18.27 the rseC gene coding for a global regulator of the sigmaE factor,
18.28 the sucA gene coding for the decarboxylase subunit of 2-ketoglutarate dehydrogenase,
18.29 the sucB gene coding for the dihydrolipoyl transsuccinase E2 subunit of 2-ketoglutarate dehydrogenase,
18.30 the sucC gene coding for the β subunit of succinyl CoA synthetase,
18.31 the sucD gene coding for the α subunit of succinyl CoA synthetase,
18.32 the aceE gene coding for the El component of the pyruvate dehydrogenase complex,
18.33 the aceF gene coding for the E2 component of the pyruvate dehydrogenase complex,
18.34 the rseB gene coding for the regulator of sigmaE factor activity, 18.35 the gene product of the yodA open reading frame (ORF) of Escherichia coli,
18.36 the gene product of the yaaU open reading frame (ORF) of Escherichia coli,
18.37 the malT gene coding for the positive transcriptional activator of the maltose regulon, and 18.38 the eno gene coding for enolase are simultaneously enhanced or, in particular, overexpressed.
19. Process according to Claims 16 to 18, wherein microorganisms are used in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partially attenuated.
20. Process according to Claim 19, characterized in that, to prepare L-threonine, microorganisms of the family Enterobacteriaceae are fermented in which additionally one or more genes selected from the group comprising:
20.1 the tdh gene coding for threonine dehydrogenase,
20.2 the dh gene coding for malate dehydrogenase, 20.3 the gene product of the yjfA open reading frame (ORF) of Escherichia coli,
20.4 the gene product of the ytfP open reading frame (ORF) of Escherichia coli,
20.5 the pckA gene coding for phosphoenolpyruvate carboxykinase, 20.6 the poxB gene coding for pyruvate oxidase,
20.7 the dgsA gene coding for the DgsA regulator of the phosphotransferase system,
20.8 the fruR gene coding for the fructose repressor, 20.9 the rpoS gene coding for the sig a38 factor, and
20.10 the aspA gene coding for aspartate ammonium lyase are simultaneously attenuated or, in particular, switched off, or the expression is reduced.
21. Process according to Claims 16 to 20, wherein the L- amino acids prepared are selected from the group comprising L-asparagine, 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-homoserine.
22. Process according to Claim 21, wherein the L-amino acids prepared are selected from the group comprising L-isoleucine, L-valine, L-methionine, L-homoserine, L- tryptophan and L-lysine.
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