WO2003023016A2 - Method for producing fine chemicals - Google Patents

Method for producing fine chemicals Download PDF

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Publication number
WO2003023016A2
WO2003023016A2 PCT/EP2002/010174 EP0210174W WO03023016A2 WO 2003023016 A2 WO2003023016 A2 WO 2003023016A2 EP 0210174 W EP0210174 W EP 0210174W WO 03023016 A2 WO03023016 A2 WO 03023016A2
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Prior art keywords
protein
dehydrogenase
microorganism according
dna
sequence
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PCT/EP2002/010174
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French (fr)
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WO2003023016A3 (en
Inventor
Brigitte Bathe
Eberhard Busker
Daniela Kruse
Mechthild Rieping
Cornel Venzago
Mike Farwick
Thomas Hermann
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Degussa Ag
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Priority to EP02797981A priority Critical patent/EP1425424A2/en
Priority to AU2002362280A priority patent/AU2002362280A1/en
Publication of WO2003023016A2 publication Critical patent/WO2003023016A2/en
Publication of WO2003023016A3 publication Critical patent/WO2003023016A3/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
    • C12N9/93—Ligases (6)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/88—Lyases (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
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to genetically modified microorganisms, in particular coryneform bacteria and Escherichia coli , in which the phosphorylation state of proteins or polypeptides has been altered such that the bacteria produce a larger amount of a desired fine chemical or of a metabolite than the wild type and to a method for producing fine chemicals or metabolites.
  • amino acids and vitamins are produced by fermentation of strains of coryneform bacteria, in particular Coryneba cteri um glutami cum, Enteroba cteria ceae , in particular Escheri chia coli or by using Bacilli , in particular Bacillus subtilis .
  • Bacilli in particular Bacillus subtilis .
  • a multiplicity of other bacteria or fungi is utilized in order to produce fine chemicals or metabolites. Due to the great importance, the production processes are constantly improved.
  • Process improvements can relate to measures regarding technical aspects of the fermentation, such as, for example, stirring and oxygen supply, or to the media composition, such as, for example, sugar concentration during fermentation, or to the work-up to give the product, for example by ion exchange chromatography, or to the intrinsic performance properties of the microorganism itself.
  • organisms can regulate their metabolic pathways very precisely.
  • the phosphorylation state of a protein affects the configuration thereof and "switches" the activity of the protein "on” or "off".
  • This object is achieved by a microorganism, in which the phosphorylatability of at least one protein has been permanently altered such that the biosynthesis of at least one fine chemical synthesized by the microorganism is increased compared to the wild type.
  • An amino acid as mentioned hereinbelow means not only the base but also any of the common salts of this amino acid, such as, for example, lysine monohydrochloride or lysine sulphate.
  • a vitamin as mentioned hereinbelow means any administerable form of the vitamin or of vitamin products.
  • nucleosides, nucleotides, antibiotics, lipids and pigments are used in the usual way but, in this case too, the present invention includes any administerable form of these substances.
  • the present invention includes, where organic acids are mentioned, both the pure acid and any of its salts and any other form of preparation.
  • a microorganism in which the phosphorylation state of at least one protein has been altered in this way compared to the protein naturally occurring in the organism used synthesizes an increased amount of a desired fine chemical.
  • fine chemicals means in the present application all metabolic products of a microorganism used, whose preparation in a pure form or as a mixture is desired.
  • Preferred fine chemicals are amino acids, in particular L-amino acids, preferably L-lysine, L-threonine, L-glutamic acid, L-methionine, L-cysteine, L-cystine, L-tyrosine, L-phenylalanine, L-tryptophan, L-valine, L-arginine, L-leucine, L-serine, L-histidine, L-aspartic acid, L-asparagine, L-glutamine, L-glycine, L-proline, L-homoserine, and L-isoleucine or amino acid mixtures; vitamins, in particular pantothenic acid, riboflavin, vitamin A, vitamin E, thiamine, biotin, folic acid, ascorbic acid, calciferol, phylloquino
  • the microorganisms according to the invention can produce the fine chemicals from various carbon sources.
  • Preferred starting substances here are glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or else acetate, glycerol, lactate or ethanol.
  • They may be representatives of prokaryotes, in particular bacteria (Gram positive or Gram negative) , for example coryneform bacteria, in particular of the genus Corynebacterium or Brevibacterium, or of E. coli , archaebacteria, in particular of the genus Thermus, yeasts, or fungi.
  • the species Corynebacterium glutamicum which is known in the art for its ability to produce L-amino acids.
  • Corynebacterium in particular of the species Corynebacterium glutamicum, are the known wild-type strains
  • Enterobacteriaceae in particular Escherichia coli
  • Enterobacteriaceae are particularly suitable for the synthesis of fine chemicals .
  • suitable L-threonine- producing strains of the genus Escherichia in particular of the species Escherichia coli , are
  • Escherichia coli B-3996 (pMW: : THY)
  • D-pantothenic acid-producing strains of the genus Escherichia in particular of the species Escherichia coli, are:
  • D-pantothenic acid-producing strains of the genus Bacillus in particular of the species Bacillus subtilis, are, inter alia, the strains mentioned in WO 01/21772.
  • Bacillus subtilis strain PA 221 is, inter alia, the strains mentioned in WO 01/21772.
  • Bacillus subtilis strain " PA 365 Bacillus subtilis strain PA 377-2
  • riboflavin-producing strains of the 'genus Bacillus in particular of the species Bacillus subtilis, are provided.
  • Bacillus subtilis B18502 Bacillus subtilis B18502 .
  • Suitable rib ' oflavin-producing microorganisms are Ashbya, in particular the species Ashbya gossypii , Eremothecium ashbyii , Candida flareri and Bacillus subtilis .
  • prokaryotic organisms for example strep.tomycetes " , actinomycetes, Bifidobacterium r Arthrobacter r ' Rhodococcus , and other Gram-positive bacterias such as Bacillus subtilis or Clostridium acetobutylicum or butyricum, or else lactobacilli such as, for example, Lactobacillus lactis , brevis r ca ' sii fermentum, bulgaricus, Lactococcus lactis or La ctobacillus acidophilus .
  • Gram-negative bacterias are Xanthomonas, Acetobacter , Pseudomonas , X.
  • campestris r P. fluorescens A. acetii or acidophilum, further Klebsiella , Enterobacter , Salmonella r Shigella , Proteus , Serra tia and Erwinia .
  • eukaryotic cells for producing desired fine chemicals .
  • all culturable eukaryotic cells capable of producing a metabolic product desired according to the invention may be used, for example Ascomycetae , Basidiomycetae, Phycomycetae and Deuteromycetae .
  • Ascomycetae are Neurospora , Sa ccharomyces r Trichoderma , Ashbya r Pichia , Hansenula , Candida , Yarrowia and Kluyveromyces .
  • Examples for Phycomyces are Zygomycetae , Oomycetae and Mucoralae, particularly Blakeslea trispora r Phycomyces blakesleanus , Rhizopus nigricans and Rhizopus oryzae .
  • Examples for Deuteromycetae are Penicillium r
  • Aspergillus and Candida particularly Aspergillus niger , oryzae, fumiga tus r flavus or wentii , Penicillium nota tum or chrysogenum and Candida flareri .
  • Particularly suitable are for example , Saccharomyces cerevisiae, Schizosaccharomyces pombe , Hansenula polymorpha , Candida bondii , Kluyveromyces lactis , Schwanniomyces occidentalis or Pichia pastoris r filamentous fungi such as Aspergillus niger, Aspergillus nidulans , Ashbya gossipii , Yarrowia lipolytica or Neurospora crassa , insect cell systems such as , for example, the baculovirus system and common cell lines of mammalian cells such as , for example, HeLa cells , COS cells or CHO cells and also algae .
  • the amount of a desired fine chemical produced is increased by altering the phosphorylation state of at least one protein of the microorganism, compared with the naturally occurring protein of the microorganism. Therefore, a microorganism of the present invention is distinguished by the fact that it contains at least one protein whose amino acid sequence differs in at least one amino acid from that of the same protein occurring naturally in this organism, it being possible for the amino acid concerned to serve as a phosphorylation site in the protein.
  • the difference to the wild-type sequence may be a mutation, in particular an amino acid exchange, or a deletion or insertion of at least one amino acid.
  • the exchange, deletion or insertion of an amino acid can remove a phosphorylation site present in the naturally occurring protein, introduce an additional phosphorylation site compared with the naturally occurring protein or permanently change the phosphorylation state of a phosphorylation site (for example, an existing phosphorylation site is permanently phosphorylated or permanently unphosphorylated) .
  • one or more amino acids which occur in the natural protein and which serve as a phosphorylation site in the protein can be exchanged for another amino acid so that the phosphorylation state which serves to regulate the protein is permanently changed. It is also possible to introduce additional phosphorylation sites into an enzyme by exchanging one or more amino acids.
  • the phosphorylation state can be changed in any manner which leads to such a change, and the phosphorylation state is changed preferably by mutagenesis of the protein-encoding DNA sequence, particularly preferably by site-directed mutagenesis of the protein-encoding DNA sequence.
  • amino acid exchanges are suitable:
  • Exchanges which remove a phosphorylation site Exchange of serine, threonine, tyrosine, aspartate, glutamate, histidine, arginine or lysine for another amino acid, meaning an exchange for any other amino acid (i.e. also for any amino acid different from those mentioned here) .
  • a particularly preferred exchange is the exchange of serine or threonine for asparagine, glutamine, glycine, alanine, cysteine, valine, methionine, isoleucine, leucin, phenylalanine, histidine, lysine, tryptophan or arginine.
  • Phosphorylation sequence herein refers to the amino acid sequence of the protein, which surrounds the amino acid which 'is reversibly phosphorylated in the wild type. Furthermore this phosphorylation sequence can comprise an amino acid which exchange changes the three dimensional structure of the protein in a way that an amino acid which can reversibly be phosphorylated in the wild type can not be phosphorylated accordingly in the mutant. These amino acid sequences are known to the skilled worker and serve to "recognize” the amino acid to be phosphorylated. Any exchange of any of the amino acids in this sequence, which results in an amino acid being no longer reversibly phosphorylatable/ dephosphorylatable as in the wild type, is to be regarded as being included in the present invention.
  • amino acids occurring in a natural protein are known to the skilled worker. According to the invention, all naturally occurring amino acids can be exchanged for one another, including the posttranslationally modified amino acids.
  • the amino acid(s) is/are located in the protein in an amino acid sequence which renders the amino acid in question accessible to a reversible phosphorylation.
  • amino acid sequences are known to the skilled worker.
  • the amino acid(s) to be exchanged may be located in the region of an active side of a protein, in particular of an enzyme, or may be located outside the active site in a region whose phosphorylation state is instrumental in controlling the activity of the protein or its affinity to other proteins .
  • the proteins whose phosphorylation state is changed according to the present invention may be enzymes, regulatory proteins, structural proteins, transport proteins, storage proteins, proteins having an immune function or components of signal transduction pathways.
  • the target protein used for a change according to the invention of the phosphorylation state may be any protein in which a change of the phosphorylation state causes an increase in the rate of synthesis of a desired fine chemical or else decreases the rate of synthesis of unwanted metabolic products.
  • proteins which are to be regarded as being within the scope of the invention are:
  • proteins of signal recognition and signal transduction including two component system(s) .
  • Nitrate Reductase Nadph
  • Nitrite Reductase Nad(P)H
  • Beta-Glucosidase 3.2.1.22 Alpha-Galactosidase
  • proteins which are involved in cell integrity and thereby maintain and/or modify the biocatalyst particular mention must be made of the proteins of DNA replication, DNA repair, transcription, translation, cell division and of the extracellular matrix.
  • DNA and RNA polymerases DNA and RNA helicases, DNA and RNA single strand-binding proteins, DNA and RNA ligases, topoisomerases and gyrases and also recombinases .
  • translation factors such as elongation factors, for example EF-Tu, EF-Ts, EF-G, initiation factors, release factors and also ribosomal proteins, tRNA synthetases, chaperones and chaperonins and also components of the extracellular matrix such as, for example, the antigen 84.
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • G proteins MAP kinases, tyrosine kinases, Janus kinases, phospholipase A, phospholipase C, protein kinase C,. and also calcium-dependent systems such as calcium-calmodulin kinases (CAM kinases) .
  • transcription factors with basic domains such as, for example, leucine-zipper factors (bZIP) , helix-loop-helix factors (bHLH) , helix-loop- helix/leucine-zipper factors (bHLH-ZIP) , NF-1 type factors, RF-X type factors, bHSH type factors.
  • Transcription factors with zinc-finger domains such as, for example, Cys 4 zinc finger, Cys2His2 zinc finger or Cys6 cysteine-zinc cluster.
  • Transcription factors with helix-turn-helix motifs such as, for example, homeo-box proteins, heat-shock factors or the tryptophan-cluster family.
  • 1 sheet structures such as, for example, the factors of the p53 family, MADS-box proteins or TATA-binding proteins .
  • proteins having storage function particular mention must be made of the systems for the synthesis of polyphosphates and storage carbohydrates such as glycogen or starch, likewise storage proteins for inorganic ions such as, for example, iron.
  • Proteins having transport function are ABC transporters, primary, secondary and binding protein- dependent transport systems and proteins facilitating diffusion. Examples are the bacterial maltose/trehalose binding proteins (malE) , multidrug-resistance proteins, components of the PTS systems for sugar uptake (HPr, enzyme I, II and III) and transport proteins secreting the components of the extracellular matrix. In addition to this, all proteins involved in the uptake or secretion of proteins and peptides.
  • Proteins involved in cellular biogenesis Proteins involved in the nitrogen metabolism, such as, for example, uridilyl transferase, or else proteins involved in homeostasis of ions.
  • Proteins which may serve as target proteins and proteins which have an altered phosphorylation pattern compared to the wild type can be found or detected by any of the methods described in the literature. Methods suitable for this purpose are those described in
  • the invention comprises the use of microorganisms in which the phosphorylation state of at least one protein has been permanently changed compared to that of the protein naturally occurring in this organism for the preparation of a fine chemical.
  • microorganisms in particular coryneform bacteria and E. coli , produce, after modification of the phosphorylation state of proteins, amino acids, in particular L-lysine and L-threonine, and vitamins, in particular pantothenic acid, in an improved manner.
  • the proteins in question whose phosphorylatability has been changed, can be introduced into the target cells by introducing plasmids which carry the protein- encoding genes or gene constructs containing the desired amino acid exchange. After the gene sequences have been introduced, they may be located in the cell either still on the plasmids or integrated into the chromosome of the microorganism.
  • Suitable plasmids are those which are replicated and expressed in the selected microorganism.
  • numerous known plasmid vectors can be used, such as, for example, pZl (Menkel et al . ,
  • plasmid vectors such as, for example, those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAGl (US-A 5, 158 , 891) may be used for coryneform bacteria in the same way.
  • Plasmid vectors which can be replicated in Enterobacteriaceae such as, for example, 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 Science USA 80 (21): 6557-6561 (1983)), may be used. It is likewise possible to transfer mutations which relate to expression of the particular genes to various strains by sequence exchange (Hamilton et al . (Journal of Bacteriology 174, 4617-4622 (1989)), conjugation or transduction.
  • the microorganisms prepared according to the invention may be cultured continuously or batchwise or in a fed batch or repeated fed batch process for the purpose of producing the fine chemicals, in particular amino acids, nucleosides, nucleotides, pigments, antibiotics or vitamins.
  • various suitable culture conditions are available.
  • the cultivation method used is not limiting to the invention.
  • a review of known culturing methods is described in the text book by Chmiel (BioprozeBtechnik 1. Einf ⁇ hrung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the text book by Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Brunswick/Wiesbaden, 1994) ) .
  • the culture medium to be used must satisfy the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms can be found in the manual "Manual of Methods for General Bacteriology” of the American Socity for Bacteriology (Washington D.C., USA, 1981).
  • Carbon sources which may be used are sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol and ethanol and organic acids such as, for example, acetic acid or amino acids such as glutamine. These substances may be used individually or as a mixture. When using photosynthetic bacteria, light may be used as energy source.
  • oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and coconut oil
  • fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid
  • alcohols such as, for example, glycerol and ethanol
  • organic acids such as, for example, acetic acid or amino acids such as glutamine
  • Nitrogen sources which may be used are 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 sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen sources may be used individually or as a mixture.
  • Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts.
  • the culture medium must contain metal salts such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth.
  • suitable precursors can be added to the culture medium.
  • the said starting materials can be added to the culture in the form of a single batch or be fed in during the cultivation in a suitable manner.
  • the pH of the culture is controlled by employing basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner.
  • Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters .
  • suitable substances having a selective effect for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, air into the culture.
  • the temperature of the culture is normally 20 °C to 45 °C and preferably 25°C to 40°C.
  • the fine chemicals produced can be analysed, for example, by anion exchange chromatography with subsequent ninhydrin derivatization, as described in Spackman et al . (Analytical Chemistry, 30, (1958), 1190) .
  • the method of the invention serves to produce, by way of fermentation, fine chemicals, in particular amino acids, nucleosides, nucleotides and vitamins.
  • fine chemicals in particular amino acids, nucleosides, nucleotides and vitamins.
  • particular preference is given to L-lysine, L-threonine, L-methionine, L-tryptophan and pantothenic acid.
  • the membrane was dried. This was followed by incubating the membrane in a blocking solution made of 1% bovine serum albumin (fraction V, Sigma, Deisenhofen, D) , 1% polyvinylpyrrolidone (Sigma, Deisenhofen, D) , 1% PEG 3350 (Sigma, Deisenhofen, D) and 0.2% Tween 20 (Sigma, Deisenhofen, D) in PBS buffer.
  • PBS buffer consisted of 274 mM NaCl, 5.4 mM KCl, 20 mM Na 2 HP0 4 and 3.6 mM KH 2 P0 4 at pH 7.2.
  • the membrane was then incubated in 12.5 ml of a solution of the primary antibody for 1 hour.
  • the primary antibody is directed against phosphorylated amino acids, in particular phosphothreonine, phosphoserine and phosphotyrosine .
  • the following antibodies were used: monoclonal anti-phosphoserine antibodies clones 1C8, 4A3, 4A9, 4H4, 7F12 and 16B4; monoclonal anti- phosphothreonine antibodies, clones 14B3, 1E11 and 4D11; monoclonal anti-phosphotyrosine antibodies 4G10 and 3B12 (Biomol, Hamburg, D) .
  • the membrane was incubated by dissolving 12.5 ⁇ g of the particular primary antibody in 1 ml of double-distilled water and adding 12.5 ml of blocking solution. The membrane was then washed five times for five minutes each in a washing solution which consisted of 10 mM Tris (Sigma, Deisenhofen, D) , 150 mM NaCl and 0.1% (v/v) Tween 20
  • the membrane was treated with 10 ml of detecting solution until a purple stain indicates the protein spots or a positive control.
  • the detecting solution used for this purpose consisted of SigmaFast BCIP/NBT buffered substrate tablets (Sigma, Deisenhofen, D) of which one was dissolved in each case in 10 ml of double-distilled water. The membrane was stained and then washed with double-distilled water, dried and stored protected from light. Purple-stained protein spots indicate phosphorylated proteins. Using molecular-weight standards and protein patterns, the spot pattern on the membranes was compared and aligned with that of Coomassie-stained two-dimensional gels
  • any other method may be employed for detecting phosphorylated proteins. Especially important is labelling of the proteins with 32 P or 33 P, for which radioactive 32 P0 4 3_ or 33 P0 4 3 ⁇ is added to growing cells. Phosphorylated proteins are then identified with the aid of autoradiography (Gooley and Packer, in: Proteome Research: New Frontiers in Functional Genomics, Springer Verlag, Berlin, 65-92, 1997) .
  • the proteins to be studied may also be isolated using any other separation method, for example capillary electrophoresis (Liu et al . , Journal of Chromatography A, 918 (2), 401-409, 2001), all types of chromatographic separation methods including thin-layer chromatography, or else by applying molecular biological methods such as, for example, His tagging.
  • capillary electrophoresis Liu et al . , Journal of Chromatography A, 918 (2), 401-409, 2001
  • all types of chromatographic separation methods including thin-layer chromatography
  • molecular biological methods such as, for example, His tagging.
  • the phosphorylated proteins were identified as described in Hermann et al . (Electrophoresis 22, 1712-1723, 2001) by excising the spots from Coomassie- stained gels, digesting the proteins with specific proteases and subsequently identifying the peptide by means of MALDI or electrospray mass spectrometry.
  • the DNA and protein sequences of suitable microorganisms which can produce the desired compounds can be found in several data bases, for example in the NCBI data base (National Center For Biotechnology Information) .
  • the data base can be found in the National Liabory of Medicine, Building 38A, Room 8N 805, Bethesda, MD 20894 USA
  • Table 1 shows some of the identified posphorylated proteins of Corynebacterium glutamicum .
  • Table 2 shows some of the identified phosphorylated proteins of Escherichia coli .
  • Phosphorylation sites may be identified in pure proteins, polypeptides or protein mixtures as described in Neubauer and Mann (Analytical Chemistry, 71(1), 235-242, 1999), Yan et al . (Journal of Chromatography A, 808 (1-2), 23-41, 1998), Oda et al. (Proceedings of the National Academy of Sciences of the USA, 96, 6591-6596, 1999), Wind et al. (Anal. Chem. 73, 29-35, 2001) , Ahn and Resing (Nature Biotechnology 19, 317-318, 2001), or Quadroni and James (in: Proteomics in Functional Genomics, irkhauser Verlag, Basle, Switzerland, P. Jolles and H.
