EP1948811A2 - Procede pour produire des acides l-amines par fermentation au moyen de bacteries coryneformes capables d'utiliser la glycerine comme source exclusive de carbone - Google Patents

Procede pour produire des acides l-amines par fermentation au moyen de bacteries coryneformes capables d'utiliser la glycerine comme source exclusive de carbone

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Publication number
EP1948811A2
EP1948811A2 EP06806854A EP06806854A EP1948811A2 EP 1948811 A2 EP1948811 A2 EP 1948811A2 EP 06806854 A EP06806854 A EP 06806854A EP 06806854 A EP06806854 A EP 06806854A EP 1948811 A2 EP1948811 A2 EP 1948811A2
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EP
European Patent Office
Prior art keywords
seq
glycerol
coryneform bacteria
amino acid
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP06806854A
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German (de)
English (en)
Inventor
Volker F. Wendisch
Doris Rittmann
Hermann Sahm
Caroline Kreutzer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Evonik Operations GmbH
Original Assignee
Evonik Degussa GmbH
Forschungszentrum Juelich GmbH
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Application filed by Evonik Degussa GmbH, Forschungszentrum Juelich GmbH filed Critical Evonik Degussa GmbH
Priority to EP11152912.9A priority Critical patent/EP2354235B1/fr
Priority to DK11152912.9T priority patent/DK2354235T3/en
Priority to PL11152912T priority patent/PL2354235T3/pl
Publication of EP1948811A2 publication Critical patent/EP1948811A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • 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
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan

Definitions

  • the invention relates to recombinant coryneform bacteria in which at least one or more of the heterologous genes of the glycerol metabolism (glycerol metabolism) selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT , glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC are expressed as well as a process for the fermentative production of L-amino acids, in particular L-lysine and L-tryptophan, wherein the medium contains glycerol as a carbon source under Use of these bacteria.
  • These bacteria show the ability to use glycerol and thus to efficiently form and accumulate the L-amino acids.
  • Chemical compounds in particular L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids, are used in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal nutrition.
  • Process improvements may include fermentation measures such as stirring and oxygen supply, or composition of nutrient media such as sugar concentration during fermentation, or work up to product form by, for example
  • Amino acid-producing strains of Corynebacterium glutamicum are used by amplifying individual amino acid biosynthesis genes and investigating the effect on L-amino acid production.
  • a summary of various aspects of Corynebacterium glutamicum genetics, metabolism and biotechnology can be found in Puhler ((chief ed.) Journal of Biotechnology 104 (1-3), 1-338 (2003)) and Eggeling and Bott (( editors) Handbook of Corynebacterium glutamicum, CRC Press, Taylor & Francis Group, Boca Raton (2005)).
  • L-amino acids preferably L-lysine and L-tryptophan
  • a technologically feasible alternative for producing the L-amino acids is to use a less expensive material as an alternative raw material for fermentation.
  • Glycerin (Propantriol) is a natural constituent of oils and fats and connects the fatty acid molecules in the triglycerides as a "bridge."
  • the glycerine molecule is highly polar and therefore readily soluble in water. RME) and is used in cosmetics, pharmaceuticals, food and technical applications. Decisive for the use of glycerine as raw material for the production of feed components is the value for money. It can be assumed that as biodiesel production increases, glycerol becomes more interesting for the production of feed additives.
  • Corynebacterium glutamicum wild-type uses a variety of monomeric and oligomeric sugars such as glucose, sucrose or maltose as the carbon source (Vahjen et al., FEMS
  • Corynebacterium glutamicum wild-type has some genes with homology to known genes of glycerol metabolism, but so far it has not been possible to explain why growth on glycerol is still not possible.
  • the inventors have set themselves the task of providing new coryneform bacteria, which are able to use glycerol as possible as sole carbon source.
  • Another directly related object was to provide an improved process for the fermentative production of L-amino acids, in particular L-lysine and L-tryptophan, with the aid of such coryneform bacteria. In particular, it should
  • Glycerol be made useful for the fermentative production of L-amino acids in the most economical manner.