  • the protein enolase can be identified as a phosphorylated protein.
  • the serine residue in position 330 represents the amino acid on which phosphorylation takes place.
  • the Corynebacterium glutamicum strain ATCC13032 is used as donor for the chromosomal DNA. Chromosomal DNA is isolated from the strain ATCC13032 by the usual methods
  • a DNA fragment carrying the eno gene is amplified with the aid of the polymerase chain reaction.
  • SEQ ID No. 1 Genenoic acid sequence (SEQ ID No. 1) (Accession number AX136862)
  • the following primer oligonucleotides are selected for the PCR:
  • the primers depicted are synthesized by MWG Biotech and the PCR reaction is carried out according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) .
  • the primers make it possible to amplify an approx. 1.3 kb DNA fragment carrying the eno gene.
  • the primers contain the sequences of cleavage sites of the restriction endonucleases BamHI (6H-enol) and, respectively, Xmal and Bglll (6H-eno2) , which are indicated by parentheses in the nucleotide sequence depicted above.
  • the amplified DNA fragment of approx. 1.3 kb, which carries the eno gene, is identified via gel electrophoresis in a 0.8% strength agarose gel and purified by the usual methods (High Pure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim) .
  • the vector pQE-30 (Qiagen, Hilden, Germany) was completely cleaved with the enzymes BamHI and Xmal and the 1.3 kb DNA fragment containing the eno gene (Accession number M89931) (Rossol and Piihler, Journal of Bacteriology 174 (9), 2968-2977 (1992)) of C. glutamicum ATCC 13032 was completely cleaved by the enzymes BamHI and Xmal.
  • the etio-carrying fragment was ligated into the vector with the aid of T4 DNA ligase.
  • the DNA was transformed into the E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)).
  • the transformants were selected on LB medium containing 100 mg/1 ampicillin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel. The DNA sequence of the amplified DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 3. The plasmid obtained is denoted pQE-30eno.
  • Site-directed mutagenesis is carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, USA) . Owing to the phosphorylation site of C. glutamicum enolase, serine at position 330, which is known from Example 2, the following primer oligonucleotides are selected for linear amplification: S330E-1 ( SEQ ID No . 13 ) :
  • the primers depicted are synthesized by MWG Biotech.
  • the codon for glutamate which is intended to replace serine at position 330 is indicated by parentheses in the nucleotide sequence depicted above.
  • the plasmid pQE-30eno described in Example 3.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase.
  • This primer extension generates a mutated plasmid with nicked circular strands.
  • the product of the linear amplification is treated with Dpnl; this endonuclease specifically cuts the methylated and semi-methylated template DNA.
  • the newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XL1 Blue cells repair the nicks in the mutated plasmid. The transformants were selected on LB medium containing 100 mg/1 ampicillin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel. The DNA sequence of the mutated DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 4. The plasmid obtained is denoted pQE-30enoS330E .
  • the plasmid pQE-30enoS330E, described in Example 3.2. is cut with the restriction enzymes EcoRI and Hindlll (Gibco Life Technologies GmbH, Düsseldorf, Germany) and, after fractionation in an agarose gel (0.8%), an approx. 0.8 kb eno fragment carrying the mutation is isolated from the agarose gel with the aid of the High Pure PCR-product purification kit (Roche, Mannheim, Germany) and used for ligation with the mobilizable cloning vector pKl ⁇ mobsacB described in Schafer et al., Gene, 14, 69-73 (1994).
  • the said cloning vector is likewise cleaved with the restriction enzymes EcoRI and Hindlll beforehand, mixed with the approx. 0.8 kb eno fragment and treated with T4 DNA ligase (Amersham- Pharmacia, Freiburg, Germany) .
  • This is followed by transforming the E . coli strain DH5 (Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) with the ligation mixture (Hanahan, in DNA cloning. A practical approach. Vol. 1, ILR Press, Cold Spring Harbor, New York, 1989) .
  • Cells containing the plasmid are selected by plating out the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor, New York, 1989) supplemented with 25 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the High Pure plasmid isolation kit from Roche and checked by restriction cleavage with the enzymes Hindlll/EcoRI and subsequent agarose gel electrophoresis.
  • the plasmid is denoted pKl8mobsacBenoS330E.
  • Figure 1 depicts a map of the plasmid.
  • the vector pK18mobsacBenoS330E was transferred into the C. glutamicum strain DSM5715 by means of electroporation (Haynes 1989, FEMS Microbiology Letters 61: 329-334). The vector cannot replicate autonomously in DSM5715 and is retained in the cell only when integrated into the chromosome.
  • Clones with integrated pK18mobsacBenoS330E are selected by plating out the electroporation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd Ed. , Cold Spring Harbor, New York, 1989) supplemented with 15 mg/1 kanamycin.
  • plasmid pK18mobsacB contains, in addition to the kanamycin-resistance gene, a copy of the sacB gene coding for Bacillus subtilis levan sucrase.
  • Sucrose- inducible expression results in the formation of levan sucrase which catalyses synthesis of the product levan which is toxic to C. glutamicum . Therefore, only those clones in which the integrated pK18mobsacBenoS330E has again been excised grow on sucrose-containing LB agar. During the excision, either the allele is exchanged and, respectively, the mutation incorporated or the original copy remains in the host chromosome, depending on the location of the second recombination event with respect to the site of mutation. Approximately 40 colonies are tested for the phenotype "growth in the presence of sucrose" and "no growth in the presence of kanamycin".
  • the LightCycler is a combination of a thermocycler and a fluorimeter.
  • LC-eno2 (SEQ ID No. 10) : 5' ACCAGTCTTGATCTGGCCAC 3'
  • the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes
  • PCR primers depicted are synthesized by MWG Biotech and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
  • the C. glutamicum strain • DSM5715enoS330E obtained in Example 3 was cultured in a medium suitable for the production of L-lysine and the L-lysine content in the culture supernatant was determined.
  • the strain was first incubated on an agar plate (brain-heart agar) at 33°C for 24 hours.
  • a preculture was inoculated (10 ml of medium in a 100 ml Erlenmeyer flask) .
  • the medium used for the preculture was the Cglll complete medium.
  • the pH was adjusted to pH 7.4.
  • the preculture was incubated on a shaker at 33 °C and 250 rpm for 24 hours.
  • a main culture was inoculated with this preculture so that the initial OD (660 nm) of the main culture was 0.1 OD.
  • MM medium was used for the main culture.
  • CSL, MOPS and the salt solution are adjusted to pH 7 with aqueous ammonia and autoclaved. Then the sterile substrate solutions and vitamin solutions and the dry- autoclaved CaC0 3 are added.
  • the cultivation is carried out in a volume of 10 ml in a 100 ml Erlenmeyer flask with baffles. The cultivation was carried out at 33 °C and 80% humidity.
  • a fragment which contains the phosphorylation site S113 of the icd gene from Escherichia coli K12 is amplified by using the polymerase chain reaction (PCR) and synthetic oligonucleotides .
  • PCR polymerase chain reaction
  • SEQ ID No. 15 the sequence known for Escherichia coli
  • primer oligonucleotides MWG Biotech, Ebersberg, Germany
  • icdA (SEQ ID No. 17) : 5 ⁇ AGA ACG TTG CGA GCT GAA TC 3 ⁇
  • the chromosomal DNA of E. coli K12 which is used for PCR is isolated by using counselQiagen Genomic-tips 100/G" (Qiagen, Hilden, Germany) according to the instructions of the producer.
  • Creagen By the specific primers an about 1.2 kbp DNA fragment can be isolated under standard conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) using Vent polymerase (New England Biolabs GmbH, Frankfurt, Germany) which contains the phosphorylation site S113 in the middle area.
  • the amplified DNA fragment is identified via gel electrophoresis in a 0.8% strength agarose gel and purified by the usual methods (High Pure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim) .
  • the cleaned PCR product is ligated with the vector pCR- Blunt II-TOPO (Zero Blunt TOPO PCR Cloning Kit, Invitrogen, Groningen, Niedermé) according to the instructions of the producer and transformed into the E. coli strain TOP10F (Invitrogen, Groningen, Netherlands) .
  • the selection of plasmid containing cells is carried out on LB agar containing 50 mg/1 kanamycin.
  • the vector is checked by means of restriction cleavage and identified in an agarose gel (0,8%) .
  • the DNA sequence of the amplified DNA fragment is checked by sequencing.
  • the sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 19.
  • the plasmid obtained is denoted pCRBlunt-icdSDM.
  • S113A-1 (SEQ ID No. 20) : 5'TTGGTGGCGGTATTCGC(GCT)CTGAACGTTGCCCTG 3'
  • the primers depicted are synthesized by MWG Biotech.
  • the codon for alanine which is intended to replace serine at position 113 is indicated by parentheses in the nucleotide sequence depicted above.
  • the plasmid pCRBlunt-icdSDM, described in Example 5.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase. This primer extension generates a mutated plasmid with nicked circular strands.
  • the product of the linear amplification is treated with Dpnl; this endonuclease specifically cuts the methylated and semi-methylated template DNA.
  • the newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XLl Blue cells repair the nicks in the mutated plasmid.
  • the transformants were selected on LB medium containing 50mg/l kanamycine .
  • the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel.
  • the DNA sequence of the mutated DNA fragment is checked by sequencing.
  • the sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 22.
  • the plasmid obtained is denoted pCRBlunt-icdS113A.
  • the plasmid pCRBlunt-icdS113A, described in Example 5.2. is cut with the restriction enzymes BamHI and Xbal (Gibco Life Technologies GmbH, Düsseldorf, Germany). After fractionation in an agarose gel (0.8%), an approx. 1.3 kb icd fragment carrying the mutation is isolated from the agarose gel with the aid of the High Pure PCR-product purification kit (Roche, Mannheim, Germany) and used for ligation with the vector pMAK705 described in Hamilton et al., Journal of Bacteriology 171, 4617 - 4622 (1989) .
  • the said cloning vector is likewise cleaved with the restriction enzymes BamHI and Xbal beforehand, mixed with the isolated icd fragment and treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) .
  • This is followed by transforming the E . coli strain DH5 ⁇ (Grant et al . , Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) with the ligation mixture (Hanahan, in DNA cloning. A practical approach. Vol. 1, ILR Press, Cold Spring Harbor, New York, 1989) .
  • Cells containing the plasmid are selected by plating out the transformation mixture on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor, New York, 1989) supplemented with 20 mg/1 chloramphenicol.
  • Plasmid DNA is isolated from a transformant with the aid of the High Pure plasmid isolation kit from Roche and checked by restriction cleavage with the enzymes BamHI, Xbal and Sail and subsequent agarose gel electrophoresis.
  • the plasmid is denoted pMAK705icdSH3A.
  • Figure 2 depicts a map of the plasmid.
  • the L-threonine producing E. coli strain MG442 is described in the patent US-A 4,278,765 and deposited as CMIM B-1628 at the Russian National Collection for industrial microorganisms (VKPM, Moskow, Russia) .
  • MG442 For the. site-specific mutagenesis in the icd gene MG442 is transformed with the plasmid pMAK705icdS113A.
  • the exchange of the gene is carried out with the selection method described by Hamilton et al. (1989) Journal of Bacteriology 171, 4617 - 4622) and is studied with the aid of the LightCycler from Roche Diagnostics (Mannheim, Germany) in order to prove the mutation of the icdS113A allele in the chromosome.
  • the LightCycler is a combination of a thermocycler and a fluorimeter.
  • icd_LCPCRl (SEQ ID No.23): 5 ' GCCTATAAAGGCGAGCGTAA 3'
  • icd_LCPCR2 (SEQ ID No.24): 5 'ACCCGCATAAATGTCTTCCG 3'
  • the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes
  • PCR primers depicted are synthesized by MWG Biotech and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
  • MG442icdS113A is grown on a minimal medium with the following composition: 3,5 g/1 Na 2 HP0 4 *2H 2 0, 1,5 g/1 KH 2 P0 4 , 1 g/1 NH 4 C1, 0,1 g/1 MgS0 4 *7H 2 0, 2 g/1 glucose, 20 g/1 Agar.
  • the production of L-theronine is checked in batch cultures of 10 ml contained in 100 ml Erlenmeyer flasks.
  • 250 ⁇ l of this starter culture are transferred into 10 ml of production medium (25 g/1 (NH 4 ) 2 S0 4 , 2 g/1 KH 2 P0 4 , 1 g/1 MgS0 4 *7H 2 0, 0,03 g/1 FeS0 4 *7H 2 0, 0,018 g/1 MnS0 4 *lH 2 0, 30 g/1 CaC0 3 , 20 g/1 glucose) and incubated for 48 hours at 37 °C. After incubation the optical density (OD) of the culture suspension is determined by using a LP2W photometer of Dr. Lange company (D ⁇ sseldorf, Germany) at a wavelength of 660 nm.
  • OD optical density
  • the concentration of produced L-threonine is determined in the sterile filtered culture supernatant by means of a amino acid analysator of Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and after column reaction with ninhydrin detection.
  • the genes sucC and sucD of E. coli K12 are amplified by using the polymerase chain reac ⁇ tion (PCR) and synthetic oligonucleotides.
  • PCR polymerase chain reac ⁇ tion
  • the sequences of the primers are modified in a manner that recognition sites for restriction enzymes are formed.
  • sucCDl-primer For the sucCDl-primer the recognition site for Xbal and for the sucCD2-primer the recognition site for Hindlll is selected, which both are indicated in the following nucleic acid sequence by parenthesis: sucCDl: 5 ⁇ - GGA (TCTAGA) CGATTACTGAAGGATGGACAGAAC - 3 ⁇ (SEQ ID No. 34)
  • sucCD2 5 ⁇ - GAG (AAGCTT) GGCGAGGGCTATTTCTTATTAC - 3 (SEQ ID No. 35)
  • the chromosomal DNA of E. coli K12 MG1655 which is used for PCR is isolated by using counselQiagen Genomic-tips 100/G" (Qiagen, Hilden, Germany) according to the instructions of the producer.
  • Creagen agen, Hilden, Germany
  • an about 2100 bp DNA fragment can be amplified under standard conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) using Pfu DNA polymerase (Promega Corporation, Madison, USA) .
  • the PCR product is cleaved by the restriction enzymes Xbal and Hindlll and and ligated with the vector pTrc99A (Pharmacia Biotech, Uppsala, Schweden) , which as well has been cut by the enzymes Xbal and Hindlll.
  • the E. coli strain MRF N (Stratagene, La Jolla, USA) is transformed with the ligation mixture and cells containing the plasmid are selected on LB agar supplemented with 50 ⁇ g/ml ampicillin. The successful cloning can be proved after plasmid DNA isolation by control cleavage with the enzymes EcoRV, Hpal and Sspl.
  • the plasmid is denoted as pTrc99AsucCD.
  • sucC-E-197-Q-l 5 ⁇ - GACCTGGCGTTGATC (CAA) ATCAACCCGCTG - 3 (SEQ ID No. 36)
  • sucC-E-197-Q-2 5 - CAGCGGGTTGAT (TTG) GATCAACGCCAGGTC - 3 ⁇ (SEQ ID No. 37)
  • the primers depicted are synthesized by MWG Biotech (Ebersberg, Germany).
  • the codon for glutamine which is intended to replace glutamic acid at position 197 of the beta subunit of the succinyl-CoA Synthetase is indicated by parentheses in the nucleotide sequence depicted above.
  • the .plasmid pTrc99AsucCD, described in Example 7.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase. This primer extension generates a mutated plasmid with nicked circular strands.
  • the product of the linear amplification is treated with Dpnl.
  • This endonuclease specifically -cuts the methylated and semi-methylated template DNA.
  • the newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XLl Blue cells repair the nicks in the mutated plasmid. The transformants were selected on LB medium containing 50 mg/1 kanamycin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel (0,8%) . The introduced mutation can be proved with the aid of the LightCycler technology from Roche Diagnostics (Mannheim, Germany) .
  • the LightCycler is a combination of a thermocycler and a fluorimeter. In the first phase, an approx. 0.5 kb DNA section containing the site of mutation is amplified by means of PCR
  • sucC-197-1 5 ⁇ - GGCAAGCGTCTGGTA - 3 ⁇ (SEQ ID No. 38]
  • sucC-197-2 5 - CTTCCTGCGACTGGT - 3 ⁇ (SEQ ID No. 39)
  • the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes (LightCycler (LC)-Red640 and fluorescein) and which hyrbidize around the site of mutation, with the aid of the fluorescence resonance energy transfer (FRET) method (Lay et al., Clinical Chemistry, 43: 2262-2267 (1997) ) .
  • fluorescent dyes LightCycler (LC)-Red640 and fluorescein
  • FRET fluorescence resonance energy transfer
  • Probe-197-1 5 - GGCGTTGATCCAAATCAACCC - Fluorescein - 3 ⁇ (SEQ ID No. 40)
  • Probe-197-2 5 ⁇ - LC Red640-CTGGTCATCACCAAACAGGG p (SEQ ID No. 41)
  • PCR primers depicted are synthesized by MWG Biotech (Ebersberg, Germany) and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
  • the plasmid is denoted pTrc99A-sucCD-El97Q.
  • the linearized plasmid is cleaved with Xbal.
  • the about 2100bp sucCD-E208Q fragment is isolated from the agarose gel by aid of QIAquick Gel Extraction Kit
  • the sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 49.
  • the sucD PCR fragment codes at positions 408 - 410 for the base trplet cytosine-adenine-guanine coding for the amino acid glutamine.
  • the plasmid obtained is denoted pCVD442sucCD-E208Q (figure 4) .
  • FIG. 1 Map of plasmid pK18mobsacBenoS330E
  • FIG. 1 Map of plasmid pMAK705icdS113A
  • FIG. 4 Map of plasmid pCVD442sucCD-E208Q
  • icdS113A mutated icd allele
  • KanR Kanamycin-resistance gene
  • sacB Levansucrase, sacB gene
  • sucC Succinyl-CoA Synthetase beta subunit, sucC gene
  • sucC E197Q mutated sucC allele
  • sucD Succinyl-CoA Synthetase alpha subunit, sucD gene
  • sucDE208Q mutated sucD allele
  • RP4mob mob region with origin of replication for transfer (oriT)
  • oriR ⁇ K origin of replication, dependend of pir gene

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Abstract

The present invention relates to genetically modified microorganisms, in particular coryneform bacteria and Escherichia coli, in which the phosphorylation state of proteins or polypeptides has been changed such that the bacteria produce a larger amount of a desired fine chemical or a metabolite than the wild type, and to a method for the production of fine chemicals or metabolites.

Description

Method for producing fine chemicals
The present invention relates to genetically modified microorganisms, in particular coryneform bacteria and Escherichia coli , in which the phosphorylation state of proteins or polypeptides has been altered such that the bacteria produce a larger amount of a desired fine chemical or of a metabolite than the wild type and to a method for producing fine chemicals or metabolites.
In recent years, there has been a distinct increase in the need of fine chemicals for a wide range of application areas, and demands on the quality and purity of the substances are very high. For example, amino acids and vitamins are applied in human medicine, in the pharmaceutical industry, in the food industry, but in particular in animal feeding. Therefore, there is a general interest in providing novel improved methods for producing amino acids, vitamins or other fine chemicals.
It is known that amino acids and vitamins are produced by fermentation of strains of coryneform bacteria, in particular Coryneba cteri um glutami cum, Enteroba cteria ceae , in particular Escheri chia coli or by using Bacilli , in particular Bacillus subtilis . In addition to this, a multiplicity of other bacteria or fungi is utilized in order to produce fine chemicals or metabolites. Due to the great importance, the production processes are constantly improved. Process improvements can relate to measures regarding technical aspects of the fermentation, such as, for example, stirring and oxygen supply, or to the media composition, such as, for example, sugar concentration during fermentation, or to the work-up to give the product, for example by ion exchange chromatography, or to the intrinsic performance properties of the microorganism itself.
In order to improve the performance properties of these microorganisms, methods of mutagenesis, selection and selection of mutants are applied. Moreover, methods of recombinant DNA technology have been employed for some years in order to improve strains of amino acid- producing Corynebacterium strains by amplifying individual genes of amino-acid biosynthesis and studying the effect on amino-acid production. Review articles on this matter can be found, inter alia, in Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44, 1991), Eggeling (Amino Acids 6:261-272, 1994), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103, 1995) and Sahm et al . (Annuals of the New York Academy of Science 782, 25-39, 1996) .
In order to meet their own needs of various substances exactly, organisms can regulate their metabolic pathways very precisely. To this end, there is on the one hand the possibility of controlling, for example, the amount of the enzymes of a synthetic pathway which are present in the cell by regulating protein synthesis (long-term control) and, on the other hand, the activity of a large variety of proteins such as, for example, many enzymes of a synthetic pathway can be controlled via their phosphorylation states (short-term control) . The phosphorylation state of a protein affects the configuration thereof and "switches" the activity of the protein "on" or "off". Regulatory proteins, too, can change their secondary, tertiary or quaternary structure and thus their affinity to the protein (s) they control by changing the phosphorylation state. As a result, the individual processes within cells can be coordinated very accurately (Proteomics - Posttranslational modifications, Vol. 1, No. 2, pages 167-364 (2001)).