  • the invention relates to recombinant coryneform bacteria which in particular already excrete L-amino acids, and in which at least one or more of the nucleotide sequence (s) coding for the heterologous gene products of the glycerol metabolism (glycerol metabolism), selected from the group glpA , glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC.
  • These bacteria show the ability to use glycerin.
  • the bacteria used include in particular coryneform bacteria in which at least one heterologous polynucleotide is encoded which codes for a polypeptide whose amino acid sequence is at least 80% or at least 90%, in particular at least 95%, preferably at least 98%, particularly preferred is at least 99% and most preferably 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID no. 8, SEQ ID no. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID no. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID no. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34 and SEQ ID NO. 36.
  • the said bacteria preferably contain at least one heterologous polynucleotide selected from the group consisting of:
  • polynucleotide having the nucleotide sequence SEQ ID no. 1, SEQ ID no. 3, SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no. 13, SEQ ID no. 15, SEQ ID no. 17, SEQ ID no. 19, SEQ ID no. 21, SEQ ID no. 23, SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO. 35 and complementary nucleotide sequences
  • SEQ ID no. 1, SEQ ID no. 3, SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no. 13, SEQ ID no. 15, SEQ ID no. 17, SEQ ID no. 19, SEQ ID no. 21, SEQ ID no. 23, SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO. 35 corresponds to the degeneration of the genetic code;
  • the stringent conditions are preferably achieved by a washing step in which the temperature extends over a range of 64 ° C to 68 ° C and the salt concentration of the buffer over a range of 2xSSC to 0, IxSSC ;
  • SEQ ID no. 3 SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no. 13, SEQ ID no. 15, SEQ ID no. 17, SEQ ID no. 19, SEQ ID no. 21, SEQ ID no. 23, SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO. 35, which contains functionally neutral sense mutations,
  • polynucleotides encode enzymes of glycerol metabolism (glycerol metabolism).
  • the invention likewise provides a process for the fermentative production of L-amino acids, in particular L-lysine and L-tryptophan, the medium containing glycerol as carbon source, using recombinant coryneform bacteria, which in particular already produce L-amino acids, and in which at least one or more of the heterologous genes of glycerol metabolism (glycerol metabolism), selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC or nucleotide sequences coding for their gene products ,
  • the bacteria according to the invention are preferably used.
  • L-amino acids or amino acids are one or more of the proteinogenic amino acids, including their salts, selected from the group L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L- Arginine and L-proline meant. Particularly preferred is L-lysine and L-tryptophan.
  • the L-amino acids also include L-homoserine.
  • Proteinogenic amino acids are the amino acids found in natural proteins, that is, in proteins of microorganisms, plants, animals and humans.
  • amino acids are mentioned below, the term also includes their salts, for example the lysine monohydrochloride or lysine sulfate in the case of the amino acid L-lysine.
  • heterologous genes or “heterologous nucleotide sequences” of the invention may be derived from any prokaryotic donor organism other than members of the genus Corynebacterium originate.
  • the genes from Escherichia coli are preferably used.
  • heterologous genes in this context describes the cloning of corresponding genes and their expression in the heterologous system, which leads to an establishment 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 generating a vector which contains the desired gene or an allele of this gene and a promoter enabling the expression of the gene and transmits by transformation, transduction or conjugation in the microorganism and optionally combines these measures.
  • alleles is meant alternative forms of a given gene.
  • the forms are characterized by differences in the nucleotide sequence.
  • Nucleotide sequence i. a gene or allele encoded protein or the encoded ribonucleic acid.
  • the subject matter of this invention is also a process for the fermentative production of L-amino acids, characterized in that the following steps are carried out:
  • the heterologous genes of the glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA,
  • Gene products encoding nucleotide sequences or alleles is or are, in a glycerol or optionally additionally one or more further C- Sources containing medium under conditions in which the desired L-amino acid is enriched in the medium or in the cells, and optionally
  • coryneform bacteria used preferably already produce the conventional one prior to the expression of one or more of the genes of the glycerol metabolism
  • Carbon sources such as glucose or sucrose L-amino acids, especially L-lysine and L-tryptophan.
  • the glycerol used can be used individually or as a mixture, wherein the proportion of glycerol should preferably be> 10 to 100%.
  • coryneform bacteria after heterologous expression of one or more of the genes of the glycerol metabolism, produce L-amino acids, in particular L-lysine and L-tryptophan, from glycerol as sole carbon source.
  • Recombinant bacteria according to the invention are produced, for example, by transformation, transduction or conjugation, or a combination of these methods, with a vector which contains the desired gene, an allele of this gene or parts thereof and a promoter which enables the expression of the gene.
  • Heterologous expression is achieved in particular by integration of the gene or alleles into the chromosome of the microorganisms or an extrachromosomally replicating vector.
  • the promoter may be its own regulatory sequence upstream of the gene, or a promoter from coryneform bacteria may be fused to the gene.
  • a promoter from coryneform bacteria may be fused to the gene.
  • Glucose sucrose, lactose, fructose, maltose, molasses, starch, cellulose or ethanol. These are representatives of coryneform bacteria.
  • the genus Corynebacterium is preferred. Particularly preferred
  • Amino acid-secreting strains based on the following types:
  • Corynebacterium efficiens such as the strain DSM44549,
  • Corynebacterium glutamicum such as strain ATCC13032,
  • thermoaminogenes such as strain FERM BP-1539, and
  • Corynebacterium ammoniagenes such as strain ATCC6871,
  • Corynebacterium glutamicum Some representatives of the species Corynebacterium glutamicum are also known in the art under other species names. These include, for example:
  • Corynebacterium glutamicum AHP-3 FermBP-7382
  • Strains designated "ATCC” can be purchased from the American Type Culture Collection (Manassas, Va.) Strains designated “DSM” can be obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). "FERM” strains are available from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) become. The cited strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A-5,250,434.
  • the nucleotide sequences of the genes or open reading frames (ORF) of Escherichia coli are known in the art and may be that described by Blattner et al. (Science 277: 1453-1462 (1997)) published genome sequence of Escherichia coli.
  • Salmonella typhimurium (Accession No .: NC 003197 (sequence of the entire genome)) and Shigella flexneri (Accession No .: NC 004337 (sequence of the entire genome)), which likewise belongs to the family Enterobacteriaceae, have the nucleotide sequence for the genes glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT and glpX are also known by way of example. Furthermore, Salmonella typhimurium
  • glycerol metabolism The genes and activities of glycerol metabolism (glycerol metabolism) are also summarized in Lin (In: Neidhardt (ed), Escherichia coli and Salmonella, American Society for Microbiology, Washington, D. C, USA: 307-342 (1996)) described.
  • the glycerol transport and metabolism-containing glycerophosphate regulone (glp) gene consists of five operons located at three different loci on the E. coli chromosome (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)). ,
  • a regulon is a unit of genes that are localized at different sites of a genome, but expressed by the same regulatory proteins is controlled.
  • An operon is a unit of co-regulated genes at a locus.
  • glpT glycerol-3-phosphate permease
  • glpQ periplasmic glycerol phosphodiesterase
  • the glpDEG operon responsible for glycerol-3-phosphate dehydrogenase GIpD is active in the presence of atmospheric oxygen (aerobic) (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)). ), the sulfur transferase GIpE (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)) and the glpG gene of unknown function (Zeng et al., Journal of Bacteriology 178: 7080-7089 (1996)). coded.
  • Phosphate dehydrogenase (anaerobic) Function In the anaerobic environment, glycerol-3
  • Phosphate for energy production from a FAD-dependent glycerol-3-phosphate Dehydrogenase oxidized to dihydroxyacetone phosphate, which can enter as an intermediate in glycolysis.
  • the reduction equivalents released in this oxidation reaction are transferred from the flavoenzyme to a membrane-associated cytochrome complex, with fumarate or nitrate serving as the terminal electron acceptor (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella, American Society for Microbiology, Washington, D.C. , USA: 307-342 (1996)).
  • the released energy is used to pump protons across the membrane from the cytoplasmic to the periplasmic side.