It was the object of the present invention to provide novel auxiliary means for the improved fermentative production of fine chemicals, in particular amino acids, nucleosides, nucleotides and vitamins.
This object is achieved by a microorganism, in which the phosphorylatability of at least one protein has been permanently altered such that the biosynthesis of at least one fine chemical synthesized by the microorganism is increased compared to the wild type.
An amino acid as mentioned hereinbelow means not only the base but also any of the common salts of this amino acid, such as, for example, lysine monohydrochloride or lysine sulphate. A vitamin as mentioned hereinbelow means any administerable form of the vitamin or of vitamin products. The terms nucleosides, nucleotides, antibiotics, lipids and pigments are used in the usual way but, in this case too, the present invention includes any administerable form of these substances. In addition, the present invention includes, where organic acids are mentioned, both the pure acid and any of its salts and any other form of preparation.
According to the invention, a microorganism in which the phosphorylation state of at least one protein has been altered in this way compared to the protein naturally occurring in the organism used synthesizes an increased amount of a desired fine chemical.
The term "fine chemicals" means in the present application all metabolic products of a microorganism used, whose preparation in a pure form or as a mixture is desired. Preferred fine chemicals are amino acids, in particular L-amino acids, preferably L-lysine, L-threonine, L-glutamic acid, L-methionine, L-cysteine, L-cystine, L-tyrosine, L-phenylalanine, L-tryptophan, L-valine, L-arginine, L-leucine, L-serine, L-histidine, L-aspartic acid, L-asparagine, L-glutamine, L-glycine, L-proline, L-homoserine, and L-isoleucine or amino acid mixtures; vitamins, in particular pantothenic acid, riboflavin, vitamin A, vitamin E, thiamine, biotin, folic acid, ascorbic acid, calciferol, phylloquinone, menaquinone, vitamin B6, vitamin B12, nicotinic acid or vitamin mixtures; nucleosides, in particular adenosine, guanosine, thymidine, uridine and cytidine, or else modified nucleosides such as inosine and xanthine, and also nucleotides, in particular adenylate, guanylate, thymidylate, uridylate and cytidylate, or else modified nucleotides such as inosinate and xanthylate; chlorophyls, bacteriochlorophyls and other tetrapyrroles; pigments such as lutein, lycopene, astaxanthin, canthaxanthin, zeaxanthin, neoxanthin, violaxanthin, phycocyanins and phycoerythrins and other carotenoids including their derivatives; antibiotics such as aminoglycosides, antifungal antibiotics, cephalosporins, beta-lactam antibiotics, chloramphenicol and its derivatives, macrolides, penicillins, tetracyclines and all other antibiotics and their derivatives and mixtures; enzymes for all applications, such as phytases, amylases and glucoamylases, pectinases, upases, penicillin acylases, acidic, neutral and basic proteases, cellulases, lactase, isomerases, invertase, pullulanase, hydantoinases, glucanases or anthocyanases; organic acids such as citric acid, lactic acid, gluconic acid, buttyric acid, acetic acid, formic acid, succinic acid, ketoglutaric acid and derivatives thereof; biologically derived polymers such as polyhydroxybuttyric acid or polyalkanoates; ergot alkaloids and other toxins; lipids such as mono-, di- and triglycerides, free saturated and unsaturated fatty acids, phospho- and glucolipids, and also sphingophospholipids, choline, betaine and sarcosine; steroids such as cholesterol including its derivatives, hopanoids, insect hormones, plant steroids such as campesterol, stigmasterol or sitosterol, cardiac glycosides, ecdysone, brassinosteroids, isoprenoids such as, for example, all terpenes, steroid hormones such as, for example, testosterone, oestrogens, progesterone, cortisone and other corticosteroids including their derivatives, aldosterone and all derivatives, gestagen and progesteron, androgens, unless already listed, bile acids and derivatives; all other metabolites. Moreover, the specific change in the phosphorylation state of proteins can be utilized in order to synthesize as much biomass as possible and thus provide, for example, single cell protein or starter cultures .
The microorganisms according to the invention can produce the fine chemicals from various carbon sources. Preferred starting substances here are glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or else acetate, glycerol, lactate or ethanol. They may be representatives of prokaryotes, in particular bacteria (Gram positive or Gram negative) , for example coryneform bacteria, in particular of the genus Corynebacterium or Brevibacterium, or of E. coli , archaebacteria, in particular of the genus Thermus, yeasts, or fungi. In the case of the genus Corynebacterium, particular mention must be made of the species Corynebacterium glutamicum which is known in the art for its ability to produce L-amino acids.
Examples of suitable strains of the genus
Corynebacterium, in particular of the species Corynebacterium glutamicum, are the known wild-type strains
Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and Brevibacterium divarica tum ATCC14020
and L-lysine-producing mutants or strains prepared therefrom, such as, for example,
Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum KCCM 10227 Corynebacterium glutamicum FERM-P 6464 and Cory.nejbacteriu.ffl glutamicum DSM5715
or strains prepared therefrom.
Moreover, Enterobacteriaceae, in particular Escherichia coli , are particularly suitable for the synthesis of fine chemicals . Examples of suitable L-threonine- producing strains of the genus Escherichia, in particular of the species Escherichia coli , are
Escherichia coli MG1655
Escherichia coli TF427 Escherichia coli H4578
Escherichia coli KY10935
Escherichia coli EL1003
Escherichia coli VNIIgenetika MG-442
Escherichia coli VNIIgenetika VL334/pYN7 Escherichia coli VNIIgenetika Ml
Escherichia coli VNIIgenetika 472T23
Escherichia coli VNIIgenetika TDH-6
Escherichia coli BKIIM B-3996 Escherichia coli BKIIM B-5318
Escherichia coli B-3996-C43
Escherichia coli B-3996-C80
Escherichia coli B-3996/pT V-pps
Escherichia coli B-3996 (pMW: : THY)
Escherichia coli B-3996/pBP5
Escherichia coli kat 13
Escherichia coli KCCM-10132
Escherichia coli H-4581
or strains prepared therefrom.
Examples of suitable L-threonine- or biotine producing strains of the genus Serratia , in particular of the species Serratia marcescens, are
Serra tia marcescens HNr21 Serra tia marcescens TLrl56 Serra tia marcescens T2000
and strains prepared therefrom.
Examples of suitable D-pantothenic acid-producing strains of the genus Escherichia , in particular of the species Escherichia coli, are
Escherichia coli FV5069 pFV31
Escherichia coli FV5069 pFV202
Escherichia coli FE6b pFE80 Escherichia coli KE3
or strains prepared therefrom.
Examples of suitable D-pantothenic acid-producing strains of the genus Bacillus, in particular of the species Bacillus subtilis, are, inter alia, the strains mentioned in WO 01/21772. Bacillus subtilis strain PA 221
Bacillus subtilis strain PA 248
Bacillus subtilis strain PA 236
Ba cillus subtilis strain PA 221/pAN429-4 Bacillus subtilis strain PA 413-4
Bacillus subtilis strain PA 236-1
Bacillus subtilis strain PA 340
Bacillus subtilis strain PA 377
Bacillus subtilis strain "PA 365 Bacillus subtilis strain PA 377-2
Bacillus subtilis strain PA 824-2
or strains prepared therefrom.
Examples of suitable riboflavin-producing strains of the 'genus Bacillus, in particular of the species Bacillus subtilis, are
Bacillus subtilis RB50 Bacillus subtilis RB58
Bacillus subtilis B18502.
or strains prepared therefrom.
Suitable rib'oflavin-producing microorganisms are Ashbya, in particular the species Ashbya gossypii , Eremothecium ashbyii , Candida flareri and Bacillus subtilis .
According to the invention, it is also possible to use other prokaryotic organisms, for example strep.tomycetes", actinomycetes, Bifidobacteriumr Arthrobacter r ' Rhodococcus , and other Gram-positive bacterias such as Bacillus subtilis or Clostridium acetobutylicum or butyricum, or else lactobacilli such as, for example, Lactobacillus lactis , brevis r ca' sii fermentum, bulgaricus, Lactococcus lactis or La ctobacillus acidophilus . Examples for Gram-negative bacterias are Xanthomonas, Acetobacter , Pseudomonas , X. campestris r P. fluorescens , A. acetii or acidophilum, further Klebsiella , Enterobacter , Salmonella r Shigella , Proteus , Serra tia and Erwinia . It is likewise possible to use eukaryotic cells for producing desired fine chemicals . To this end, all culturable eukaryotic cells capable of producing a metabolic product desired according to the invention may be used, for example Ascomycetae , Basidiomycetae, Phycomycetae and Deuteromycetae . Examples of Ascomycetae are Neurospora , Sa ccharomyces r Trichoderma , Ashbya r Pichia , Hansenula , Candida , Yarrowia and Kluyveromyces . Examples for Phycomyces are Zygomycetae , Oomycetae and Mucoralae, particularly Blakeslea trispora r Phycomyces blakesleanus , Rhizopus nigricans and Rhizopus oryzae . Examples for Deuteromycetae are Penicilliumr
Aspergillus and Candida, particularly Aspergillus niger , oryzae, fumiga tus r flavus or wentii , Penicillium nota tum or chrysogenum and Candida flareri . Particularly suitable are for example , Saccharomyces cerevisiae, Schizosaccharomyces pombe , Hansenula polymorpha , Candida bondii , Kluyveromyces lactis , Schwanniomyces occidentalis or Pichia pastoris r filamentous fungi such as Aspergillus niger, Aspergillus nidulans , Ashbya gossipii , Yarrowia lipolytica or Neurospora crassa , insect cell systems such as , for example, the baculovirus system and common cell lines of mammalian cells such as , for example, HeLa cells , COS cells or CHO cells and also algae .
According to the present invention, the amount of a desired fine chemical produced is increased by altering the phosphorylation state of at least one protein of the microorganism, compared with the naturally occurring protein of the microorganism. Therefore, a microorganism of the present invention is distinguished by the fact that it contains at least one protein whose amino acid sequence differs in at least one amino acid from that of the same protein occurring naturally in this organism, it being possible for the amino acid concerned to serve as a phosphorylation site in the protein. The difference to the wild-type sequence may be a mutation, in particular an amino acid exchange, or a deletion or insertion of at least one amino acid. The exchange, deletion or insertion of an amino acid can remove a phosphorylation site present in the naturally occurring protein, introduce an additional phosphorylation site compared with the naturally occurring protein or permanently change the phosphorylation state of a phosphorylation site (for example, an existing phosphorylation site is permanently phosphorylated or permanently unphosphorylated) .
In order to change the phosphorylatability, in particular to permanently change the phosphorylation state of a protein, one or more amino acids which occur in the natural protein and which serve as a phosphorylation site in the protein can be exchanged for another amino acid so that the phosphorylation state which serves to regulate the protein is permanently changed. It is also possible to introduce additional phosphorylation sites into an enzyme by exchanging one or more amino acids.
The phosphorylation state can be changed in any manner which leads to such a change, and the phosphorylation state is changed preferably by mutagenesis of the protein-encoding DNA sequence, particularly preferably by site-directed mutagenesis of the protein-encoding DNA sequence.
The following amino acid exchanges are suitable:
1. Exchanges which remove a phosphorylation site: Exchange of serine, threonine, tyrosine, aspartate, glutamate, histidine, arginine or lysine for another amino acid, meaning an exchange for any other amino acid (i.e. also for any amino acid different from those mentioned here) . A particularly preferred exchange is the exchange of serine or threonine for asparagine, glutamine, glycine, alanine, cysteine, valine, methionine, isoleucine, leucin, phenylalanine, histidine, lysine, tryptophan or arginine.
2. Exchanges which introduce a phosphorylation site:
Exchange of an amino acid which is not serine, threonine, tyrosine, aspartic acid, glutamic acid, histidine, arginine or lysine for any of these amino acids .
3. Exchanges, after which an existing potential phosphorylation site can no longer be regulated, due to the fact that permanent phosphorylation or permanent dephosphorylation ensues:
Exchange of serine, threonine, histidine or tyrosine for glutamic acid, aspartic acid, alanine, glycine or valine.
4. Exchanges in the "phosphorylation sequence" region:
"Phosphorylation sequence" herein refers to the amino acid sequence of the protein, which surrounds the amino acid which 'is reversibly phosphorylated in the wild type. Furthermore this phosphorylation sequence can comprise an amino acid which exchange changes the three dimensional structure of the protein in a way that an amino acid which can reversibly be phosphorylated in the wild type can not be phosphorylated accordingly in the mutant. These amino acid sequences are known to the skilled worker and serve to "recognize" the amino acid to be phosphorylated. Any exchange of any of the amino acids in this sequence, which results in an amino acid being no longer reversibly phosphorylatable/ dephosphorylatable as in the wild type, is to be regarded as being included in the present invention.
The amino acids occurring in a natural protein are known to the skilled worker. According to the invention, all naturally occurring amino acids can be exchanged for one another, including the posttranslationally modified amino acids.
According to the invention, the amino acid(s) is/are located in the protein in an amino acid sequence which renders the amino acid in question accessible to a reversible phosphorylation. These amino acid sequences are known to the skilled worker. The amino acid(s) to be exchanged may be located in the region of an active side of a protein, in particular of an enzyme, or may be located outside the active site in a region whose phosphorylation state is instrumental in controlling the activity of the protein or its affinity to other proteins .
The proteins whose phosphorylation state is changed according to the present invention may be enzymes, regulatory proteins, structural proteins, transport proteins, storage proteins, proteins having an immune function or components of signal transduction pathways.
The target protein used for a change according to the invention of the phosphorylation state may be any protein in which a change of the phosphorylation state causes an increase in the rate of synthesis of a desired fine chemical or else decreases the rate of synthesis of unwanted metabolic products. Examples of proteins which are to be regarded as being within the scope of the invention are:
proteins of the biosynthetic pathway of the appropriate fine chemical whose synthesis is desired, in particular key enzymes of the synthetic pathway,
proteins of biochemical pathways,
- proteins which regulate biosynthesis pathways of desired fine chemicals (feed-forward or feedback) ,
proteins of signal recognition and signal transduction, including two component system(s) ,