  • the proton gradient produced on the membrane changes both the electrical and the chemical potential, which drives the membrane-bound ATPase and thereby generates ATP.
  • glpB gene Name: membrane anchor subunit of sn-glycerol-3-phosphate dehydrogenase (anaerobic) Function: see glpA
  • glpC gene name: small subunit of sn-glycerol-3
  • glycerol-3-phosphate is used to generate energy from this FAD-dependent glycerol-3-phosphate dehydrogenase (GIpD)
  • Flavoenzym on a membrane-associated cytochrome complex with molecular oxygen or nitrate serving as the terminal electron acceptor (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella, American Society for Microbiology, Washington, D.C., USA: 307-342 (1996)).
  • the released energy is used to pump protons across the membrane from the cytoplasmic to the periplasmic side.
  • the proton gradient produced on the membrane changes both the electrical and the chemical potential, which drives the membrane-bound ATPase and thereby generates ATP.
  • the open reading frame of the glpD gene consists of 501 codons and the translated sequence encodes a protein of molecular weight 57 kDa (Austin and Larson, Journal of Bacteriology 173: 101-107 (1991)).
  • glpE gene name: sulfur transferase; acidic, cytoplasmic rhodanese
  • GIpE has been identified as a sulfur transferase (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)).
  • the glpE gene Encoded acidic, cytoplasmic rhodanese with a molecular weight of 12 kDa catalyzes as a dimer the transfer of sulfur to the sulfur acceptor thioredoxin 1 (Ray et al., Journal of Bacteriology 182: 2277-2284 (2000)).
  • Glycerol facilitator GIpF Function Facilitated diffusion of glycerol from the nutrient medium is catalyzed by the glycerol facilitator GIpF (Borgnia and Agre, Proc. Natl. Acad.
  • gene of the glp regulons Function The physiological function of glpG is still unknown.
  • the glpG gene product is a basic, cytoplasmic or membrane-associated protein having a molecular weight of 28 kDa (Zeng et al., Journal of Bacteriology 178: 7080-7089 (1996)).
  • Cytoplasmic glycerol is immediately phosphorylated by the ATP-dependent glycerol kinase K, which in its enzymatically active form is associated with the glycerol fibril promoter GIpF (Voegele et al., Journal of Bacteriology 175: 1087-1094 (1993)).
  • Glycerophosphodiesterase Function Glycerophosphate diesters, the deacetylated degradation products of phospholipids (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella, American Society for Microbiology, Washington, D.C, USA: 307-342 (1996)), are incorporated herein by reference Periplasm of the localized phosphodiesterase GIpQ too Alcohol and glycerol-3-phosphate hydrolyzed (Larson et al., Journal of Biological Chemistry 258: 5428-5432
  • the gene product derived from the heterologous glpQ gene in the claimed bacteria must comprise a leader peptide, as is typical of excreted Gram-positive bacteria
  • glpT gene name: glycerol-3-phosphate permease function: glycerol-3-phosphate is produced by the permease GIpT (Eiglmeier et al., Molecular Microbiology 1: 251-258 (1987); Larson et al., Journal of Bacteriology 152: 1008-1021 (1982)) into the cell interior in exchange for inorganic phosphate (Auer et al., Biochemistry 40: 6628-6635 (2001)). The energy needed for transport is provided by this antiport and at the same time accumulation of the toxic phosphate is prevented (Xavier et al., Journal of Bacteriology
  • glpX gene name: fructose-1, 6-bisphosphatase II
  • GIpX Role of the enzyme GIpX can be found.
  • gldA gene name: glycerol dehydrogenase (NAD)
  • Glycerol dehydrogenase catalyzes the reversible NAD-dependent reaction of glycerol to dihydroxyacetone (Truniger and Boos, Journal of Bacteriology 176 (6): 1796-1800 (1994))
  • Dihydroxyacetone kinase catalyzes the reaction of dihydroxyacetone as a phosphoenolpyruvate (PEP) -dependent dihydroxyacetone phosphotransferase
  • the dihydroxyacetone kinase catalyzes the function of the PEP-dependent dihydroxyacetone phosphotransferase
  • DhaL carries ADP as a cofactor for the transfer of DhaM phosphate to dihydroxyacetone.