proteins of gene regulation,
proteins involved in cell integrity, thereby maintaining and/or modifying the biocatalyst,
components of the protein secretion apparatus,
proteins with immune functions,
proteins with storage function,
membrane-bound or membrane-associated proteins
- proteins with transport function.
In particular, it is possible to select as a target protein one or more enzymes from the following list of proteins. Shown are proteins from Corynebacterium glutamicum, Escherichia coli , Bacillus subtilis, Lactococcus lactis, Clostridium acetobutylicum,
Sa ccharomyces cerevisiae with their EC number: 1.1.1.1 alcohol dehydrogenase
1.1.1.100 3-Oxoacyl- [Acyl-Carrier-Protein] Reductase 1.1.1.103 L-Threonine 3-Dehydrogenase 1.1.1.105 Retinol Dehydrogenase 1.1.1.122 D-Threo-Aldose 1-Dehydrogenase
1.1.1.125 2-Deoxy-D-Gluconate 3-Dehydrogenase 1.1.1.132 Gdp-mannose 6-Dehydrogenase 1.1.1.14 L-Iditol 2-Dehydrogenase 1.1.1.140 Sorbitol-6-Phosphate 2-Dehydrogenase 1.1.1.145 3beta-Hydroxy-Delta5-Steroid Dehydrogenase 1.1.1.146 llbeta-Hydroxysteroid Dehydrogenase 1.1.1.153 Sepiapterin Reductase
1.1.1.157 3-Hydroxybutyryl-Coa Dehydrogenase
1.1.1.158 Udp-N-Acetylmuramate Dehydrogenase 1.1.1.159 7alpha-Hydroxysteroid Dehydrogenase
1.1.1.169 2-Dehydropantoate 2-Reductase
1.1.1.17 Mannitol-1-Phosphate 5-Dehydrogenase 1.1.1.179 D-Xylose 1-Dehydrogenase (Nadp+)
1.1.1.18 Myo-Inositol 2-Dehydrogenase 1.1.1.184 Carbonyl Reductase (Nadph)
1.1.1.188 Prostaglandin-F Synthase 1.1.1.195 Cinnamyl-Alcohol Dehydrogenase
1.1.1.2 Alcohol Dehydrogenase (Nadp+) 1.1.1.202 1, 3-Propanediol Dehydrogenase 1.1.1.204 Xanthine Dehydrogenase 1.1.1.205 Imp Dehydrogenase
1.1.1.21 Aldehyde Reductase
1 . 1 . 1 . 211 Long-Chain-3-Hydroxyacyl-Coa Dehydrogenase 1 . 1 . 1 . 218 Morphine 6-Dehydrogenase 1 . 1 . 1 . 219 Dihydrokaempferol 4-Reductase
1.1.1.22 Udpglucose 6-Dehydrogenase
1.1.1.23 Histidinol Dehydrogenase 1.1.1.233 N-Acylmannosamine 1-Dehydrogenase 1.1.1.236 Tropinone Reductase 1.1.1.24 Quinate 5-Dehydrogenase 1.1.1.244 Methanol Dehydrogenase 1.1.1.25 Shikimate 5-Dehydrogenase
1.1.1.27 L-Lactate Dehydrogenase
1.1.1.28 D-Lactate Dehydrogenase 1.1.1.29 Glycerate Dehydrogenase
1.1.1.3 Homoserine Dehydrogenase 1.1.1.30 3-Hydroxybutyrate Dehydrogenase
1.1.1.34 Hydroxymethylglutaryl-Coa Reductase (Nadph)
1.1.1.35 3-Hydroxyacyl-Coa Dehydrogenase 1.1.1.36 Acetoacetyl-Coa Reductase
1.1.1.37 Malate Dehydrogenase
1.1.1.38 Malate Dehydrogenase (Oxaloacetate- Decarboxylating)
1.1.1.4 (R,R) -Butanediol Dehydrogenase 1.1.1.40 Malate Dehydrogenase (Oxaloacetate- Decarboxylating) (Nadp+)
1.1.1.41 Isocitrate Dehydrogenase (Nad+)
1.1.1.42 Isocitrate Dehydrogenase (Nadp+) 1.1.1.44 Phosphogluconate Dehydrogenase
(Decarboxylating)
1.1.1.47 Glucose 1-Dehydrogenase
1.1.1.48 Galactose 1-Dehydrogenase
1.1.1.49 Glucose-6-Phosphate 1-Dehydrogenase 1.1.1.53 3alpha (Or 20beta) -Hydroxysteroid Dehydrogenase
1.1.1.56 Ribitol 2-Dehydrogenase
1.1.1.57 Fructuronate Reductase
1.1.1.58 Tagaturonate Reductase 1.1.1.6 Glycerol Dehydrogenase 1.1.1.61 4-Hydroxybutyrate Dehydrogenase
1.1.1.62 Estradiol 17beta-Dehydrogenase
1.1.1.69 Gluconate 5-Dehydrogenase
1.1.1.73 Octanol Dehydrogenase
1.1.1.77 Lactaldehyde Reductase 1.1.1.8 Glycerol-3-Phosphate Dehydrogenase (Nad+)
1.1.1.81 Hydroxypyruvate Reductase
1.1.1.83 D-Malate Dehydrogenase (Decarboxylating)
1.1.1.85 3-Isopropylmalate Dehydrogenase
1.1.1.86 Ketol-Acid Reductoisomerase 1.1.1.88 Hydroxymethylglutaryl-Coa Reductase
1.1.1.9 D-Xylulose Reductase
1.1.1.90 Aryl-Alcohol Dehydrogenase
1.1.1.91 Aryl-Alcohol Dehydrogenase (Nadp+) 1.1.1.93 Tartrate Dehydrogenase 1.1.1.95 Phosphoglycerate Dehydrogenase
1.1.2.3 L-Lactate Dehydrogenase (Cytochrome)
1.1.2.4 D-Lactate Dehydrogenase (Cytochrome) 1.1.3.13 Alcohol Oxidase
1.1.3.15 (S)-2-Hydroxy-Acid Oxidase 1.1.3.22 Xanthine Oxidase
1.1.3.24 L-Galactonolactone Oxidase 1.1.3.4 Glucose Oxidase 1.1.3.8 L-Gulonolactone Oxidase
1.1.99.1 Choline Dehydrogenase 1.1.99.10 Glucose Dehydrogenase (Acceptor)
1.1.99.17 Glucose Dehydrogenase (Pyrroloquinoline-
Quinone)
1.1.99.23 Polyvinyl-Alcohol Dehydrogenase (Acceptor)
1.1.99.5 Glycerol-3-Phosphate Dehydrogenase 1.1.99.6 D-2-Hydroxy-Acid Dehydrogenase
1.1.99.8 Alcohol Dehydrogenase (Acceptor)
1.10.2.2 Ubiquinol—Cytochrome-C Reductase
1.10.3.1 Catechol Oxidase
1.10.3.2 Laccase 1.10.3.3 L-Ascorbate Oxidase 1.10.99.1 Plastoquinol--Plastocyanin Reductase
1.11.1.1 Nadh Peroxidase
1.11.1.10 Chloride Peroxidase
1.11.1.11 L-Ascorbate Peroxidase 1.11.1.12 Phospholipid-Hydroperoxide Glutathione Peroxidase
1.11.1.5 Cytochrome-C Peroxidase
1.11.1.6 Catalase
1.11.1.7 Peroxidase 1.11.1.9 Glutathione Peroxidase
1.12.1.2 Hydrogen Dehydrogenase 1.12.1.2 Hydrogen Dehydrogenase 1.12.2.1 Cytochrome-C3 Hydrogenase 1.12.99.1 Coenzyme F420 Hydrogenase 1.13.11.1 Catechol 1, 2-Dioxygenase
1.13.11.27 4-Hydroxyphenylpyruvate Dioxygenase 1.13.11.32 2-Nitropropane Dioxygenase 1.13.11.37 Hydroxyquinol 1, 2-Dioxygenase 1.13.11.42 Indoleamine-Pyrrole 2, 3-Dioxygenase 1.13.11.6 3-Hydroxyanthranilate 3, 4-Dioxygenase 1.13.11.8 Protocatechuate 4, 5-Dioxygenase
1.13.12.4 Lactate 2-Monooxygenase 1.14.11.14 6beta-Hydroxyhyoscyamine Epoxidase 1.14.12.10 Benzoate 1, 2-Dioxygenase 1.14.12.11 Toluene Dioxygenase
1.14.12.12 Naphthalene 1, 2-Dioxygenase 1.14.12.3 Benzene 1, 2-Dioxygenase
1.14.13.1 Salicylate 1-Monooxygenase
1.14.13.2 4-Hydroxybenzoate 3-Monooxygenase 1.14.13.20 2, 4-Dichlorophenol 6-Monooxygenase
1.14.13.20 2, 4-Dichlorophenol 6-Monooxygenase 1.14.13.25 Methane Monooxygenase
1.14.13.3 4-Hydroxyphenylacetate 3-Monooxygenase 1.14.13.39 Nitric-Oxide Synthase 1.14.13.7 Phenol 2-Monooxygenase
1.14.13.8 Dimethylaniline Monooxygenase (N-Oxide-
Forming)
1.14.14.1 Unspecific Monooxygenase
1.14.14.3 Alkanal Monooxygenase (Fmn-Linked) 1.14.99.3 Heme Oxygenase (Decyclizing)
1.14.99.5 Stearoyl-Coa Desaturase 1.14.99.7 Squalene Monooxygenase 1.15.1.1 Superoxide Dismutase
1.16.1.1 Mercury (Ii) Reductase 1.17.4.1 Ribonucleoside-Diphosphate Reductase
1.17.4.2 Ribonucleoside-Triphosphate Reductase
1.18.1.1 Rubredoxin—Nad+ Reductase
1.18.1.2 Ferredoxin—Nadp+ Reductase
1.18.1.3 Ferredoxin—Nad+ Reductase 1.18.1.4 Rubredoxin—Nad(P)+ Reductase 1.18.6.1 Nitrogenase 1.18.99.1 Hydrogenase
1.2.1.1 Formaldehyde Dehydrogenase (Glutathione) 1.2.1.10 Acetaldehyde Dehydrogenase (Acetylating) 1.2.1.11 Aspartate-Semialdehyde Dehydrogenase
1.2.1.12 Glyceraldehyde-3-Phosphate Dehydrogenase ( Phosphorylating)
1.2.1.13 Glyceraldehyde-3-Phosphate Dehydrogenase (Nadp+) (Phosphorylating) 1.2.1.16 Succinate-Semialdehyde Dehydrogenase (Nad(P)+) 1.2.1.19 Aminobutyraldehyde Dehydrogenase
1.2.1.2 Formate Dehydrogenase 1.2.1.22 Lactaldehyde Dehydrogenase
1.2.1.27 Methylmalonate-Semialdehyde Dehydrogenase (Acylating)
1.2.1.3 Aldehyde Dehydrogenase (Nad+)
1.2.1.3 Aldehyde Dehydrogenase (Nad+)
1.2.1.31 L-Aminoadipate-Semialdehyde Dehydrogenase 1.2.1.38 N-Acetyl-Gamma-Glutamyl-Phosphate Reductase 1.2.1.39 Phenylacetaldehyde Dehydrogenase
1.2.1.4 Aldehyde Dehydrogenase (Nadp+)
1.2.1.40 3alpha, 7alpha, 12alpha-Trihydroxycholestan-26- Al
1.2.1.41 Glutamate-5-Semialdehyde Dehydrogenase 1.2.1.46 Formaldehyde Dehydrogenase
1.2.1.5 Aldehyde Dehydrogenase (Nad(P)+)
1.2.1.8 Betaine-Aldehyde Dehydrogenase
1.2.1.9 Glyceraldehyde-3-Phosphate Dehydrogenase (Nadp+) 1.2.2.2 Pyruvate Dehydrogenase (Cytochrome) 1.2.3.1 Aldehyde Oxidase 1.2.3.3 Pyruvate Oxidase
1.2.4.1 Pyruvate Dehydrogenase (Lipoamide)
1.2.4.2 Oxoglutarate Dehydrogenase (Lipoamide) 1.2.4.4 3-Methyl-2-Oxobutanoate Dehydrogenase
(Lipoamide)
1.2.7.1 Pyruvate Synthase
1.2.99.2 Carbon Monoxide Dehydrogenase 1.2.99.4 Formaldehyde Dismutase 1.2.99.5 Formylmethanofuran Dehydrogenase
1.3.1.12 Prephenate Dehydrogenase
1.3.1.13 Prephenate Dehydrogenase (Nadp+)
1.3.1.19 Cis-1, 2-Dihydrobenzene-l, 2-Diol Dehydrogenase
1.3.1.2 Dihydropyrimidine Dehydrogenase (Nadp+) 1.3.1.23 Cholestenone 5beta-Reductase
1.3.1.24 Biliverdin Reductase 1.3.1.26 Dihydrodipicolinate Reductase 1.3.1.28 2, 3-Dihydro-2, 3-Dihydroxybenzoate Dehydrogenase 1.3.1.29 Cis-1, 2-Dihydro-l, 2-Dihydroxynaphthalene Dehydrogenase
1.3.1.32 Maleylacetate Reductase 1.3.1.34 2, 4-Dienoyl-Coa Reductase (Nadph) 1.3.1.35 Phosphatidylcholine Desaturase
1.3.1.42 12-0xophytodienoate Reductase
1.3.1.43 Cyclohexadienyl Dehydrogenase 1.3.1.45 2 ' -Hydroxyisoflavone Reductase 1.3.1.8 Acyl-Coa Dehydrogenase (Nadp+) 1.3.1.9 Enoyl- [Acyl-Carrier Protein] Reductase (Nadh)
1.3.3.1 Dihydroorotate Oxidase
1.3.3.3 Coproporphyrinogen Oxidase
1.3.3.4 Protoporphyrinogen Oxidase
1.3.3.5 Bilirubin Oxidase 1.3.3.6 Acyl-Coa Oxidase
1.3.5.1 Succinate Dehydrogenase (Ubiquinone) 1.3.99.1 Succinate Dehydrogenase 1.3.99.10 Isovaleryl-Coa Dehydrogenase 1.3.99.13 Long-Chain-Acyl-Coa Dehydrogenase 1.3.99.3 Acyl-Coa Dehydrogenase
1.3.99.4 3-Oxosteroid 1-Dehydrogenase
1.3.99.5 3-Oxo-5alpha-Steroid 4-Dehydrogenase
1.4.1.1 Alanine Dehydrogenase 1.4.1.13 Glutamate Synthase (Nadph) 1.4.1.14 Glutamate Synthase (Nadh)
1.4.1.16 Diaminopimelate Dehydrogenase
1.4.1.2 Glutamate Dehydrogenase 1.4.1.20 Phenylalanine Dehydrogenase
1.4.1.3 Glutamate Dehydrogenase (Nad(P)+) 1.4.1.4 Glutamate Dehydrogenase (Nadp+)
1.4.1.8 Valine Dehydrogenase (Nadp+)
1.4.1.9 Leucine Dehydrogenase 1.4.3.16 L-Aspartate Oxidase 1.4.3.2 L-Amino-Acid Oxidase 1.4.3.4 Amine Oxidase (Flavin-Containing) 1.4.3.5 Pyridoxamine-Phosphate Oxidase
1.4.3.5 Pyridoxamine-Phosphate Oxidase
1.4.3.6 Amine Oxidase (Copper-Containing) 1.4.4.2 Glycine Dehydrogenase (Decarboxylating) 1.4.4.2 Glycine Dehydrogenase (Decarboxylating)
1.4.7.1 Glutamate Synthase (Ferredoxin) 1.4.99.1 D-Amino-Acid Dehydrogenase
1.5.1.10 Saccharopine Dehydrogenase (Nadp+, L-Glutamate Forming) 1.5.1.12 l-Pyrroline-5-Carboxylate Dehydrogenase 1.5.1.12 l-Pyrroline-5-Carboxylate Dehydrogenase 1.5.1.15 Methylenetetrahydrofolate Dehydrogenase (Nad+)
1.5.1.2 Pyrroline-5-Carboxylate Reductase 1.5.1.20 Methylenetetrahydrofolate Reductase (Nadph) 1.5.1.24 N5- (Carboxyethyl) Ornithine Synthase 1.5.1.3 Dihydrofolate Reductase
1.5.1.5 Methylenetetrahydrofolate Dehydrogenase (Nadp+)
1.5.1.7 Saccharopine Dehydrogenase (Nad+, L-Lysine Forming) 1.5.3.1 Sarcosine Oxidase
1.5.99.2 Dimethylglycine Dehydrogenase
1.5.99.4 Nicotine Dehydrogenase
1.5.99.7 Trimethylamine Dehydrogenase
1.5.99.8 Proline Dehydrogenase 1.6.1.1 Nad(P)+ Transhydrogenase (B-Specific) 1.6.2.2 Cytochrome-B5 Reductase
1.6.2.4 Nadph—Ferrihemoprotein Reductase
1.6.4.2 Glutathione Reductase (Nadph)
1.6.4.5 Thioredoxin Reductase (Nadph) 1.6.5.3 Nadh Dehydrogenase (Ubiquinone)
1.6.5.4 Monodehydroascorbate Reductase (Nadh)
1.6.5.5 Nadph : Quinone Reductase 1.6.6.1 Nitrate Reductase (Nadh)
1.6.6.3 Nitrate Reductase (Nadph) 1.6.6.4 Nitrite Reductase (Nad(P)H)
1.6.6.8 Gmp Reductase
1.6.6.9 Trimethylamine-N-Oxide Reductase
1.6.8.1 Nad(P)H Dehydrogenase (Fmn)
1.6.8.2 Nadph Dehydrogenase (Flavin) 1.6.99.1 Nadph Dehydrogenase
1.6.99.2 Nad(P)H Dehydrogenase (Quinone)
1.6.99.3 Nadh Dehydrogenase
1.7.3.3 Urate Oxidase
1.7.7.1 Ferredoxin--Nitrite Reductase 1.7.99.4 Nitrate Reductase
1.7.99.5 5, 10-Methylenetetrahydrofolate Reductase (Fadh2)
1.7.99.7 Nitric-Oxide Reductase
1.8.1.2 Sulfite Reductase (Nadph) 1.8.1.4 Dihydrolipoamide Dehydrogenase
1.8.4.5 Methionine-S-Oxide Reductase
1.8.4.6 Protein-Methionine-S-Oxide Reductase 1.8.4.6 Protein-Methionine-S-Oxide Reductase 1.8.7.1 Sulfite Reductase (Ferredoxin) 1.8.99.2 Adenylylsulfate Reductase 1.9.3.1 Cytochrome-C Oxidase
1.97.1.4 [Pyruvate Formate-Lyase] Activating Enzyme 2.1.1.104 Caffeoyl-Coa O-Methyltransferase 2.1.1.107 Uroporphyrin-Iii C-Methyltransferase 2.1.1.113 Site-Specific Dna-Methyltransferase (Cytosine-N4-Specific)
2.1.1.13 5-Methyltetrahydrofolate—Homocysteine S- Methyltransferase
2.1.1.14 5-Methyltetrahydropteroyltriglutamate— Homocysteine 2.1.1.17 Phosphatidylethanolamine N-Methyltransferase 2.1.1.31 Trna (Guanine-Nl-) -Methyltransferase 2.1.1.35 Trna (Uracil-5-) -Methyltransferase 2.1.1.37 Dna (Cytosine-5-) -Methyltransferase 2.1.1.41 24-Sterol C-Methyltransferase 2.1.1.45 Thymidylate Synthase
2.1.1.48 Rrna (Adenine-N6-) -Methyltransferase 2.1.1.63 Methylated-Dna—Protein-Cysteine S- Methyltransferase 2.1.1.64 3-Demethylubiquinone-9 3-O-Methyltransferase
2.1.1.71 Phosphatidyl-N-Methylethanolamine N- Methyltransferase
2.1.1.72 Site-Specific Dna-Methyltransferase (Adenine- Specific) 2.1.1.73 Site-Specific Dna-Methyltransferase (Cytosine-
Specific )
2.1.1.77 Protein-L-Isoaspartate (D-Aspartate) 0-
Methyltransferase
2.1.1.79 Cyclopropane-Fatty-Acyl-Phospholipid Synthase 2.1.1.80 Protein-Glutamate O-Methyltransferase
2.1.1.98 Diphthine Synthase
2.1.2.1 Glycine Hydroxymethyltransferase
2.1.2.10 Aminomethyltransferase
2.1.2.11 3-Methyl-2-Oxobutanoate Hydroxymethyltransferase
2.1.2.2 Phosphoribosylglycinamide Formyltransferase
2.1.2.3 Phosphoribosylaminoimidazolecarboxamide Formyltransferase
2.1.2.5 Glutamate Formiminotransferase 2.1.2.9 Methionyl-Trna Formyltransferase
2.1.3.2 Aspartate Carbamoyltransferase
2.1.3.3 Ornithine Carbamoyltransferase
2.2.1.1 Transketolase
2.2.1.2 Transaldolase 2.3.1.1 Amino-Acid N-Acetyltransferase
2.3.1.117 2, 3, 4, 5-Tetrahydropyridine-2-Carboxylate N- Succinyltransferase
2.3.1.118 N-Hydroxyarylamine O-Acetyltransferase
2.3.1.12 Dihydrolipoamide S-Acetyltransferase ' 2.3.1.128 Ribosomal-Protein-Alanine N-Acetyltransferase 2.3.1.129 Acyl- [Acyl-Carrier-Protein] —Udp-N-
2.3.1.15 Glycerol-3-Phosphate O-Acyltransferase
2.3.1.16 Acetyl-Coa C-Acyltransferase
2.3.1.18 Galactoside O-Acetyltransferase 2.3.1.19 Phosphate Butyryltransferase
2.3.1.21 Carnitine O-Palmitoyltransferase 2.3.1.28 Chloramphenicol O-Acetyltransferase
2.3.1.29 Glycine C-Acetyltransferase
2.3.1.30 Serine O-Acetyltransferase 2.3.1.31 Homoserine O-Acetyltransferase 2.3.1.35 Glutamate N-Acetyltransferase
2.3.1.37 5-Aminolevulinate Synthase
2.3.1.38 [Acyl-Carrier-Protein] S-Acetyltransferase
2.3.1.39 [Acyl-Carrier-Protein] S-Malonyltransferase 2.3.1.41 3-Oxoacyl- [Acyl-Carrier-Protein] Synthase
2.3.1.43 Phosphatidylcholine—Sterol O-Acyltransferase
2.3.1.46 Homoserine O-Succinyltransferase
2.3.1.47 8-Amino-7-Oxononanoate Synthase
2.3.1.48 Histone Acetylfransferase 2.3.1.5 Arylamine N-Acetyltransferase
2.3.1.50 Serine C-Palmitoyltransferase
2.3.1.51 l-Acylglycerol-3-Phosphate O-Acyltransferase 2.3.1.54 Formate C-Acetyltransferase
2.3.1.57 Diamine N-Acetyltransferase 2.3.1.6 Choline O-Acetyltransferase
2.3.1.60 Gentamicin 3 ' -N-Acetyltransferase
2.3.1.61 Dihydrolipoamide S-Succinyltransferase
2.3.1.7 Carnitine O-Acetyltransferase 2.3.1.74 Naringenin-Chalcone Synthase 2.3.1.8 Phosphate Acetylfransferase
2.3.1.81 Aminoglycoside N3 ' -Acetylfransferase
2.3.1.84 Alcohol O-Acetyltransferase
2.3.1.85 Fatty-Acid Synthase
2.3.1.86 Fatty-Acyl-Coa Synthase 2.3.1.9 Acetyl-Coa C-Acetyltransf erase
2.3.1.9 Acetyl-Coa C-Acetyltransf erase 2.3.1.97 Glycylpeptide N-Tetradecanoyltransf erase 2.3.2.13 Protein-Glutamine Gamma-Glutamyltransf erase 2.3.2.2 Gamma-Glutamyltransf erase 2.3.2.5 Glutaminyl-Peptide Cyclotransf erase 2.3.2.6 Leucyltransf erase 2.3.2.8 Arginyltransf erase 2.4.1.1 Phosphorylase
2.4.1.10 Levansucrase 2.4.1.109 Dolichyl-Phosphate-Mannose—Protein Mannosyltransferase
2.4.1.11 Glycogen (Starch) Synthase
2.4.1.117 Dolichyl-Phosphate Beta-Glucosyltransferase 2.4.1.119 Dolichyl-Diphosphooligosaccharide—Protein Glycosyltransferase
2.4.1.12 Cellulose Synthase (Udp-Forming) 2.4.1.131 Glycolipid 2-Alpha-Mannosyltransferase 2.4.1.15 Alpha, Alpha-Trehalose-Phosphate Synthase (Udp- Forming) 2.4.1.16 Chitin Synthase
2.4.1.17 Glucuronosyltransferase
2.4.1.18 1, 4-Alpha-Glucan Branching Enzyme 2.4.1.182 Lipid-A-Disaccharide Synthase 2.4.1.186 Glycogenin Glucosyltransferase 2.4.1.19 Cyclomaltodextrin Glucanotransferase 2.4.1.21 Starch Synthase