  • dhaM gene name: PTS protein subunit of
  • Dihydroxyacetone kinase catalyzes the function of PEP-dependent dihydroxyacetone phosphotransferase
  • DhaM consists of three domains with similarity to the three domains of the
  • Phosphoenolpyruvate-dependent phosphotransferase system Phosphorylated DhaM transfers the phosphate to the DhaL bound ADP.
  • Activator of the dha operon (dhaKLM) Function transcriptional activator, stimulates transcription of the dha operon from a sigma70 promoter (Bachler et al., The EMBO Journal
  • fsa gene name: fructose 6-phosphate aldolase I
  • Fructose-6-phosphate aldolase I catalyzes aldol cleavage of fructose-6-phosphate, substrates of the enzyme are dihydroxyacetone and fructose-6-phosphate and
  • Glyceraldehyde-3-phosphate not used are fructose, fructose-1-phosphate, fructose-1, 6-bisphosphate or dihydroxyacetone-phosphate (Schurmann and Sprenger, Journal of Biological
  • Fructose 6-phosphate aldolase II catalyzes aldol cleavage of the
  • the talC gene is located next to the gldA gene.
  • NCBI National Library of Biotechnology Information
  • the open reading frames described in the specified text can be used according to the invention. Furthermore, alleles of the genes or open reading frames can be used, which result from the degeneracy of the genetic code or by function-neutral sense mutations ("sense mutations").
  • the alleles of the treated genes which contain function-neutral sense mutations include, among others, those which have no more than 13 or no more than 10, preferably no more than 7 or no more than 5, very preferably no more than 3 or no more than 2 or at least conservative Amino acid exchange in the protein they encode lead.
  • the aromatic amino acids are called conservative exchanges when phenylalanine, tryptophan and tyrosine are exchanged.
  • the hydrophobic amino acids are called conservative exchanges when leucine, isoleucine and valine are exchanged.
  • the polar amino acids are called conservative exchanges when glutamine and asparagine are exchanged.
  • the basic amino acids are called conservative exchanges when arginine, lysine and histidine are exchanged.
  • the acidic amino acids are called conservative exchanges when aspartic acid and glutamic acid are exchanged.
  • the hydroxyl-containing amino acids are called conservative exchanges when serine and threonine are interchanged.
  • nucleotide sequences which code for variants of the proteins mentioned which additionally have an N- or C-terminus Lengthening or shortening by at least one (1) amino acid included.
  • This extension or truncation is not more than 13, 10, 7, 5, 3 or 2 amino acids or amino acid residues.
  • aminopeptidases the terminal methionine is removed during protein synthesis.
  • Suitable alleles include those that encode proteins in which at least one (1) amino acid is inserted (insertion) or deleted (deletion).
  • the maximum number of such changes referred to as indices, may be 2, 3, 4, 5 but in no case more than 6 amino acids.
  • suitable alleles include those obtained by hybridization, in particular under stringent conditions using SEQ ID NO. 1, SEQ ID no. 3, SEQ ID no.
  • SEQ ID no. 23 SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO. 35 or
  • Hybridization "by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and by Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). Hybridization takes place under stringent conditions, that is to say, only Hybrids are formed in which the probe and target sequence, ie the polynucleotides treated with the probe, are at least 70% identical It is known that the stringency of the hybridization including the washing steps by Varying the buffer composition, the temperature and the salt concentration is influenced or determined. The hybridization reaction is generally performed at relatively low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).
  • probes can also hybridize with polynucleotides that are less than 70%
  • Hybridization temperature in steps of about 1 - 2 0 C from 50 ° C to 68 ° C polynucleotide fragments can be isolated, for example, at least 70% or at least 80% or at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence of the probe used or to the in SEQ ID NO. 1, SEQ ID no. 3, SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no. 13, SEQ ID no. 15, SEQ ID no. 17, SEQ ID no. 19, SEQ ID no. 21, SEQ ID no. 23, SEQ ID no.
  • SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 SEQ ID no. 33 or SEQ ID NO. 35 have shown nucleotide sequences. Further instructions for hybridization are available on the market in the form of so-called kits (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).
  • polynucleotides of Bacillus subtilis (Accession No .: NC 000964 (sequence of the entire genome)) and Streptomyces coelicolor (Accession No .: NC 003888 (sequence of the entire genome)), for example, at least 70% or at least 80% or at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% Identity to the in SEQ ID NO. 1, SEQ ID no. 3, SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no.
  • SEQ ID no. 15 SEQ ID no. 17, SEQ ID no. 19, SEQ ID no. 21, SEQ ID no. 23, SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO.
  • NCBI National Library of Medicine National Database
  • the expression of the glycerol metabolism genes can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software.
  • a common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712-23 (2001)).
  • the protein concentration can also and subsequent optical evaluation with: by western blot hybridization with an antibody specific for the protein to antibody (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 A Laboratory Manual 2 nd Ed Sambrook et al., Molecular Cloning.). corresponding software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5: 32-39; Lottspeich
  • DNA-binding proteins can be measured by DNA band-shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology 183: 2151-2155).
  • DNA band-shift assays also referred to as gel retardation
  • the effect of DNA-binding proteins on the expression of other genes can be prepared by various well-described methods of reporter gene assays are detected (Sambrook et al, Molecular cloning. A laboratory manual 2 nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor.. , NY, 1989).
  • the intracellular enzymatic activities can be determined by various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627, Ray et al.
  • the heterologous genes are expressed, for example, using episomal plasmids.
  • Suitable plasmids are those which are replicated in coryneform bacteria.
  • Numerous known plasmid vectors such as pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLI or pGAl.
  • plasmid vectors such as those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (US-A 5,158,891) can be used be used in the same way.
  • pCG4 US-A 4,489,160
  • pNG2 Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)
  • pAG1 US-A 5,158,891
  • plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon or in WO03 / 040373.
  • the gene is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum.
  • vectors examples include pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega Corporation , Madison, WI, USA), pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry 269: 32678-84, US-A 5, 487, 993), pCR® Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEMI (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342) in question.
  • pSUP301 Synmon et al., Bio / Technology 1, 784-791 (1983)
  • the plasmid vector containing the heterologous gene to be amplified, optionally including the expression and / or regulatory signals, and the peripheral regions of a nonessential homologous gene is then converted by conjugation or transformation into the desired strain of C. glutamicum.
  • the method of conjugation is described by Shufer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiology Letters 123, 343-347 (1994)).
  • the resulting strain contains a copy of the heterologous gene, including the plasmid vector, at the desired locus of the C. glutamicum chromosome, which was predetermined via the homologous nucleotide sequences on the plasmid.
  • a suitable second excision-causing cross-over event in the target gene or in the target sequence the incorporation of only the heterologous gene is achieved, leaving no nucleotide sequence capable of episomal replication in microorganisms at the respective natural gene locus, not capable of transposing Nucleotide sequence and no resistance to antibiotics mediating nucleotide sequence.
  • L-amino acids in addition to the functional expression of one or more of the heterologous genes of glycerol metabolism (glycerol metabolism) selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, one or more metabolic pathway enzymes that increase or decrease the formation of the desired amino acid, such as the biosynthetic pathway, the glycolysis, the anaplerotic, the citric acid cycle, the pentose phosphate cycle, the amino acid export and optionally regulatory proteins either to express, in particular overexpress, or mitigate, in particular to reduce expression.
  • the heterologous genes of glycerol metabolism glycerol metabolism
  • amplification or “amplification” in this context 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, for example by the copy number of the gene or the Gene increases, a strong promoter or a gene or allele used for that corresponding enzyme or protein with a high activity and optionally combines these measures.
  • the activity or concentration of the corresponding protein is generally increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%. , Up to 1000% or 2000% based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism increased.
  • the copy number of the respective genes may be increased, or the promoter and regulatory region or ribosome binding site located upstream of the structural gene may be mutated.
  • expression cassettes act, which are installed upstream of the structural gene.
  • inducible promoters it is additionally possible to increase expression in the course of fermentative amino acid production. Measures to extend the lifetime of m-RNA also improve expression.
  • enzyme activity is also enhanced.
  • the genes or gene constructs may either be present in different copy number plasmids or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by altering the composition of the medium and culture.
  • JP-A-10-229891 to Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), to Makrides (Microbiological Reviews 60: 512-538 (1996)) and to known textbooks of genetics and molecular biology.
  • genes are overexpressed, for example with the aid of episomal plasmids.
  • Suitable plasmids are those which are replicated in coryneform bacteria.
  • Numerous known plasmid vectors e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107 : 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLI or pGAl.
  • Other plasmid vectors e.g.
  • plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum.
  • vectors examples include PSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega Corporation , Madison, WI, USA), pCR2.1-T0P0 (Shuman (1994), Journal of Biological Chemistry 269: 32678-84, US-A 5,487,993), pCR® Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEMI (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342).
  • the plasmid vector containing the gene to be amplified is then converted by conjugation or transformation into the desired strain of C
  • WO03 / 040373 isolates the nucleotide sequence of the desired ORF, gene or allele, optionally including the expression and / or regulatory signals, and two copies, preferably in tandem, into a non-replicative vector for C. glutamicum such as pK18mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174:
  • the vector is then converted by transformation or conjugation into the desired coryneform bacterium. After homologous recombination by means of a first integration-causing cross-over event and a suitable second excision-causing cross-over event in the target gene or in the target sequence to achieve the incorporation of the additional gene copy. Thereafter, those bacteria are isolated in which two copies of the ORF, gene or allele are present at the respective natural location in place of the original single copy present. At the respective natural gene locus, no nucleotide sequence capable of episomal replication in microorganisms remains, no nucleotide sequence capable of transposing it, and no nucleotide sequence conferring resistance to antibiotics.
  • the term "attenuation” in this context describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or by using a gene or allele, which codes for a corresponding enzyme or protein with a low activity or inactivates the corresponding gene or enzyme (protein) and optionally combines these measures.
  • the activity or concentration of the corresponding protein is generally 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, respectively the activity or concentration of the protein in the initial microorganism lowered.
  • the increase or decrease in the protein concentration can be detected by the previously mentioned methods (Hermann et al., Electrophoresis, 22: 1712-23 (2001); Lohaus and Meyer, Biospektrum 5: 32-39 (1998); Lottspeich,
  • endogenous genes refers to the genes or nucleotide sequences present in the population of a species.
  • the heterologous genes of the glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC
  • the genes selected from the group of genes or alleles of lysine production amplified, in particular overexpressed.
  • “Genes or alleles of lysine production are understood as meaning all, preferably endogenous, open reading frames, genes or alleles whose amplification / overexpression can bring about an improvement in lysine production.
  • genes or alleles include, but are not limited to, the following genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh , gnd, lysC, lysC FBR , lysE, msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pycP458S, sigC, sigD, sigE, sigH, sigM,
  • L-lysine in addition to the functional expression of one or more of the heterologous genes of glycerol metabolism (glycerol metabolism), selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, simultaneously one or more of the genes selected from the group of genes or alleles that are not essential for growth or lysine production , attenuates, in particular, turns off or reduces its expression.
  • the heterologous genes of glycerol metabolism glycerol metabolism
  • genes or alleles include, among others, the following open reading frames, genes or alleles: aecD, ccpAl, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysRI, lysR2, lysR3, menE, mqo, pck, pgi, poxB and zwa2, which are summarized and explained in Table 2.
  • Endogenous polynucleotides of Corynebacterium glutamicum (Accession No .: NC 006958 or NC 003450 (sequence of the entire genome)), for example, at least 45% or at least 50% or at least 60% or at least 70% or at least 80% or at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% Identity to the in SEQ ID NO. 1, SEQ ID no. 3, SEQ ID no. 5, SEQ ID no. 7, SEQ ID no. 9, SEQ ID NO. 11, SEQ ID no. 13, SEQ ID no. 15, SEQ ID no. 17, SEQ ID no.