2.4.1.24 1, 4-Alpha-Glucan 6-Alpha-Glucosyltransferase
2.4.1.25 4-Alpha-Glucanotransferase 2.4.1.34 1,3-Beta-Glucan Synthase 2.4.1.44 Lipopolysaccharide Galactosyltransferase
2.4.1.46 1,2-Diacylglycerol 3-Beta-
Galactosyltransferase
2.4.1.52 Poly (Glycerol-Phosphate) Alpha-
Glucosyltransferase 2.4.1.56 Lipopolysaccharide N-
Acetylglucosaminyltransferase
2.4.1.58 Lipopolysaccharide Glucosyltransferase I
2.4.1.7 Sucrose Phsophorylase
2.4.1.83 Dolichyl-Phosphate Beta-D-Mannosyltransferase 2.4.2.1 Purine-Nucleoside Phosphorylase
2.4.2.10 Orotate Phosphoribosyltransferase
2.4.2.11 Nicotinate Phosphoribosyltransferase
2.4.2.14 Amidophosphoribosyltransferase 2.4.2.17 Atp Phosphoribosyltransferase 2.4.2.18 Anthranilate Phosphoribosyltransferase 2.4.2.19 Nicotinate-Nucleotide Pyrophosphorylase (Carboxylating)
2.4.2.2 Pyrimidine-Nucleoside Phosphorylase
2.4.2.21 Nicotinate-Nucleotide--Dimethylbenzimidazole Phosphoribosyl
2.4.2.22 Xanthine Phosphoribosyltransferase
2.4.2.28 5 ' -Methylthioadenosine Phosphorylase
2.4.2.29 Queuine Trna-Ribosyltransferase
2.4.2.3 Uridine Phosphorylase 2.4.2.4 Thymidine Phosphorylase
2.4.2.7 Adenine Phosphoribosyltransferase
2.4.2.8 Hypoxanthine Phosphoribosyltransferase
2.4.2.9 Uracil Phosphoribosyltransferase
2.4.99.1 Beta-Galactoside Alpha-2, 6-Sialyltransferase 2.5.1.1 Dimethylallyltranstransferase
2.5.1.10 Geranyltranstransferase
2.5.1.15 Dihydropteroate Synthase
2.5.1.16 Spermidine Synthase
2.5.1.17 Cob(I)Alamin Adenosyltransferase 2.5.1.18 Glutathione Transferase
2.5.1.19 3-Phosphoshikimate 1-Carboxyvinyltransferase
2.5.1.21 Farnesyl-Diphosphate Farnesyltransferase
2.5.1.22 Spermine Synthase
2..5.1.26 Alkylglycerone-Phosphate Synthase 2.5.1.29 Farnesyltranstransferase
2.5.1.3 Thiamin-Phosphate Pyrophosphorylase
2.5.1.30 Trans-Hexaprenyltranstransferase
2.5.1.31 Di-Trans, Poly-Cis-Decaprenylcistransferase
2.5.1.32 Geranylgeranyl-Diphosphate Geranylgeranyltransferase 2.5.1.33 Trans-Pentaprenyltranstransferase
2.5.1.6 Methionine Adenosyltransferase
2.5.1.7 Udp-N-Acetylglucosamine 1- Carboxyvinyltransferase 2.5.1.8 Trna Isopentenyltransferase 2.5.1.9 Riboflavin Synthase 2.6.1.1 Aspartate Transaminase
2.6.1.1 Aspartate Transaminase 2.6.1.11 Acetylornithine Transaminase 2.6.1.13 Ornithine--Oxo-Acid Transaminase 2.6.1.14 Asparagine—Oxo-Acid Transaminase 2.6.1.16 Glutamine--Fructose-6-Phosphate Transaminase (Isomerizing) 2.6.1.18 Beta-Alanine—Pyruvate Transaminase 2.6.1.19 4-Aminobutyrate Transaminase
2.6.1.2 Alanine Transaminase 2.6.1.21 D-Alanine Transaminase 2.6.1.36 L-Lysine 6-Transaminase
2.6.1.42 Branched-Chain-Amino-Acid Transaminase 2.6.1.44 Alanine—Glyoxylate Transaminase 2.6.1.45 Serine—Glyoxylate Transaminase 2.6.1.5 Tyrosine Transaminase
2.6.1.51 Serine--Pyruvate Transaminase
2.6.1.52 Phosphoserine Transaminase 2.6.1.57 Aromatic-Amino-Acid Transaminase
2.6.1.62 Adenosylmethionine--8-Amino-7-Oxononanoate
Transaminase
2.6.1.64 Glutamine--Phenylpyruvate Transaminase
2.6.1.66 Valine--Pyruvate Transaminase 2.6.1.7 Kynurenine—Oxoglutarate Transaminase
2.6.1.9 Histidinol-Phosphate Transaminase
2.7.1.1 Hexokinase
2.7.1.105 6-Phosphofructo-2-Kinase
2.7.1.107 Diacylglycerol Kinase 2.7.1.109 [Hydroxymethylglutaryl-Coa Reductase (Nadph) ;
Kinase
2.7.1.11 6-Phosphofructokinase
2.7.1.112 Protein-Tyrosine Kinase
2.7.1.113 Deoxyguanosine Kinase 2.7.1.115 [3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) ] Kinase
2.7.1.116 [Isocitrate Dehydrogenase (Nadp+) ] Kinase
2.7.1.117 Myosin-Light-Chain Kinase
2.7.1.12 Gluconokinase 2.7.1.123 Ca2+/Calmodulin-Dependent Protein Kinase
2.7.1.128 [Acetyl-Coa Carboxylase] Kinase
2.7.1.129 Myosin-Heavy-Chain Kinase 2.7.1.135 Tau-Protein Kinase 2.7.1.137 1-Phosphatidylinositol 3-Kinase 2.7.1.141 [Rna-Polymerase] -Subunit Kinase 2.7.1.15 Ribokinase
2.7.1.16 Ribulokinase
2.7.1.17 Xylulokinase
2.7.1.19 Phosphoribulokinase 2.7.1.2 Glucokinase
2.7.1.20 Adenosine Kinase
2.7.1.21 Thymidine Kinase
2.7.1.25 Adenylylsulfate Kinase
2.7.1.26 Riboflavin Kinase 2.7.1.3 Ketohexokinase
2.7.1.30 Glycerol Kinase
2.7.1.32 Choline Kinase
2.7.1.33 Pantothenate Kinase 2.7.1.35 Pyridoxal Kinase 2.7.1.36 Mevalonate Kinase
2.7.1.37 Protein Kinase
2.7.1.38 Phosphorylase Kinase
2.7.1.39 Homoserine Kinase 2.7.1.4 Fructokinase 2.7.1.40 Pyruvate Kinase
2.7.1.45 2-Dehydro-3-Deoxygluconokinase
2.7.1.47 D-Ribulokinase
2.7.1.48 Uridine Kinase
2.7.1.49 Hydroxymethylpyrimidine Kinase 2.7.1.5 Rha nulokinase
2.7.1.50 Hydroxyethylthiazole Kinase
2.7.1.52 Fucokinase
2.7.1.53 L-Xylulokinase 2.7.1.56 1-Phosphofructokinase 2.7.1.6 Galactokinase
2.7.1.67 1-Phosphatidylinositol 4-Kinase
2.7.1.69 Protein- (Pai) -Phosphohistidine—Sugar
Phosphotransferase
2.7.1.71 Shikimate Kinase 2.7.1.73 Inosine Kinase
2.7.1.76 Deoxyadenosine Kinase
2.7.1.95 Kanamycin Kinase
2.7.1.99 [Pyruvate Dehydrogenase (Lipoamide)] Kinase
2.7.2.1 Acetate Kinase 2.7.2.11 Glutamate 5-Kinase
2.7.2.2 Carbamate Kinase
2.7.2.3 Phosphoglycerate Kinase
2.7.2.4 Aspartate Kinase 2.7.2.7 Butyrate Kinase 2.7.2.8 Acetylglutamate Kinase 2.7.3.3 Arginine Kinase
2.7.3.9 Phosphoenolpyruvate—Protein Phosphotransferase 2.7.4.1 Polyphosphate Kinase
2.7.4.10 Nucleoside-Triphosphate—Adenylate Kinase 2.7.4.14 Cytidylate Kinase 2.7.4.16 Thiamin-Phosphate Kinase
2.7.4.2 Phosphomevalonate Kinase
2.7.4.3 Adenylate Kinase
2.7.4.6 Nucleoside-Diphosphate Kinase 2.7.4.8 Guanylate Kinase
2.7.4.9 Dt p Kinase
2.7.6.1 Ribose-Phosphate Pyrophosphokinase
2.7.6.2 Thiamin Pyrophosphokinase
2.7.6.3 2-Amino-4-Hydroxy-6- Hydroxymethyldihydropteridine Pyro- 2.7.6.5 Gtp Pyrophosphokinase
2.7.7.10 Utp--Hexose-l-Phosphate Uridylyltransferase 2.7.7.12 Udpglucose—Hexose-1-Phosphate Uridylyltransferase 2.7.7.13 Mannose-1-Phosphate Guanylyltransferase
2.7.7.14 Ethanolamine-Phosphate Cytidylyltransferase
2.7.7.15 Choline-Phosphate Cytidylyltransferase 2.7.7.19 Polynucleotide Adenylyltransferase 2.7.7.2 Fmn Adenylyltransferase 2.7.7.22 Mannose-1-Phosphate Guanylyltransferase (Gdp)
2.7.7.24 Glucose-1-Phosphate Thymidylyltransferase
2.7.7.25 Trna Adenylyltransferase
2.7.7.27 Glucose-1-Phosphate Adenylyltransferase
2.7.7.38 3-Deoxy-Manno-Octulosonate Cytidylyltransferase
2.7.7.39 Glycerol-3-Phosphate Cytidylyltransferase
2.7.7.4 Sulfate Adenylyltransferase
2.7.7.41 Phosphatidate Cytidylyltransferase
2.7.7.42 [Glutamate--Ammonia-Ligase] Adenylyltransferase
2.7.7.49 Rna-Directed Dna Polymerase
2.7.7.5 Sulfate Adenylyltransferase (Adp) 2.7.7.50 Mrna Guanylyltransferase 2.7.7.53 Atp Adenylyltransferase 2.7.7.56 Trna Nucleotidyltransferase
2.7.7.59 [Protein-Pii] Uridylyltransferase
2.7.7.6 Dna-Directed Rna Polymerase
2.7.7.7 Dna-Directed Dna Polymerase
2.7.7.8 Polyribonucleotide Nucleotidyltransferase 2.7.7.9 Utp—Glucose-1-Phosphate Uridylyltransferase 2.7.8.1 Ethanolaminephosphotransferase
2.7.8.11 Cdpdiacylglycerol—Inositol 3- Phosphatidyltransferase
2.7.8.12 Cdpglycerol Glycerophosphotransferase 2.7.8.12 Cdpglycerol Glycerophosphotransferase
2.7.8.13 Phospho-N-Acetylmuramoyl-Pentapeptide- Transferase
2.7.8.15 Udp-N-Acetylglucosamine—Dolichyl- 2.7.8.19 Udpglucose--Glycoprotein Glucosephosphotransferase 2.7.8.2 Diacylglycerol Cholinephosphotransferase 2.7.8.20 Phosphatidylglycerol—Membrane-Oligosaccharide 2.7.8.5 Cdpdiacylglycerol--Glycerol-3-Phsophate 3- Phosphatidyltransferase 2.7.8.6 Undecaprenyl-Phosphate Galactosephosphotransferase
2.7.8.7 Holo- [Acyl-Carrier Protein] Synthase
2.7.8.8 Cdpdiacylglycerol--Serine 0- Phosphatidyltransferase 2.7.9.2 Pyruvate, ater Dikinase
2.8.1.1 Thiosulfate Sulfurtransferase
2.8.1.5 Thiosulfate--Dithiol Sulfurtransferase
2.8.3.5 3-Oxoacid Coa-Transferase
2.8.3.9 Butyrate—Acetoacetate Coa-Transferase 2.9.1.1 L-Seryl-Trna (Ser) Seleniumtransferase
3.1.1.1 Carboxylesterase
3.1.1.10 Tropinesterase
3.1.1.11 Pectinesterase
3.1.1.13 Sterol Esterase 3.1.1.2 Arylesterase
3.1.1.29 Aminoacyl-Trna Hydrolase
3.1.1.3 Triacylglycerol Lipase 3.1.1.32 Phospholipase Al 3.1.1.41 Cephalosporin-C Deacetylase 3.1.1.45 Carboxymethylenebutenolidase
3.1.1.5 Lisophospholipase 3.1.1.59 Juvenile-Hormone Esterase 3.1.1.61 Protein-Glutamate Methylesterase 3.1.1.8 Cholinesterase 3.1.11.1 Exodeoxyribonuclease 1
3.1.11.2 Exodeoxyribonuclease Iii
3.1.11.2 Exodeoxyribonuclease Iii
3.1.11.5 Exodeoxyribonuclease V
3.1.11.6 Exodeoxyribonuclease Vii 3.1.13.1 Exoribonuclease Ii
3.1.13.4 Poly (A) -Specific Ribonuclease 3.1.2.1 Acetyl-Coa Hydrolase
3.1.2.14 Oleoyl- [Acyl-Carrier-Protein] Hydrolase
3.1.2.15 Ubiquitin Thiolesterase 3.1.2.2 Palmitoyl-Coa Hydrolase
3.1.2.6 Hydroxyacylglutathione Hydrolase
3.1.21.1 Deoxyribonuclease I
3.1.21.2 Deoxyribonuclease Iv (Phage-T4-Induced)
3.1.21.3 Type I Site-Specific Deoxyribonuclease 3.1.21.4 Type Ii Site-Specific Deoxyribonuclease
3.1.21.5 Type Iii Site-Specific Deoxyribonuclease
3.1.22.4 Crossover Junction Endoribonuclease 3.1.25.1 Deoxyribonuclease (Pyrimidine Dimer) 3.1.26.3 Ribonuclease Iii 3.1.26.4 Calf Thymus Ribonuclease H 3.1.26.5 Ribonuclease P 3.1.27.1 Ribonuclease T2
3.1.27.6 Enterobacter Ribonuclease
3.1.3.1 Alkaline Phosphatase 3.1.3.10 Glucose-1-Phosphatase
3.1.3.11 Fructose-Bisphosphatase
3.1.3.12 Trehalose-Phosphatase
3.1.3.15 Histidinol-Phosphatase
3.1.3.16 Phosphoprotein Phosphatase 3.1.3.17 [Phosphorylase] Phosphatase
3.1.3.18 Phosphoglycolate Phosphatase
3.1.3.2 Acid Phosphatase
3.1.3.25 Myo-Inositol-1 (Or 4) -Monophosphatase 3.1.3.27 Phosphatidylglycerophosphatase 3.1.3.3 Phosphoserine Phosphatase 3.1.3.41 4-Nitrophenylphosphatase 3.1.3.43 [Pyruvate Dehydrogenase (Lipoamide) ] - Phosphatase
3.1.3.46 Fructose-2, 6-Bisphosphate 2-Phosphatase 3.1.3.48 Protein-Tyrosine-Phosphatase
3.1.3.5 5 ' -Nucleotidase
3.1.3.7 3 ' (2 ' ) , 5 ' -Bisphosphate Nucleotidase
3.1.3.8 3-Phytase
3.1.31.1 Micrococcal Nuclease 3.1.4.1 Phosphodiesterase I
3.1.4.11 l-Phosphatidylinositol-4, 5-Bisphosphate Phosphodiesterase
3.1.4.12 Sphingomyelin Phosphodiesterase 3.1.4.14 [Acyl-Carrier-Protein] Phosphodiesterase 3.1.4.16 2 ', 3 ' -Cyclic-Nucleotide 2 ' -Phosphodiesterase
3.1.4.17 3 ', 5 ' -Cyclic-Nucleotide Phosphodiesterase
3.1.4.3 Phospholipase C
3.1.4.46 Glycerophosphodiester Phosphodiesterase 3.1.4.50 Glycoprotein Phospholipase D 3.1.5.1 Dgtpase
3.1.6.1 Arylsulfatase
3.1.6.13 Iduronate-2-Sulfatase
3.1.6.14 N-Acetylglucosamine-6-Sulfatase
3.1.6.2 Steryl-Sulfatase 3.1.6.4 N-Acetylgalactosamine-6-Sulf atase
3.1.6.8 Cerebroside-Sulf atase
3.1.7.2 Guanosine-3 ', 5 ' -Bis (Diphosphate) 3'-
Pyrophosphatase
3.2.1.1 Alpha-Amylase 3.2.1.10 Oligo-1, 6-Glucosidase
3.2.1.113 Mannosyl-Oligosaccharide 1,2-Alpha-
Mannosidase
3.2.1.135 Neopullulanase
3.2.1.14 Chitinase 3.2.1.15 Polygalacturonase 3 . 2 . 1 . 17 Lysozyme
3.2.1.18 Exo-Alpha-Sialidase
3.2.1.20 Alpha-Glucosidase
3.2.1.21 Beta-Glucosidase 3.2.1.22 Alpha-Galactosidase
3.2.1.23 Beta-Galactosidase
3.2.1.24 Alpha-Mannosidase
3.2.1.25 Beta-Mannosidase
3.2.1.26 Beta-Fructofuranosidase 3.2.1.28 Alpha, Alpha-Trehalase
3.2.1.3 Glucan 1, 4-Alpha-Glucosidase 3.2.1.31 Beta-Glucuronidase 3.2.1.33 Amylo-1, 6-Glucosidase 3.2.1.37 Xylan 1, 4-Beta-Xylosidase 3.2.1.39 Glucan Endo-1, 3-Beta-D-Glucosidase
3.2.1.4 Cellulase
3.2.1.41 Alpha-Dextrin Endo-1, 6-Alpha-Glucosidase
3.2.1.48 Sucrose Alpha-Glucosidase
3.2.1.52 Beta-N-Acetylhexosaminidase 3.2.1.54 Cyclomaltodextrinase
3.2.1.55 Alpha-N-Arabinofuranosidase
3.2.1.58 Glucan 1, 3-Beta-Glucosidase
3.2.1.60 Glucan 1, 4-Alpha-Maltotetraohydrolase
3.2.1.65 Levanase 3.2.1.68 Isoamylase
3.2.1.7 Inulinase
3.2.1.70 Glucan 1, 6-Alpha-Glucosidase
3.2.1.73 Licheninase
3.2.1.74 Glucan 1, 4-Beta-Glucosidase 3.2.1.78 Mannan Endo-1, 4-Beta-Mannosidase
3.2.1.8 Endo-1, 4-Beta-Xylanase 3.2.1.82 Exo-Poly-Alpha-Galacturonosidase 3.2.1.85 6-Phospho-Beta-Galactosidase 3.2.1.91 Cellulose 1, 4-Beta-Cellobiosidase 3.2.1.93 Alpha, Alpha-Phosphotrehalase
3.2.1.96 Mannosyl-Glycoprotein Endo-Beta-N-
Acetylglucosaminidase
3.2.2.17 Deoxyribodipyrimidine Endonucleosidase
3.2.2.20 Dna-3-Methyladenine Glycosidase I 3.2.2.21 Dna-3-Methyladenine Glycosidase Ii
3.2.2.23 Formamidopyrimidine-Dna Glycosidase
3.2.2.24 Adp-Ribosyl- [Dinitrogen Reductase] Hydrolase 3.2.2.4 Amp Nucleosidase
3.3.1.1 Adenosylhomocysteinase 3.3.2.1 Isochorismatase
3.3.2.3 Epoxide Hydrolase
3.3.2.6 Leukotriene-A4 Hydrolase
3.4.11.1 Leucyl Aminopeptidase
3.4.11.1 Leucyl Aminopeptidase 3.4.11.10 Bacterial Leucyl Aminopeptidase 3.4.11.15 Lysyl Aminopeptidase
3.4.11.18 Methionyl Aminopeptidase
3.4.11.19 D-Stereospecific Aminopeptidase
3.4.11.2 Membrane Alanyl Aminopeptidase 5 3.4.11.5 Prolyl Aminopeptidase
3.4.11.7 Glutamyl Aminopeptidase
3.4.11.9 X-Pro Aminopeptidase
3.4.13.18 Cytosol Non-Specific Dipeptidase
3.4.13.3 X-His Dipeptidase 10 3.4.13.9 X-Pro Dipeptidase
3.4.14.11 Xaa-Pro Dipeptidyl-Peptidase 3.4.14.5 Dipeptidyl-Peptidase Iv 3.4.15.5 Peptidyl-Dipeptidase Dcp 3.4.16.1 Serine-Type Carboxypepetidase 15 3.4.16.4 Serine-Type D-Ala-D-Ala Carboxypeptidase
3.4.17.1 Carboxypeptidase A 3.4.17.15 Carboxypeptidase A2
3.4.17.2 Carboxypeptidase B
3.4.17.4 Gly-X Carboxypeptidase
20 3.4.17.8 Muramoylpentapeptide Carboxypeptidase 3.4.19.1 Acylaminoacyl-Peptidase
3.4.19.3 Pyroglutamyl-Peptidase I 3.4.21.1 Chymotrypsin
3.4.21.26 Prolyl Oligopeptidase 25 3.4.21.4 Trypsin
3.4.21.48 Cerevisin 3.4.21.53 Endopeptidase La
3.4.21.61 Kexin
3.4.21.62 Subtilisin 30 3.4.21.66 Thermitase
3.4.21.83 Oligopeptidase B 3.4.21.87 Omptin 3.4.21.92 Endopeptidase Clp 3.4.22.17 Calpain 35 3.4.23.18 Aspergillopepsin I
3.4.23.24 Candidapepsin
3.4.23.25 Saccharopepsin 3.4.23.33 Xanthomonapepsin
3.4.23.36 Signal Peptidase Ii ■40 3.4.23.5 Cathepsin D
3.4.24.11 Neprilysin
3.4.24.13 Iga-Specific Metalloendopeptidase
3.4.24.15 Thimet Oligopeptidase
3.4.24.27 Thermolysin 45 3.4.24.28 Bacillolysin
3.4.24.37 Saccharolysin 3.4.24.55 Pitrilysin
3 . 4 . 24 . 57 O-Syaloglycoprotein Endopeptidase' 3 . 4 . 99 . 36 Leader Peptidase 50 3 . 4 . 99 . 41 Mitochondrial Processing Peptidase 3.4.99.44 Pitrilysin
3.4.99.45 Insulinase
3.4.99.46 Multicatalytic Endopeptidase Complex
3.5.1.1 Asparaginase 3.5.1.10 Formyltetrahydrofolate Deformylase
3.5.1.11 Penicillin Amidase
3.5.1.13 Aryl-Acylamidase
3.5.1.14 Aminoacylase
3.5.1.16 Acetylornithine Deacetylase 3.5.1.18 Succinyl-Diaminopimelate Desuccinylase
3.5.1.2 Glutaminase
3.5.1.24 Choloylglycine Hydrolase
3.5.1.25 N-Acetylglucosamine-6-Phosphate Deacetylase
3.5.1.26 N4- (Beta-N-Acetylglucosaminyl) -L-Asparaginase 3.5.1.27 N-Formylmethionylaminoacyl-Trna Deformylase
3.5.1.28 N-Acetylmuramoyl-L-Alanine Amidase 3.5.1.32 Hippurate Hydrolase
3.5.1.38 Glutamin- (Asparagin-) Ase
3.5.1.4 Amidase 3.5.1.41 Chitin Deacetylase
3.5.1.5 Urease
3.5.1.59 N-Carbamoylsarcosine Amidase
3.5.2.12 6-Aminohexanoate-Cyclic-Dimer Hydrolase 3.5.2.14 N-Methylhydantoinase (Atp-Hydrolysing) 3.5.2.2 Dihydropyrimidinase
3.5.2.3 Dihydroorotase
3.5.2.5 Allantoinase
3.5.2.6 Beta-Lactamase 3.5.3.1 Arginase 3.5.3.11 Agmatinase
3.5.3.19 Ureidoglycolate Hydrolase
3.5.3.4 Allantoicase 3.5.3.6 Arginine Deiminase
3.5.4.1 Cytosine Deaminase 3.5.4.10 Imp Cyclohydrolase
3.5.4.12 Dcmp Deaminase
3.5.4.16 Gtp Cyclohydrolase I
3.5.4.19 Phosphoribosyl-Amp Cyclohydrolase
3.5.4.2 Adenine Deaminase 3.5.4.23 Blasticidin-S Deaminase 3.5.4.25 Gtp Cyclohydrolase Ii
3.5.4.4 Adenosine Deaminase
3.5.4.5 Cytidine Deaminase
3.5.4.6 Amp Deaminase 3.5.4.9 Methenyltetrahydrofolate Cyclohydrolase 3.5.5.1 Nitrilase
3.6.1.1 Inorganic Pyrophosphatase 3.6.1.11 Exopolyphosphatase
3.6.1.17 Bis (5 ' -Nucleosyl) -Tetraphosphatase (Asymmetrical) 3.6.1.23 Dutp Pyrophosphatase
3.6.1.26 Cdpdiacylglycerol Pyrophosphatase
3.6.1.3 Adenosinetriphosphatase
3.6.1.31 Phosphoribosyl-Atp Pyrophosphatase 3.6.1.32 Myosin Atpase
3.6.1.33 Dynein Atpase
3.6.1.34 H+-Transporting Atp Synthase
3.6.1.35 H+-Transporting Atpase
3.6.1.36 H+/K+-Exchanging Atpase 3.6.1.37 Na+/K+-Exchanging Atpase
3.6.1.38 Ca2H—Transporting Atpase
3.6.1.41 Bis (5 ' -Nucleosyl) -Tetraphosphatase (Symmetrical)
3.6.1.42 Guanosine-Diphosphatase 3.6.1.45 Udp-Sugar Siphosphatase
3.6.1.45 Udp-Sugar Siphosphatase
3.6.1.5 Apyrase
3.6.1.7 Acylphosphatase
3.6.1.9 Nucleotide Pyrophosphatase 3.7.1.3 Kynureninase
3.7.1.8 2, 6-Dioxo-6-Phenylhexa-3-Enoate Hydrolase
3.7.1.9 2-Hydroxymuconate-Semialdehyde Hydrolase 3.8.1.3 Haloacetate Dehalogenase
3.8.1.6 4-Chlorobenzoate Dehalogenase 4.1.1.1 Pyruvate Decarboxylase
4.1.1.11 Aspartate 1-Decarboxylase 4.1.1.15 Glutamate Decarboxylase
4.1.1.17 Ornithine Decarboxylase
4.1.1.18 Lysine Decarboxylase 4.1.1.19 Arginine Decarboxylase
4.1.1.20 Diaminopimelate Decarboxylase
4.1.1.21 Phosphoribosylammoimidazole Carboxylase 4.1.1.23 Orotidine-5 ' -Phosphate Decarboxylase 4.1.1.3 Oxaloacetate Decarboxylase 4.1.1.31 Phosphoenolpyruvate Carboxylase
4.1.1.32 Phosphoenolpyruvate Carboxykinase (Gtp)
4.1.1.33 Diphosphomevalonate Decarboxylase
4.1.1.37 Uroporphyrinogen Decarboxylase
4.1.1.39 Ribulose-Bisphosphate Carboxylase 4.1.1.41 Methylmalonyl-Coa Decarboxylase
4.1.1.44 4-Carboxymuconolactone Decarboxylase
4.1.1.47 Tartronate-Semialdehyde Synthase
4.1.1.48 Indole-3-Glycerol-Phosphate Synthase
4.1.1.49 Phosphoenolpyruvate Carboxykinase (Atp) 4.1.1.5 Acetolactate Decarboxylase
4.1.1.50 Adenosylmethionine Decarboxylase
4.1.1.64 2, 2-Dialkylglycine Decarboxylase (Pyruvate)
4.1.1.65 Phosphatidylserine Decarboxylase 4.1.1.71 2-0xoglutarate Decarboxylase 4.1.1.73 Tartrate Decarboxylase 4.1.1.8 Oxalyl-Coa Decarboxylase
4.1.2.13 Fructose-Bisphosphate Aldolase
4.1.2.14 2-Dehydro-3-Deoxyphosphogluconate Aldolase 4.1.2.16 2-Dehydro-3-Deoxyphosphooctonate Aldolase 4.1.2.17 L-Fuculose-Phosphate Aldolase 4.1.2.19 Rhamnulose-1-Phosphate Aldolase 4.1.2.25 Dihydroneopterin Aldolase 4.1.2.4 Deoxyribose-Phosphate Aldolase
4.1.3.1 Isocitrate Lyase 4.1.3.12 2-Isopropylmalate Synthase
4.1.3.16 4-Hydroxy-2-Oxoglutarate Aldolase 4.1.3.18 Acetolactate Synthase
4.1.3.2 Malate Synthase
4.1.3.21 Homocitrate Synthase 4.1.3.27 Anthranilate Synthase
4.1.3.3 N-Acetylneuraminate Lyase
4.1.3.36 Naphthoate Synthase
4.1.3.4 Hydroxymethylglutaryl-Coa Lyase
4.1.3.5 Hydroxymethylglutaryl-Coa Synthase '4.1.3.6 Citrate Lyase
4.1.3.7 Citrate (Si) -Synthase
4.1.99.1 Tryptophanase
4.1.99.2 Tyrosine Phenol-Lyase
4.1.99.3 Deoxyribodipyrimidine Photo-Lyase 4.1.99.3 Deoxyribodipyrimidine Photo-Lyase
4.1.99.4 1-Aminocyclopropane-l-Carboxylate Deaminase
4.2.1.1 Carbonate Dehydratase
4.2.1.10 3-Dehydroquinate Dehydratase
4.2.1.11 Phosphopyruvate Hydratase 4.2.1.12 Phosphogluconate Dehydratase
4.2.1.13 L-Serine Dehydratase
4.2.1.14 D-Serine Dehydratase
4.2.1.16 Threonine Dehydratase
4.2.1.17 Enoyl-Coa Hydratase 4.2.1.19 Imidazoleglycerol-Phosphate Dehydratase
4.2.1.2 Fumarate Hydratase 4.2.1.20 Tryptophan Synthase
4.2.1.22 Cystathionine Beta-Synthase 4.2.1.24 Porphobilinogen Synthase 4.2.1.3 Aconitate Hydratase
4.2.1.32 L(+)-Tartrate Dehydratase
4.2.1.33 3-Isopropylmalate Dehydratase 4.2.1.36 Homoaconitate Hydratase 4.2.1.36 Homoaconitate Hydratase 4.2.1.40 Glucarate Dehydratase
4.2.1.41 5-Dehydro-4-Deoxyglucarate Dehydratase
4.2.1.46 Dtdpglucose 4, 6-Dehydratase
4.2.1.49 Urocanate Hydratase
4.2.1.51 Prephenate Dehydratase 4.2.1.52 Dihydrodipicolinate Synthase 4.2.1.60 3-Hydroxydecanoyl- [Acyl-Carrier-Protein] Dehydratase
4.2.1.61 3-Hydroxypalmitoyl- [Acyl-Carrier-Protein] Dehydratase 4.2.1.69 Cyanamide Hydratase
4.2.1.7 Altronate Dehydratase 4.2.1.70 Pseudouridylate Synthase
4.2.1.74 Long-Chain-Enoyl-Coa Hydratase
4.2.1.75 Uroporphyrinogen-Iii Synthase 4.2.1.8 Mannonate Dehydratase
4.2.1.89 Carnitine Dehydratase
4.2.1.9 Dihydroxy-Acid Dehydratase 4.2.1.96 Tetrahydrobiopterin Dehydratase
4.2.2.10 Pectin Lyase 4.2.2.2 Pectate Lyase
4.2.99.11 Methylglyoxal Synthase
4.2.99.18 Dna- (Apurinic Or Apyrimidinic Site) Lyase 4.2.99.2 Threonine Synthase 4.2.99.8 Cysteine Synthase 4.2.99.9 O-Succinylhomoserine (Thiol) -Lyase 4.3.1.1 Aspartate Ammonia-Lyase
4.3.1.12 Ornithine Cyclodeaminase
4.3.1.3 Histidine Ammonia-Lyase
4.3.1.4 Formiminotetrahydrofolate Cyclodeaminase 4.3.1.7 Ethanolamine Ammonia-Lyase
4.3.1.8 Hydroxymethylbilane Synthase
4.3.2.1 Argininosuccinate Lyase
4.3.2.2 Adenylsuccinate Lyase 4.3.99.1 Cyanate Lyase 4.4.1.1 Cystathionine Gamma-Lyase
4.4.1.11 Methionine Gamma-Lyase
4.4.1.14 1-Aminocyclopropane-l-Carboxylate Synthase 4.4.1.17 Holocytochrome-C Synthase
4.4.1.5 Lactoylglutathione Lyase 4.4.1.8 Cystathionine Beta-Lyase
4.5.1.5 S-Carboxymethylcysteine Synthase 4.6.1.1 Adenylate Cyclase
4.6.1.10 6-Pyruvoil Tetrahydrobiopterin Synthase
4.6.1.3 3-Dehydroquinate Synthase 4.6.1.4 Chorismate Synthase
4.99.1.1 Ferrochelatase
5.1.1.1 Alanine Racemase
5.1.1.11 Phenylalanine Racemase (Atp-Hydrolysing)
5.1.1.13 Aspartate Racemase 5.1.1.3 Glutamate Racemase
5.1.1.7 Diaminopimarate Epimerase
5.1.2.2 Mandelate Racemase
5.1.2.3 3-Hydroxybutyryl-Coa Epimerase 5.1.3.1 Ribulose-Phosphate 3-Epimerase 5.1.3.13 Dtdp-4-Dehydrorhamnose 3, 5-Epimerase 5.1.3.14 Udp-N-Acetylglucosamine 2-Epimerase
5.1.3.2 Udpglucose 4-Epimerase
5.1.3.3 Aldose 1-Epimerase
5.1.3.4 L-Ribulose-Phosphate 4-Epimerase 5.2.1.8 Peptidylprolyl Isomerase
5.3.1.1 Triose-Phosphate Isomerase
5.3.1.10 Glucosamine-6-Phosphate Isomerase
5.3.1.12 Glucuronate Isomerase
5.3.1.14 L-Rhamnose Isomerase 5.3.1.16 N- (5 ' -Phospho-D-Ribosylformimino) -5-Amino-l-
5.3.1.17 4-Deoxy-L-Threo-5-Hexosulose-Uronate Ketol-
Isomerase
5.3.1.24 Phosphoribosylanthranilate Isomerase
5.3.1.4 L-Arabinose Isomerase 5.3.1.5 Xylose Isomerase
5.3.1.6 Ribose-5-Phosphate Epimerase
5.3.1.8 Mannose-6-Phosphate Isomerase •
5.3.1.9 Glucose-6-Phosphate Isomerase
5.3.3.1 Steroid Delta-Isomerase 5.3.3.10 5-Carboxymethyl-2-Hydroxymuconate Delta- Isomerase
5.3.3.2 Isopentenyl-Diphosphate Delta-Isomerase 5.3.3.8 Dodecenoyl-Coa Delta-Isomerase
5.3.4.1 Protein Disulfide-Isomerase 5.4.2.1 Phosphoglycerate Mutase
5.4.2.2 Phosphoglucomutase
5.4.2.3 Phosphoacetylglucosamine Mutase
5.4.2.4 Bisphosphoglycerate Mutase
5.4.2.6 Beta-Phosphoglucomutase 5.4.2.7 Phosphopentomutase
5.4.2.8 Phosphomannomutase
5.4.2.9 Phosphoenolpyruvate Mutase
5.4.3.8 Glutamate-1-Semialdehyde 2, 1-Aminomutase 5.4.99.12 Trna-Pseudouridine Synthase I 5.4.99.2 Methylmalonyl-Coa Mutase
5.4.99.5 Chorismate Mutase
5.4.99.6 Isochorismate Synthase
5.4.99.7 Lanosterol Synthase
5.4.99.8 Cycloartenol Synthase 5.5.1.1 Muconate Cycloisomerase
5.5.1.2 3-Carboxy-Cis, Cis-Muconate Cycloisomerase 5.5.1.4 Myo-Inositol-1-Phosphate Synthase
5.5.1.7 Chloromuconate Cycloisomerase 5.99.1.2 Dna Topoisomerase 5.99.1.3 Dna Topoisomerase (Atp-Hydrolysing) 6.1.1.1 Tyrosine—Trna Ligase
6.1.1.10 Methionine—Trna Ligase
6.1.1.11 Serine--Trna Ligase
6.1.1.12 Aspartate—Trna Ligase 6.1.1.14 Glycine—Trna Ligase 6.1.1.15 Proline--Trna Ligase
6.1.1.16 Cysteine—Trna Ligase
6.1.1.17 Glutamate—Trna Ligase
6.1.1.18 Glutamine—Trna Ligase 6.1.1.19 Arginine—Trna Ligase
6.1.1.2 Tryptophan--Trna Ligase
6.1.1.20 Phenylalanine—Trna Ligase
6.1.1.21 Histidine—Trna Ligase
6.1.1.22 Asparagine—Trna Ligase 6.1.1.3 Threonine—Trna Ligase
6.1.1.4 Leucine--Trna Ligase
6.1.1.5 Isoleucine—Trna Ligase
6.1.1.6 Lysine—Trna Ligase
6.1.1.7 Alanine—Trna Ligase 6.1.1.9 Valine—Trna Ligase
6.2.1.1 Acetate—Coa Ligase 6.2.1.12 4-Coumarate—Coa Ligase
6.2.1.14 6-Carboxyhexanoate—Coa Ligase 6.2.1.22 Citrate (Pro-3s) -Lyase Ligase 6.2.1.26 O-Succinylbenzoate—Coa Ligase 6.2.1.27 4-Hydroxybenzoate--Coa Ligase
6.2.1.3 Long-Chain-Fatty-Acid—Coa Ligase
6.2.1.4 Succinate--Coa Ligase (Gdp-Forming) 6.3.1.1 Aspartate--Ammonia Ligase 6.3.1.2 Glutamate--Ammonia Ligase
6.3.1.5 Nad+ Synthase
6.3.2.1 Pantoate—Beta-Alanine Ligase
6.3.2.12 Dihydrofolate Synthase
6.3.2.13 Udp-N-Acetylmuramoylalnyl-D-Glutamate—2 , 6- Diaminopimelate Ligase
6.3.2.15 Udp-N-Acetylmuramoylalanyl-D-Glutamyl-2 , 6- 6.3.2.17 Tetrahydrofolylpolyglutamate Synthase
6.3.2.19 Ubiquitin—Protein Ligase
6.3.2.2 Glutamate—Cysteine Ligase 6.3.2.25 Tublin-Tyrosine Ligase
6.3.2.3 Glutathione Synthase
6.3.2.4 D-Alanine-D-Alanine Ligase
6.3.2.6 Phosphoribosylaminoimidazolesuccinocarboxyamide Synthase 6.3.2.8 Udp-N-Acetylmuramate—Alanine Ligase
6.3.2.9 Udp-N-Acetylmuramoylalanine—D-Glutamate Ligase
6.3.3.1 Phosphoribosylformylglycinamidine Cyclo-Ligase
6.3.3.2 5-Formyltetrahydrofolate Cyclo-Ligase
6.3.3.3 Dethiobiotin Synthase 6.3.4.13 Phosphoribosylamine—Glycine Ligase
6.3.4.14 Biotin Carboxylase
6.3.4.15 Biotin-- [Acetyl-Coa-Carboxylase] Ligase
6.3.4.16 Carbamoyl-Phosphate Synthase (Ammonia) 6.3.4.2 Ctp Synthase 6.3.4.3 Formate—Tetrahydrofolate Ligase 6.3.4.4 Adenylosuccinate Synthase
6.3.4.5 Argininosuccinate Synthase
6.3.4.6 Urea Carboxylase
6.3.5.1 Nad+ Synthase (Glutamine-Hydrolysing) 6.3.5.2 Gmp Synthase (Glutamine-Hydrolysing)
6.3.5.3 Phosphoribosylfor ylglycinamidine Synthase
6.3.5.4 Asparagine Synthase (Glutamine-Hydrolysing)
6.3.5.5 Carbamoyl-Phosphate Synthase (Glutamine- Hydrolysing) 6.4.1.1 Pyruvate Carboxylase
6.4.1.2 Acetyl-Coa Carboxylase
6.4.1.3 Propionyl-Coa Carboxylase
6.5.1.1 Dna Ligase (Atp)
6.5.1.2 Dna Ligase (Nad+) 6.5.1.4 Rna-3 ' -Phosphate Cyclase
Of the proteins which are involved in cell integrity and thereby maintain and/or modify the biocatalyst, particular mention must be made of the proteins of DNA replication, DNA repair, transcription, translation, cell division and of the extracellular matrix. These are in particular DNA and RNA polymerases, DNA and RNA helicases, DNA and RNA single strand-binding proteins, DNA and RNA ligases, topoisomerases and gyrases and also recombinases . Likewise, translation factors such as elongation factors, for example EF-Tu, EF-Ts, EF-G, initiation factors, release factors and also ribosomal proteins, tRNA synthetases, chaperones and chaperonins and also components of the extracellular matrix such as, for example, the antigen 84.
Of the proteins of signal recognition and signal transduction, particular mention must be made of the histidine kinases and response regulators of the prokaryotic two-component systems, likewise of protein kinases and protein phosphatases . In the case of the eukaryotic signal transduction pathways, those systems must be mentioned, in which the following components are involved: inositol triphosphate (IP3) , diacylglycerol (DAG), G proteins, MAP kinases, tyrosine kinases, Janus kinases, phospholipase A, phospholipase C, protein kinase C,. and also calcium- dependent systems such as calcium-calmodulin kinases (CAM kinases) .
Of the proteins of prokaryotic gene regulation, particular mention must be made of the following families of transcription factors:
sigma(70), sigma(70) ECF family, sigma(54), types derived from sigma(54), MarR type, Lad type, GntR type, LysR type, ArsR type, DeoD type, AraC type, GerE type, Crp type, Xre type, MerR type, TetR type, AsnC type, LexA type, Hth6 type, response regulators, (two- component systems), Fur type, Bgl-antitermination types, iclR type, GreA/B type, Fe-dependent transcription factors, HrcA type, Arg-repressor type.
Of the proteins of eukaryotic gene regulation, particular mention must be made of the following transcription factors: transcription factors with basic domains such as, for example, leucine-zipper factors (bZIP) , helix-loop-helix factors (bHLH) , helix-loop- helix/leucine-zipper factors (bHLH-ZIP) , NF-1 type factors, RF-X type factors, bHSH type factors. Transcription factors with zinc-finger domains such as, for example, Cys 4 zinc finger, Cys2His2 zinc finger or Cys6 cysteine-zinc cluster. Transcription factors with helix-turn-helix motifs such as, for example, homeo-box proteins, heat-shock factors or the tryptophan-cluster family. Furthermore, transcription factors with beta-
1 sheet structures such as, for example, the factors of the p53 family, MADS-box proteins or TATA-binding proteins .
Of the proteins having storage function, particular mention must be made of the systems for the synthesis of polyphosphates and storage carbohydrates such as glycogen or starch, likewise storage proteins for inorganic ions such as, for example, iron.
Proteins having transport function are ABC transporters, primary, secondary and binding protein- dependent transport systems and proteins facilitating diffusion. Examples are the bacterial maltose/trehalose binding proteins (malE) , multidrug-resistance proteins, components of the PTS systems for sugar uptake (HPr, enzyme I, II and III) and transport proteins secreting the components of the extracellular matrix. In addition to this, all proteins involved in the uptake or secretion of proteins and peptides.
Proteins involved in cellular biogenesis. Proteins involved in the nitrogen metabolism, such as, for example, uridilyl transferase, or else proteins involved in homeostasis of ions.
Proteins which may serve as target proteins and proteins which have an altered phosphorylation pattern compared to the wild type can be found or detected by any of the methods described in the literature. Methods suitable for this purpose are those described in
Wind, M. et al., Anal. Chem. 2001, 73, 29-35, those in Ahn, N. & Resing, K. , Nature Biotechnology 2001, 19,
317-318, and in Quadroni, M. & James, P., Proteomics in functional Genomics, 2000 (Birkhauser Verlag, Basle,
Switzerland, P. Jolles and H. Jornvall, editors) which can be used for detecting phosphorylation of the protein or any method for protein or DNA analysis, which can detect the amino acid exchange in comparison with the wild-type protein.
The invention comprises the use of microorganisms in which the phosphorylation state of at least one protein has been permanently changed compared to that of the protein naturally occurring in this organism for the preparation of a fine chemical. When working on the present invention, it was found that microorganisms, in particular coryneform bacteria and E. coli , produce, after modification of the phosphorylation state of proteins, amino acids, in particular L-lysine and L-threonine, and vitamins, in particular pantothenic acid, in an improved manner.
The proteins in question, whose phosphorylatability has been changed, can be introduced into the target cells by introducing plasmids which carry the protein- encoding genes or gene constructs containing the desired amino acid exchange. After the gene sequences have been introduced, they may be located in the cell either still on the plasmids or integrated into the chromosome of the microorganism.
Instructions for site-directed mutagenesis and for introducing plasmids into the microorganisms can be found by the skilled worker, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in the European Patent EPS 0 472 869, in US Patent 4,601,893, in Schwarzer and Pϋhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al . (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246, in Malumbres et al . (Gene 134, 15-24 (1993)), in the Japanese Patent JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in well-known text books of genetics and molecular biology.
Suitable plasmids are those which are replicated and expressed in the selected microorganism. For coryneform bacteria, for example, numerous known plasmid vectors can be used, such as, for example, pZl (Menkel et al . ,
Applied and Environmental Microbiology (1989) 64:
549-554), pEKExl (Eikmanns et al . , Gene 102: 93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107: 69-74
(1991) ) , which are based on the cryptic plasmids pHMl519, pBLl or pGAl . Other plasmid vectors such as, for example, those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAGl (US-A 5, 158 , 891) may be used for coryneform bacteria in the same way.