  • SEQ ID no. 21, SEQ ID no. 23, SEQ ID no. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID no. 33 or SEQ ID NO. 35 have nucleotide sequences shown and have the functions described, can be amplified. Those known from genome projects
  • NCBI National Library of Medicine National Database
  • Coryneform bacteria were also found to be common upon overexpression of the endogenous polynucleotides glpK (58% identity to SEQ ID No 15) encoding the
  • the bacteria according to the invention can be used continuously - as described, for example, in PCT / EP2004 / 008882 - or discontinuously in the batch process (batch culturing) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing L-amino acids be cultivated.
  • the culture medium to be used must suitably satisfy the requirements of the respective strain. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology, of the American Society for Bacteriology (Washington, DC, USA, 1981).
  • the carbon source used is glycerol. This can be used singly or as a mixture.
  • Sugar and carbohydrates e.g. Glucose,
  • oils and fats such as soya oil, sunflower oil, peanut oil and coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as ethanol and methanol and organic acids such as acetic acid, wherein the proportion of glycerol at least greater than or equal to ( ⁇ ) 10%, or
  • organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate,
  • Ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used.
  • the nitrogen sources can be used singly or as a mixture.
  • Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • the culture medium must further contain salts of metals, e.g. Magnesium sulfate or iron sulfate necessary for growth.
  • essential growth factors such as amino acids and vitamins can be used in addition to the above-mentioned substances.
  • suitable precursors can be added to the culture medium.
  • the said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.
  • basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used.
  • anti-foaming agents such as fatty acid polyglycol esters can be used.
  • suitable selective substances such as antibiotics may be added to the medium.
  • oxygen or oxygen containing gas mixtures such as air in the culture.
  • the temperature of the culture is normally from 20 0 C to 45 ° C and preferably at 25 ° C to 40 0 C. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes longer cultivation times are possible.

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Abstract

La présente invention concerne un procédé pour produire des acides L-aminés comprenant les étapes suivantes: a) culture des bactéries coryneformes recombinées produisant l'acide L-aminé souhaité, dans lesquelles est/sont exprimé(s) au moins un ou plusieurs des polynucléotides hétérologues du métabolisme de la glycérine (métabolisme du glycérol), choisi(s) dans le groupe comprenant glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa et talC, dans un milieu contenant de la glycérine ou éventuellement en supplément une ou plusieurs autres sources de C, dans des conditions dans lesquelles le milieu ou les cellules s'enrichissent en acide L-aminé souhaité; et éventuellement b) isolation de l'acide L-aminé souhaité, des constituants du milieu de fermentation et/ou de la biomasse pouvant éventuellement subsister en intégralité ou en parties (> 0 à 100%) dans le produit final. Le procédé comprend également l'utilisation de bactéries dans lesquelles ont amplifie en supplément d'autres gènes du processus de biosynthèse de l'acide L-aminé souhaité, ou dans lesquelles les processus de métabolisme qui limitent la formation des acides L-aminés, sont au moins partiellement stoppés.
EP06806854A 2005-10-05 2006-09-27 Procede pour produire des acides l-amines par fermentation au moyen de bacteries coryneformes capables d'utiliser la glycerine comme source exclusive de carbone Withdrawn EP1948811A2 (fr)

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DK11152912.9T DK2354235T3 (en) 2005-10-05 2006-09-27 Method for fermative preparation of L-amino acids using coryneform bacteria
PL11152912T PL2354235T3 (pl) 2005-10-05 2006-09-27 Sposób fermentacyjnego wytwarzania L-aminokwasów z zastosowaniem bakterii maczugowatych

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PL2354235T3 (pl) 2015-04-30
US20080293100A1 (en) 2008-11-27
DK2354235T3 (en) 2015-02-16
DE102005047596A1 (de) 2007-04-12
WO2007039532A3 (fr) 2007-08-02
ES2529107T3 (es) 2015-02-16
WO2007039532A2 (fr) 2007-04-12
EP2354235A1 (fr) 2011-08-10
EP2354235B1 (fr) 2014-11-19
US9150827B2 (en) 2015-10-06

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