Plasmid vectors which can be replicated in Enterobacteriaceae, such as, for example, 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 Science USA 80 (21): 6557-6561 (1983)), may be used. It is likewise possible to transfer mutations which relate to expression of the particular genes to various strains by sequence exchange (Hamilton et al . (Journal of Bacteriology 174, 4617-4622 (1989)), conjugation or transduction.
The microorganisms prepared according to the invention may be cultured continuously or batchwise or in a fed batch or repeated fed batch process for the purpose of producing the fine chemicals, in particular amino acids, nucleosides, nucleotides, pigments, antibiotics or vitamins. For each cell type, various suitable culture conditions are available. The cultivation method used is not limiting to the invention. A review of known culturing methods is described in the text book by Chmiel (BioprozeBtechnik 1. Einfϋhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the text book by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Brunswick/Wiesbaden, 1994) ) .
The culture medium to be used must satisfy the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms can be found in the manual "Manual of Methods for General Bacteriology" of the American Socity for Bacteriology (Washington D.C., USA, 1981). Carbon sources which may be used are sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol and ethanol and organic acids such as, for example, acetic acid or amino acids such as glutamine. These substances may be used individually or as a mixture. When using photosynthetic bacteria, light may be used as energy source. Nitrogen sources which may be used are 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 sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts. Moreover, the culture medium must contain metal salts such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, it is possible, in addition to the abovementioned substances, to employ essential growth substances such as amino acids and vitamins. In addition to this, suitable precursors can be added to the culture medium. The said starting materials can be added to the culture in the form of a single batch or be fed in during the cultivation in a suitable manner.
The pH of the culture is controlled by employing basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters . To maintain the stability of plasmids, it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, air into the culture. The temperature of the culture is normally 20 °C to 45 °C and preferably 25°C to 40°C.
The fine chemicals produced can be analysed, for example, by anion exchange chromatography with subsequent ninhydrin derivatization, as described in Spackman et al . (Analytical Chemistry, 30, (1958), 1190) .
The method of the invention serves to produce, by way of fermentation, fine chemicals, in particular amino acids, nucleosides, nucleotides and vitamins. Of the fine chemicals produced, particular preference is given to L-lysine, L-threonine, L-methionine, L-tryptophan and pantothenic acid.
Examples
The present invention is described in more detail hereinbelow on the basis of exemplary embodiments.
Example 1 :
Detection of phosphorylated proteins by radioactive labelling or antibodies
To detect phosphorylated proteins, first cells from shaker-flask experiments or fermentations were grown at particular times or conditions. Corynebacterium glutamicum cells were then disrupted as described in Hermann et al . (Electrophoresis 19, 3217-3221, 1998), Hermann et al . (Electrophoresis 21, 654-659, 2000) and Hermann et al . (Electrophoresis 22, 1712-1723, 2001) and the proteins were purified and fractionated. The corresponding purification of proteins from cell cultures of Escherichia coli was carried out as described in Molloy et al . (Electrophoresis 19, 837-844, 1998). The proteins were then fractionated according to their isoelectric point and their molecular weight with the aid of two-dimensional gel electrophoresis, as described in Hermann et al .
(Electrophoresis 22, 1712-1723, 2001) in the case of
Corynebacterium glutamicum and in Molloy et al . (Electrophoresis 19, 837-844, 1998) in the case of Escheri chia coli . After electrophoresis, the proteins were blotted onto a PVDF membrane with the aid of the semi-dry blotting method (Immobilon-P, pore diameter 0.45 μm, Millipore, Eschborn, D) . For this purpose, the gel was first equilibrated in anode buffer consisting of 20 mM Tris and 150 mM glycine, pH 8.3, for 30 minutes. This was followed by soaking six layers of filter paper (Novablot PKG/500, Pharmacia Biotech, Freiburg, D) in anode buffer and placing them on the anode of the . blotting apparatus (Multiphor II and Nova Blot, Pharmacia Biotech, Freiburg, D) . The PVDF membrane was laid on top of this and the gel to be blotted in turn on top of the membrane. Then six layers of filter paper were soaked in cathode buffer which contained, compared to the anode buffer, additionally 0.1% SDS and the layers were placed on top of the gel. Finally, the graphite cathode was placed on the top. The proteins were transferred at room temperature and 10 V, 0.8 mA/cm2 and 5 W for approx. one to two hours. After the blotting, the membrane was dried. This was followed by incubating the membrane in a blocking solution made of 1% bovine serum albumin (fraction V, Sigma, Deisenhofen, D) , 1% polyvinylpyrrolidone (Sigma, Deisenhofen, D) , 1% PEG 3350 (Sigma, Deisenhofen, D) and 0.2% Tween 20 (Sigma, Deisenhofen, D) in PBS buffer. PBS buffer consisted of 274 mM NaCl, 5.4 mM KCl, 20 mM Na2HP04 and 3.6 mM KH2P04 at pH 7.2. The membrane was then incubated in 12.5 ml of a solution of the primary antibody for 1 hour. The primary antibody is directed against phosphorylated amino acids, in particular phosphothreonine, phosphoserine and phosphotyrosine . The following antibodies were used: monoclonal anti-phosphoserine antibodies clones 1C8, 4A3, 4A9, 4H4, 7F12 and 16B4; monoclonal anti- phosphothreonine antibodies, clones 14B3, 1E11 and 4D11; monoclonal anti-phosphotyrosine antibodies 4G10 and 3B12 (Biomol, Hamburg, D) . The membrane was incubated by dissolving 12.5 μg of the particular primary antibody in 1 ml of double-distilled water and adding 12.5 ml of blocking solution. The membrane was then washed five times for five minutes each in a washing solution which consisted of 10 mM Tris (Sigma, Deisenhofen, D) , 150 mM NaCl and 0.1% (v/v) Tween 20
(Sigma, Deisenhofen, D) and had been adjusted to pH 7.4 with HCl. The washed membrane is then incubated in
100 ml of a solution of the secondary antibody for one hour. For this purpose, depending on the type of primary antibody, 0.6 mg of AffiniPure goat anti-mouse IgG or IgM in the form of an alkaline-phosphatase conjugate (Dianova, Hamburg, D) was dissolved in 1 ml of double-distilled water and diluted 1:5 000 to 1:50 000 in double-distilled water. The membrane was then again washed five times for five minutes in a washing solution consisting of 10 mM Tris (Sigma,
Deisenhofen, D) , 150 mM NaCl and 0.1% (v/v) Tween 20
(Sigma, Deisenhofen, D) , and adjusted to pH 7.4 with HCl. Finally, the membrane was treated with 10 ml of detecting solution until a purple stain indicates the protein spots or a positive control. The detecting solution used for this purpose consisted of SigmaFast BCIP/NBT buffered substrate tablets (Sigma, Deisenhofen, D) of which one was dissolved in each case in 10 ml of double-distilled water. The membrane was stained and then washed with double-distilled water, dried and stored protected from light. Purple-stained protein spots indicate phosphorylated proteins. Using molecular-weight standards and protein patterns, the spot pattern on the membranes was compared and aligned with that of Coomassie-stained two-dimensional gels
(Neuhoff et al., Electrophoresis 9, 255-262, 1988).
Besides the detection via antibodies described herein, any other method may be employed for detecting phosphorylated proteins. Especially important is labelling of the proteins with 32P or 33P, for which radioactive 32P04 3_ or 33P04 3~ is added to growing cells. Phosphorylated proteins are then identified with the aid of autoradiography (Gooley and Packer, in: Proteome Research: New Frontiers in Functional Genomics, Springer Verlag, Berlin, 65-92, 1997) .
In addition to two-dimensional gel electrophoresis, the proteins to be studied may also be isolated using any other separation method, for example capillary electrophoresis (Liu et al . , Journal of Chromatography A, 918 (2), 401-409, 2001), all types of chromatographic separation methods including thin-layer chromatography, or else by applying molecular biological methods such as, for example, His tagging.
The phosphorylated proteins were identified as described in Hermann et al . (Electrophoresis 22, 1712-1723, 2001) by excising the spots from Coomassie- stained gels, digesting the proteins with specific proteases and subsequently identifying the peptide by means of MALDI or electrospray mass spectrometry.
The DNA and protein sequences of suitable microorganisms which can produce the desired compounds can be found in several data bases, for example in the NCBI data base (National Center For Biotechnology Information) . The data base can be found in the National Liabory of Medicine, Building 38A, Room 8N 805, Bethesda, MD 20894 USA
(http://www.ncbi.nlm.nih.gov). Another data base in which the according sequences can be obtained is the Swiss Prot and Trembl data base of the Swiss Institute of Bioinformatics CMO-Rue Michelle-ser ve 1, 1211 Geneve 4, Switzerland (http://www.expasy.ch). Yet another data base is the data base PIR, the Protein Information Researse Database of the National Biomedical Research Foundation, 3900 Reservoir Road, NW.
Table 1 shows some of the identified posphorylated proteins of Corynebacterium glutamicum .
Table 1
Figure imgf000047_0001
Figure imgf000048_0001
Table 2 shows some of the identified phosphorylated proteins of Escherichia coli .
Tabelle 2
Figure imgf000048_0002
Figure imgf000049_0001
Example 2 :
Detection of phosphorylation sites in bacterial proteins by ESI-MS and ICP-MS
Phosphorylation sites may be identified in pure proteins, polypeptides or protein mixtures as described in Neubauer and Mann (Analytical Chemistry, 71(1), 235-242, 1999), Yan et al . (Journal of Chromatography A, 808 (1-2), 23-41, 1998), Oda et al. (Proceedings of the National Academy of Sciences of the USA, 96, 6591-6596, 1999), Wind et al. (Anal. Chem. 73, 29-35, 2001) , Ahn and Resing (Nature Biotechnology 19, 317-318, 2001), or Quadroni and James (in: Proteomics in Functional Genomics, irkhauser Verlag, Basle, Switzerland, P. Jolles and H. Jδrnvall, editors, 2000). A review of methods for identifying phosphorylation sites in proteins or peptides can furthermore be found in Gooley and Packer (in: Proteome Research: New Frontiers in functional Genomics, Springer Verlag, Berlin, 65-92, 1997) . Example 3 :
Modification of the phosphorylation site in Corynebacterium glutamicum enolase by site-directed mutagenesis
By using the methods described in examples 1 and 2 the protein enolase can be identified as a phosphorylated protein. The serine residue in position 330 (S330) represents the amino acid on which phosphorylation takes place.
3.1. Obtaining the eno-encoding DNA
The Corynebacterium glutamicum strain ATCC13032 is used as donor for the chromosomal DNA. Chromosomal DNA is isolated from the strain ATCC13032 by the usual methods
(Eikmanns et al., Microbiology 140: 1817-1828 (1994)).
A DNA fragment carrying the eno gene is amplified with the aid of the polymerase chain reaction. Owing to the known sequence of the C. glutamicum eno gene (SEQ ID No. 1) (Accession number AX136862), the following primer oligonucleotides are selected for the PCR:
6H-enol (SEQ ID No. 7):
5 ' CGC ( GGA TCO GCT GAA ATC ATG CAC GTA TTC 3 '
6H-eno2 ( SEQ ID No . 8 ) :
5' GCG(AGA TCT) (CCC GGG) TTA GCC CTG AAA GCG TGG C 3'
The primers depicted are synthesized by MWG Biotech and the PCR reaction is carried out according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) . The primers make it possible to amplify an approx. 1.3 kb DNA fragment carrying the eno gene. Moreover, the primers contain the sequences of cleavage sites of the restriction endonucleases BamHI (6H-enol) and, respectively, Xmal and Bglll (6H-eno2) , which are indicated by parentheses in the nucleotide sequence depicted above.
The amplified DNA fragment of approx. 1.3 kb, which carries the eno gene, is identified via gel electrophoresis in a 0.8% strength agarose gel and purified by the usual methods (High Pure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim) .
The vector pQE-30 (Qiagen, Hilden, Germany) was completely cleaved with the enzymes BamHI and Xmal and the 1.3 kb DNA fragment containing the eno gene (Accession number M89931) (Rossol and Piihler, Journal of Bacteriology 174 (9), 2968-2977 (1992)) of C. glutamicum ATCC 13032 was completely cleaved by the enzymes BamHI and Xmal. The etio-carrying fragment was ligated into the vector with the aid of T4 DNA ligase. The DNA was transformed into the E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). The transformants were selected on LB medium containing 100 mg/1 ampicillin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel. The DNA sequence of the amplified DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 3. The plasmid obtained is denoted pQE-30eno.
3.2. Site-specific mutagenesis, exchange of Ser for Glu
Site-directed mutagenesis is carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, USA) . Owing to the phosphorylation site of C. glutamicum enolase, serine at position 330, which is known from Example 2, the following primer oligonucleotides are selected for linear amplification: S330E-1 ( SEQ ID No . 13 ) :
5 ' CGCTAAGAAGGCTGCCAAC (GAG) ATCCTGGTTAAGGTGAACC 3 '
S330E-2 (SEQ ID No. 14) :
5 ' GGTTCACCTTAACCAGGAT (CTC) GTTGGCAGCCTTCTTAGCG 3'
The primers depicted are synthesized by MWG Biotech. The codon for glutamate which is intended to replace serine at position 330 is indicated by parentheses in the nucleotide sequence depicted above. The plasmid pQE-30eno, described in Example 3.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase. This primer extension generates a mutated plasmid with nicked circular strands. The product of the linear amplification is treated with Dpnl; this endonuclease specifically cuts the methylated and semi-methylated template DNA. The newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XL1 Blue cells repair the nicks in the mutated plasmid. The transformants were selected on LB medium containing 100 mg/1 ampicillin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel. The DNA sequence of the mutated DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 4. The plasmid obtained is denoted pQE-30enoS330E .
3.3. Incorporation of the eno allele into the chromosome The eno allele obtained in Example 3.2. is incorporated into the C. glutamicum chromosome at the eno-gene locus by means of exchange mutagenesis with the aid of the sacB system described in Schafer et al . , Gene, 14, 69-73 (1994). This system allows the skilled worker identification or selection of allele exchanges which take place during homologous recombination.
3.3.1. Construction of the exchange vector pK18mobsacBenoS330E
The plasmid pQE-30enoS330E, described in Example 3.2. is cut with the restriction enzymes EcoRI and Hindlll (Gibco Life Technologies GmbH, Karlsruhe, Germany) and, after fractionation in an agarose gel (0.8%), an approx. 0.8 kb eno fragment carrying the mutation is isolated from the agarose gel with the aid of the High Pure PCR-product purification kit (Roche, Mannheim, Germany) and used for ligation with the mobilizable cloning vector pKlδmobsacB described in Schafer et al., Gene, 14, 69-73 (1994). The said cloning vector is likewise cleaved with the restriction enzymes EcoRI and Hindlll beforehand, mixed with the approx. 0.8 kb eno fragment and treated with T4 DNA ligase (Amersham- Pharmacia, Freiburg, Germany) . This is followed by transforming the E . coli strain DH5 (Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) with the ligation mixture (Hanahan, in DNA cloning. A practical approach. Vol. 1, ILR Press, Cold Spring Harbor, New York, 1989) . Cells containing the plasmid are selected by plating out the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, New York, 1989) supplemented with 25 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of the High Pure plasmid isolation kit from Roche and checked by restriction cleavage with the enzymes Hindlll/EcoRI and subsequent agarose gel electrophoresis. The plasmid is denoted pKl8mobsacBenoS330E. Figure 1 depicts a map of the plasmid.
3.3.2. Exchange of the eno allele in the C. glutamicum strain DSM5715
The vector pK18mobsacBenoS330E, mentioned in Example 3.3.1, was transferred into the C. glutamicum strain DSM5715 by means of electroporation (Haynes 1989, FEMS Microbiology Letters 61: 329-334). The vector cannot replicate autonomously in DSM5715 and is retained in the cell only when integrated into the chromosome. Clones with integrated pK18mobsacBenoS330E are selected by plating out the electroporation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor, New York, 1989) supplemented with 15 mg/1 kanamycin. Grown clones are streaked out on LB agar plates containing 25 mg/1 kanamycin and incubated at 33 °C for 16 hours. Mutants in which the plasmid containing the wild-type copy has been excised as the result of a second recombination event (according to Schafer et al . , 1990, Journal of Microbiology 172: 1663-1666) are selected by cultivating the clones, after incubation in LB liquid medium for 16 hours, on LB agar containing 10% sucrose. The plasmid pK18mobsacB contains, in addition to the kanamycin-resistance gene, a copy of the sacB gene coding for Bacillus subtilis levan sucrase. Sucrose- inducible expression results in the formation of levan sucrase which catalyses synthesis of the product levan which is toxic to C. glutamicum . Therefore, only those clones in which the integrated pK18mobsacBenoS330E has again been excised grow on sucrose-containing LB agar. During the excision, either the allele is exchanged and, respectively, the mutation incorporated or the original copy remains in the host chromosome, depending on the location of the second recombination event with respect to the site of mutation. Approximately 40 colonies are tested for the phenotype "growth in the presence of sucrose" and "no growth in the presence of kanamycin". Two colonies which have the phenotype "growth in the presence of sucrose" and "no growth in the presence of kanamycin" are studied with the aid of the LightCycler from Roche Diagnostics (Mannheim, Germany) in order to detect the mutation of the eno_S330E allele in the chromosome. The LightCycler is a combination of a thermocycler and a fluorimeter.
In the first phase, an approx. 0.3 kb DNA section containing the site of mutation is amplified by means of PCR (Innis et al., PCR protocols. A guide to methods and applications, 1990, Academic Press) using the following primer oligonucleotides.
LC-enol (SEQ ID No. 9) :
5' TGCAGGAAGATGACTGGGAG 3'
LC-eno2 (SEQ ID No. 10) : 5' ACCAGTCTTGATCTGGCCAC 3'
In the second phase, the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes
(LightCycler (LC)-Red640 and fluorescein) and which hyrbidize around the site of mutation, with the aid of the fluorescence resonance energy transfer (FRET) method (Lay et al . , Clinical Chemistry, 43: 2262-2267 (1997) ) .
eno330EC (SEQ ID No. 11):
5' LC-Red640 - AACCAGGATCTCGTTGGCAGC - (P) 3' eno330-A ( SEQ ID No . 12 ) :
5' CGAAGGTCTCGGTGAGGGTACCGATCTGGTTCACC - fluorescein 3'
The PCR primers depicted are synthesized by MWG Biotech and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
In this way, a clone was identified which contains the base triplet guanine-adenine-guanine at positions 988-990 of the coding region of the eno gene and thus has the eno_S330E allele (SEQ ID No. 5) . This clone was referred to as C. glutamicum DSM5715enoS330E strain.
Example 4 :
Effect of the mutagenesis carried out in Example 3 on the production of lysine in a model strain
Production of L-lysine
The C. glutamicum strain • DSM5715enoS330E obtained in Example 3 was cultured in a medium suitable for the production of L-lysine and the L-lysine content in the culture supernatant was determined.
For this purpose, the strain was first incubated on an agar plate (brain-heart agar) at 33°C for 24 hours. Starting from this agar-plate culture, a preculture was inoculated (10 ml of medium in a 100 ml Erlenmeyer flask) . The medium used for the preculture was the Cglll complete medium.
Cglll medium
NaCl 2.5 g/1
Bacto peptone 10 g/1 Bacto yeast extract 10 g/1
Glucose (autoclaved separately) 2% (w/v)
The pH was adjusted to pH 7.4.
The preculture was incubated on a shaker at 33 °C and 250 rpm for 24 hours. A main culture was inoculated with this preculture so that the initial OD (660 nm) of the main culture was 0.1 OD. MM medium was used for the main culture.
MM medium
CSL (corn steep liquor) 5 g/1
MOPS 20 g/1
Glucose (autoclaved separately) 50 g/1
Salts:
(NH4)2S04 25 g/1
KH2P04 0.1 g/1
MgS04 * 7H20 1.0 g/1
CaCl2 * 2H20 10 mg/1
FeS04 * 7H20 10 mg/1
MnS04 * H20 5.0 mg/1
Biotin (sterile-filtered) 0.3 mg/1
Thiamine * HCl (sterile-filtered) 0.2 mg/1 Leucine (sterile-filtered) 0.1 g/1
CaCO, 25 g/1
CSL, MOPS and the salt solution are adjusted to pH 7 with aqueous ammonia and autoclaved. Then the sterile substrate solutions and vitamin solutions and the dry- autoclaved CaC03 are added.
The cultivation is carried out in a volume of 10 ml in a 100 ml Erlenmeyer flask with baffles. The cultivation was carried out at 33 °C and 80% humidity.
After 72 hours, the OD at a wavelength of 660 nm was measured in a Biomek 1000 (Beckman Instruments GmbH,
Munich) . The amount of L-lysine formed was determined in an amino acid analyser from Eppendorf-BioTronik
(Hamburg, Germany) by ion exchange chromatography and after-column derivatization with ninhydrin detection.
Table 3 shows the result of the experiment.
Table 3
Figure imgf000058_0001
Example 5 :
Modification of the phosphorylation site in Isocitrate Dehydrogenase of Escherichia coli by site-directed mutagenesis 5.1. Obtaining the icd coding DNA
A fragment which contains the phosphorylation site S113 of the icd gene from Escherichia coli K12 (Hurley et al . , Proceedings of the National Academy of Sciences of the United States of America (22) 8635-8639 (1989)) is amplified by using the polymerase chain reaction (PCR) and synthetic oligonucleotides . Starting from the icd gene sequence known for Escherichia coli (SEQ ID No. 15, Accession Number AE000213) the following primer oligonucleotides (MWG Biotech, Ebersberg, Germany) are selected for the PCR:
icdA (SEQ ID No. 17) : 5 λ AGA ACG TTG CGA GCT GAA TC 3 Λ
icdE (SEQ ID No. 18) :
5 GTC ACC GTT CAG GTT CAT AC 3 λ
The chromosomal DNA of E. coli K12 which is used for PCR is isolated by using „Qiagen Genomic-tips 100/G" (Qiagen, Hilden, Germany) according to the instructions of the producer. By the specific primers an about 1.2 kbp DNA fragment can be isolated under standard conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) using Vent polymerase (New England Biolabs GmbH, Frankfurt, Germany) which contains the phosphorylation site S113 in the middle area.
The amplified DNA fragment is identified via gel electrophoresis in a 0.8% strength agarose gel and purified by the usual methods (High Pure PCR Product Purification Kit, Roche Diagnostics GmbH, Mannheim) . The cleaned PCR product is ligated with the vector pCR- Blunt II-TOPO (Zero Blunt TOPO PCR Cloning Kit, Invitrogen, Groningen, Niederlande) according to the instructions of the producer and transformed into the E. coli strain TOP10F (Invitrogen, Groningen, Netherlands) . The selection of plasmid containing cells is carried out on LB agar containing 50 mg/1 kanamycin. After plasmid isolation the vector is checked by means of restriction cleavage and identified in an agarose gel (0,8%) . The DNA sequence of the amplified DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 19. The plasmid obtained is denoted pCRBlunt-icdSDM.
5.2. Site-specific mutagenesis, exchange of a serine codon for a alanine codon
Site-directed mutagenesis is carried out using the
QuikChange site-directed mutagenesis kit (Stratagene,
La Jolla, USA) . Owing to the known phosphorylation site of Escherichia coli K12 Isocitrate Dehydrogenase
(Hurley et al . , Proceedings of the National Academy of Sciences of the United States of America (22) 8635-8639 (1989)), serine at position 113, the following primer oligonucleotides are selected for linear amplification:
S113A-1 (SEQ ID No. 20) : 5'TTGGTGGCGGTATTCGC(GCT)CTGAACGTTGCCCTG 3'
S113A-2 (SEQ ID No. 21) :
5 ' CAGGGCAACGTTCAG ( AGC ) GCGAATACCGCCACCAA 3 ' The primers depicted are synthesized by MWG Biotech. The codon for alanine which is intended to replace serine at position 113 is indicated by parentheses in the nucleotide sequence depicted above. The plasmid pCRBlunt-icdSDM, described in Example 5.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase. This primer extension generates a mutated plasmid with nicked circular strands. The product of the linear amplification is treated with Dpnl; this endonuclease specifically cuts the methylated and semi-methylated template DNA. The newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XLl Blue cells repair the nicks in the mutated plasmid. The transformants were selected on LB medium containing 50mg/l kanamycine . After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel. The DNA sequence of the mutated DNA fragment is checked by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 22. The plasmid obtained is denoted pCRBlunt-icdS113A.
5.3 Construction of the exchange vector pMAK705icdS113A
The plasmid pCRBlunt-icdS113A, described in Example 5.2. is cut with the restriction enzymes BamHI and Xbal (Gibco Life Technologies GmbH, Karlsruhe, Germany). After fractionation in an agarose gel (0.8%), an approx. 1.3 kb icd fragment carrying the mutation is isolated from the agarose gel with the aid of the High Pure PCR-product purification kit (Roche, Mannheim, Germany) and used for ligation with the vector pMAK705 described in Hamilton et al., Journal of Bacteriology 171, 4617 - 4622 (1989) . The said cloning vector is likewise cleaved with the restriction enzymes BamHI and Xbal beforehand, mixed with the isolated icd fragment and treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) . This is followed by transforming the E . coli strain DH5α (Grant et al . , Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) with the ligation mixture (Hanahan, in DNA cloning. A practical approach. Vol. 1, ILR Press, Cold Spring Harbor, New York, 1989) . Cells containing the plasmid are selected by plating out the transformation mixture on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, New York, 1989) supplemented with 20 mg/1 chloramphenicol.
Plasmid DNA is isolated from a transformant with the aid of the High Pure plasmid isolation kit from Roche and checked by restriction cleavage with the enzymes BamHI, Xbal and Sail and subsequent agarose gel electrophoresis. The plasmid is denoted pMAK705icdSH3A. Figure 2 depicts a map of the plasmid.
5.4. Site-specific mutagenesis of the phosphoserine codon of the Isocitrate Dehydrogenase in the E. coli strain MG442
The L-threonine producing E. coli strain MG442 is described in the patent US-A 4,278,765 and deposited as CMIM B-1628 at the Russian National Collection for industrial microorganisms (VKPM, Moskow, Russia) .
For the. site-specific mutagenesis in the icd gene MG442 is transformed with the plasmid pMAK705icdS113A. The exchange of the gene is carried out with the selection method described by Hamilton et al. (1989) Journal of Bacteriology 171, 4617 - 4622) and is studied with the aid of the LightCycler from Roche Diagnostics (Mannheim, Germany) in order to prove the mutation of the icdS113A allele in the chromosome. The LightCycler is a combination of a thermocycler and a fluorimeter.
In the first phase, an approx. 0.3 kb DNA section containing the site of mutation is amplified by means of PCR (Innis et al., PCR protocols. A guide to methods and applications, 1990, Academic Press) using the following primer oligonucleotides.
icd_LCPCRl (SEQ ID No.23): 5 ' GCCTATAAAGGCGAGCGTAA 3'
icd_LCPCR2 (SEQ ID No.24): 5 'ACCCGCATAAATGTCTTCCG 3'
In the second phase, the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes
(LightCycler (LC) -Red640 and fluorescein) and which hyrbidize around the site of mutation, with the aid of the fluorescence resonance energy transfer (FRET) method (Lay et al., Clinical Chemistry, 43: 2262-2267
(1997) ) .
icd/113-C (SEQ ID No.25): 5"LC-Red640 - GGTATTCGCGCTCTGAACGTT - (P) 3'
icd/113-A (SEQ ID No.26): 5'TTGCCATTAAAGGTCCGCTGACCACTCCGGTTGGT - Fluorescein 3'
The PCR primers depicted are synthesized by MWG Biotech and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
In this way, a clone was identified which contains a guanosine base instead of a thymidine base at position 595 of the DNA sequence (SEQ ID No. 27) . Therefore the idc allele codes at position 337-339 for the base triplet Guanine-Cytosine-Thymine and thus contains the icd_S113A allele (SEQ ID No.27). This clone is referred to as MG442icdS113A strain.
Example 6:
Production of L-treonine with the strain MG442icdS113A
MG442icdS113A is grown on a minimal medium with the following composition: 3,5 g/1 Na2HP04*2H20, 1,5 g/1 KH2P04, 1 g/1 NH4C1, 0,1 g/1 MgS04*7H20, 2 g/1 glucose, 20 g/1 Agar. The production of L-theronine is checked in batch cultures of 10 ml contained in 100 ml Erlenmeyer flasks. Therefore 10 ml of culture medium with the following composition: 2 g/1 yeast extract, 10 g/1 (NH4)2S04, 1 g/1 KH2P04, 0,5 g/1 MgS04*7H20, 15 g/1 CaC03, 20 g/1 glucose is inoculated and incubated for 16 hours at 37C and 180 rpm on a ESR incubator of Kϋhner AG company (Birsfelden, Swiss) . 250 μl of this starter culture are transferred into 10 ml of production medium (25 g/1 (NH4)2S04, 2 g/1 KH2P04, 1 g/1 MgS04*7H20, 0,03 g/1 FeS04*7H20, 0,018 g/1 MnS04*lH20, 30 g/1 CaC03, 20 g/1 glucose) and incubated for 48 hours at 37 °C. After incubation the optical density (OD) of the culture suspension is determined by using a LP2W photometer of Dr. Lange company (Dϋsseldorf, Germany) at a wavelength of 660 nm.
Thereafter the concentration of produced L-threonine is determined in the sterile filtered culture supernatant by means of a amino acid analysator of Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and after column reaction with ninhydrin detection.
Table 4 shows the result of this experiment.
Table 4
Figure imgf000065_0001
Example 7 :
Construction of exchange vectors for a site-specific mutagenesis of the alpha- and beta-subunit of the succinyl-CoA Synthetase (sucC and SucD genes) of Escherichia coli .
7.1. Obtaining the sucCD coding DNA
The genes sucC and sucD of E. coli K12 are amplified by using the the polymerase chain reac<tion (PCR) and synthetic oligonucleotides. Starting from the nucleic acid sequence of the genes sucC and sucD in E. coli K12 MG1655 (SEQ ID No. 29, Accession Number AE000176, Blattner et al . (Science 277: 1453-1462 (1997)) PCR primers are synthesized (MWG Biotech, Ebersberg, Germany) . The sequences of the primers are modified in a manner that recognition sites for restriction enzymes are formed. For the sucCDl-primer the recognition site for Xbal and for the sucCD2-primer the recognition site for Hindlll is selected, which both are indicated in the following nucleic acid sequence by parenthesis: sucCDl: 5Λ - GGA (TCTAGA) CGATTACTGAAGGATGGACAGAAC - 3Λ (SEQ ID No. 34)
sucCD2: 5Λ - GAG (AAGCTT) GGCGAGGGCTATTTCTTATTAC - 3 (SEQ ID No. 35)
The chromosomal DNA of E. coli K12 MG1655 which is used for PCR is isolated by using „Qiagen Genomic-tips 100/G" (Qiagen, Hilden, Germany) according to the instructions of the producer. By the specific primers an about 2100 bp DNA fragment can be amplified under standard conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) using Pfu DNA polymerase (Promega Corporation, Madison, USA) . The PCR product is cleaved by the restriction enzymes Xbal and Hindlll and and ligated with the vector pTrc99A (Pharmacia Biotech, Uppsala, Schweden) , which as well has been cut by the enzymes Xbal and Hindlll. The E. coli strain MRFN (Stratagene, La Jolla, USA) is transformed with the ligation mixture and cells containing the plasmid are selected on LB agar supplemented with 50μg/ml ampicillin. The successful cloning can be proved after plasmid DNA isolation by control cleavage with the enzymes EcoRV, Hpal and Sspl. The plasmid is denoted as pTrc99AsucCD.
7.2. Site-specific mutagenesis, exchange of the glutamic acid codon for a glutamine codon in the sucC gene coding for the according amino acid at position 197 of the beta subunit of the succinyl-CoA Synthetase.
By QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) the site-specific mutagenesis is carried out. For linear amplification the following primer oligonucleotides are selected:
sucC-E-197-Q-l: 5λ - GACCTGGCGTTGATC (CAA) ATCAACCCGCTG - 3 (SEQ ID No. 36) sucC-E-197-Q-2: 5 - CAGCGGGTTGAT (TTG) GATCAACGCCAGGTC - 3Λ (SEQ ID No. 37)
The primers depicted are synthesized by MWG Biotech (Ebersberg, Germany). The codon for glutamine which is intended to replace glutamic acid at position 197 of the beta subunit of the succinyl-CoA Synthetase is indicated by parentheses in the nucleotide sequence depicted above. The .plasmid pTrc99AsucCD, described in Example 7.1., is employed together with the two primers each of which is complementary to one strand of the plasmid for linear amplification by means of PfuTurbo DNA polymerase. This primer extension generates a mutated plasmid with nicked circular strands. The product of the linear amplification is treated with Dpnl. This endonuclease specifically -cuts the methylated and semi-methylated template DNA. The newly synthesized nicked mutated vector DNA is transformed into the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, the XLl Blue cells repair the nicks in the mutated plasmid. The transformants were selected on LB medium containing 50 mg/1 kanamycin. After isolating the DNA, the plasmid obtained is checked by means of restriction cleavage and identified in an agarose gel (0,8%) . The introduced mutation can be proved with the aid of the LightCycler technology from Roche Diagnostics (Mannheim, Germany) . The LightCycler is a combination of a thermocycler and a fluorimeter. In the first phase, an approx. 0.5 kb DNA section containing the site of mutation is amplified by means of PCR
(Innis et al . , PCR protocols. A guide to methods and applications, 1990, Academic Press) using the following primer oligonucleotides:
sucC-197-1: 5Λ - GGCAAGCGTCTGGTA - 3Λ (SEQ ID No. 38] sucC-197-2: 5 - CTTCCTGCGACTGGT - 3Λ (SEQ ID No. 39)
In the second phase, the presence of the mutation is detected by a melting-curve analysis using two additional oligonucleotides of different length, which have been labelled with different fluorescent dyes (LightCycler (LC)-Red640 and fluorescein) and which hyrbidize around the site of mutation, with the aid of the fluorescence resonance energy transfer (FRET) method (Lay et al., Clinical Chemistry, 43: 2262-2267 (1997) ) .
Probe-197-1: 5 - GGCGTTGATCCAAATCAACCC - Fluorescein - 3 λ (SEQ ID No. 40)
Probe-197-2: 5 Λ - LC Red640-CTGGTCATCACCAAACAGGG p (SEQ ID No. 41)
The PCR primers depicted are synthesized by MWG Biotech (Ebersberg, Deutschland) and the hybridization oligonucleotides depicted are synthesized by TIB MOLBIOL (Berlin, Germany) .
The plasmid is denoted pTrc99A-sucCD-El97Q.
7.5. Construction of the exchange vector pCVD442-sucCD- E208Q
The plasmid pTrc99A-sucCD-E208Q as described in example 7.4 ist cut with the restriction enzyme Hindlll and thereafter the 5 v -overhanging ends are treated with Klenow enzyme. After purification with QIAquick PCR Purification Kit (Product No. 28106, Qiagen, Hilden, Germany) the linearized plasmid is cleaved with Xbal. After separation in an agarose gel (0,8%) the about 2100bp sucCD-E208Q fragment is isolated from the agarose gel by aid of QIAquick Gel Extraction Kit
(Product No. 28706, Qiagen, Hilden, Germany) and is ligated with the plasmid pCVD442 (Donnenberg & Kaper
(1991) Infect. Immun. 59, 4310 - 4317) which has been cut with Smal and Xbal. THe strain DH5αλpir, a lambda- lysogenic strain containing the pir gene, is transformed with the ligation mixture and plasmid containing cells are selected on LB agar containing 50 ml/1 ampicillin. The successful cloning can be proved after plasmid DNA isolation by control cleavage with the enzyme EcoRI. A 474 bp DNA fragment can be amplified with the primers sucD-208-1 (SEQ ID No. 45) and sucD-208-2 (SEQ ID No. 46) under standard conditions and proved by sequencing. The sequence of the PCR product agrees with the sequence depicted in SEQ ID NO. 49. The sucD PCR fragment codes at positions 408 - 410 for the base trplet cytosine-adenine-guanine coding for the amino acid glutamine. The plasmid obtained is denoted pCVD442sucCD-E208Q (figure 4) .
The following figures are attached:
Figure 1: Map of plasmid pK18mobsacBenoS330E
Figure 2: Map of plasmid pMAK705icdS113A
Figure 3: Map of plasmid pCVD442sucCD-E197Q
Figure 4: Map of plasmid pCVD442sucCD-E208Q
Designations of lengths have to be considered as approximatly . The abbreviations and names used have the following meaning:
bla: ampicillin resistance gene
cat: chloamphenicol resistance gene enoS330E: mutated eno allele
icdS113A: mutated icd allele
KanR: Kanamycin-resistance gene
BamHI: Cleavage site of restriction enzyme BamHI
Hindlll: Cleavage site of restriction enzyme Hindlll
EcoRI: Cleavage site of restriction enzyme EcoRI
Sail: Cleavage site of restriction enzyme Sail
Xbal: Cleavage site of restriction enzyme Xbal
sacB: Levansucrase, sacB gene
sucC: Succinyl-CoA Synthetase beta subunit, sucC gene
sucC E197Q: mutated sucC allele
sucD: Succinyl-CoA Synthetase alpha subunit, sucD gene
sucDE208Q: mutated sucD allele
RP4mob: mob region with origin of replication for transfer (oriT)
oriRδK: origin of replication, dependend of pir gene
oriV: Origin of replication V
rep-ts: temperature sensitive replication region of the plasmid pSClOl

Claims

Patent claims
1. Microorganism, in which the phosphorylatability of at least one protein has been permanently altered such that the biosynthesis of at least one fine chemical synthesized by the microorganism is increased compared to the wild type.
2. Microorganism according to Claim 1, characterized in that the microorganism has been genetically modified.
3. Microorganism according to Claim 1, characterized in that the microorganism is a prokaryote, a eukaryote or an archaebacterium.
4. Microorganism according to Claim 3, characterized in that it is a prokaryote selected from the group of gram positive or gram negative bacteria.
Microorganism according to Claim 4, characterized in that it is selected from the group of coryneform or coliform bacteria, nonsporogenous bacilli, endosporogenous bacilli and cocci.
6. Microorganism according to any of Claims 1 to 5, characterized in that the protein is an enzyme, a protein of the biosynthetic pathway for the appropriate fine chemical whose synthesis is desired, in particular a key enzyme of the synthetic pathway, a protein of a biochemical pathway, a regulatory protein, a component of a signal transduction pathway, including two component system (s), a protein mediating cell integrity, a membrane-bound or membrane-associated protein, a storage protein, a transport protein or an immunoprotein.
7. Microorganism according to any of Claims 1 to 6, characterized in that the fine chemical produced is an amino acid, a vitamin, a nucleoside, a nucleotide, a pigment or a protein.
8. Microorganism according to any of Claims 1 to 7, characterized in that the phosphorylatability of the protein has been altered due to a mutation in at least one amino acid of the protein compared to the wild-type sequence.
9. Microorganism according to Claim 8, characterized in that the mutation is an amino-acid exchange, a deletion or an insertion of at least one amino acid.
10. Microorganism according to Claim 8, characterized in that an exchange of an amino acid which can be reversibly phosphorylated in the wild type or an amino-acid exchange in the region of the phosphorylation sequence of a protein or any mutation which changes the three dimensional structure of the protein in a manner that the phosphorylation- / dephosphorylation properties are changed has taken place.
11. Microorganism according to Claim 10, characterized in that a serine, threonine, tyrosine, histidine, glutamate, aspartate, arginine or lysine, which is phosphorylatable in the wild type, has been exchanged for a different amino acid.
12. Microorganism according to Claim 10, characterized in that an amino acid which is not phosphorylatable in the wild type has been exchanged for a serine, threonine, tyrosine, aspartate, glutamate, histidine, arginine or lysine .
13. Method for producing fine chemicals or metabolites by using a microorganism according to any of Claims 1 to 11.
14. Use of a DNA sequence coding for a protein which contains a phosphorylation site, whereby said sequence contains such a mutation that the protein is changed in its phosphorylatability for the production of a microorganism according to any of claims 1 to 12.
15. Use of a DNA sequence coding for a protein which contains a phosphorylation site, whereby said sequence contains such a mutation that the protein is changed in its phosphorylatability for the production of fine chemicals.
16. Use of a DNA sequence according to any of claims 14 or 15, whereby the sequence is selected from a sequence which encodes a protein which is an enzyme, a protein of the biosynthetic pathway for the appropriate fine chemical whose synthesis is desired, in particular a key enzyme of the synthetic pathway, a protein of a biochemical pathway, a regulatory protein, a component of a signal transduction pathway, including two component system (s), a protein mediating cell integrity, a membrane-bound or membrane-associated protein, a storage protein, a transport protein or an immunoprotein.
17. Use of a DNA sequence according to claim 16, characterized in that the sequence is selected from enoS330E (SEQ ID No: 4), icdSH3A (SEQ ID No: 27), a mutant of the sucC gene (SEQ ID No: 30) or a mutant of the sucD gene (SEQ ID No: 32) .
PCT/EP2002/010174 2001-09-13 2002-09-11 Method for producing fine chemicals WO2003023016A2 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071094A3 (en) * 2004-01-23 2005-11-24 Degussa Method for producing l-threonine using recombinant enterobacteriaceae with incre ased enolase activity
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
EP2665826B1 (en) 2011-01-20 2017-11-01 Evonik Degussa GmbH Process for the fermentative production of sulphur-containing amino acids

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Publication number Priority date Publication date Assignee Title
WO2008042338A2 (en) 2006-09-28 2008-04-10 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
DE102008032882A1 (en) * 2008-07-14 2010-01-21 Schäfer, Volker, Dr. New monomeric, oligomeric or polymeric organo-zinc cluster coordinately fixed with aliphatic and/or aromatic, primary, secondary or tertiary amine, useful e.g. for preventing fish smell and as acid scavengers in industrial and motor oil

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DE19947791A1 (en) * 1999-10-05 2001-04-12 Degussa New nucleotide sequences coding for the eno gene

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071094A3 (en) * 2004-01-23 2005-11-24 Degussa Method for producing l-threonine using recombinant enterobacteriaceae with incre ased enolase activity
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US9909130B2 (en) 2005-03-18 2018-03-06 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi
EP2665826B1 (en) 2011-01-20 2017-11-01 Evonik Degussa GmbH Process for the fermentative production of sulphur-containing amino acids

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