US20020081673A1 - Novel nucleotide sequences coding for the glbO gene - Google Patents

Novel nucleotide sequences coding for the glbO gene Download PDF

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US20020081673A1
US20020081673A1 US09/813,932 US81393201A US2002081673A1 US 20020081673 A1 US20020081673 A1 US 20020081673A1 US 81393201 A US81393201 A US 81393201A US 2002081673 A1 US2002081673 A1 US 2002081673A1
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polynucleotide
gene
codes
sequence
amino acid
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Bettina Mockel
Achim Marx
Walter Pfefferle
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Priority to US09/813,932 priority Critical patent/US20020081673A1/en
Priority to EP01940376A priority patent/EP1287143B1/de
Priority to DE60127116T priority patent/DE60127116T2/de
Priority to CN01810385.5A priority patent/CN1432066A/zh
Priority to AU2001273977A priority patent/AU2001273977A1/en
Priority to PCT/EP2001/004792 priority patent/WO2001094569A2/en
Priority to AT01940376T priority patent/ATE356210T1/de
Priority to US10/139,520 priority patent/US6759218B2/en
Publication of US20020081673A1 publication Critical patent/US20020081673A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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

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  • the invention provides nucleotide sequences coding for the glb0 gene and a process for the fermentative production of L-amino acids, in particular L-lysine, using coryneform bacteria in which the glb0 gene is amplified. All references cited herein are expressly incorporated by reference. Incorporation by reference is also designated by the term “I.B.R.” following any citation.
  • L-amino acids in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but especially in animal nutrition.
  • L-amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up to yield the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
  • strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant metabolites and produce L-lysine.
  • antimetabolites such as for example the lysine analogue S-2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant metabolites and produce L-lysine.
  • An object of the invention is to provide new measures for improved fermentative preparation of L-amino acids, in particular L-lysine.
  • Amino acids, in particular L-lysine are used in human medicine, in the pharmaceuticals industry and, in particular, in animal nutrition. Therefore, there is a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
  • L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e. g. lysine monohydrochloride or lysine sulfate, are also meant by this.
  • the new DNA sequence of C. glutamicum which codes for the glb0 gene and which as a constituent of the present invention is SEQ ID NO 1 and related sequences.
  • the amino acid sequence of the corresponding gene product of the glb0 gene has furthermore been derived from the present DNA sequence.
  • the resulting amino acid sequence of the glb0 gene product is SEQ ID NO 2 and related sequences.
  • FIG. 1 is a map of the plasmid pEC-K18mob2.
  • FIG. 2 is a map of the plasmid pEC-K18mob2glb0exp.
  • the invention provides an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group consisting of:
  • polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2,
  • the invention also provides a polynucleotide which is a preferably recombinant DNA replicable in coryneform bacteria.
  • the invention also provides a polynucleotide which is an RNA.
  • the invention also provides a polynucleotide as described above, wherein it preferably comprises a replicable DNA containing:
  • the relative degree of substitution or mutation in the polynucleotide or amino acid sequence to produce a desired percentage of sequence identity can be established or determined by well-known methods of sequence analysis. These methods are disclosed and demonstrated in Bishop, et al. “DNA & Protein Sequence Analysis (A Practical Approach”), Oxford Univ. Press, Inc. (1997) I.B.R. and by Steinberg, Michael “Protein Structure Prediction” (A Practical Approach), Oxford Univ. Press, Inc. (1997) I.B.R. Hybridization of complementary sequences can occur at varying degrees of stringency. Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R.
  • Hybridization of complementary sequences can occur at varying degrees of stringency.
  • Sambrook et al. Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R.
  • Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993)I.B.R. and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260) I.B.R.
  • Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, missense mutations or nonsense mutations are referred to. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely. Deletions of several codons typically lead to a complete loss of the enzyme activity.
  • the invention also provides a vector containing one of the stated polynucleotides and coryneform bacteria acting as host cell which contain the vector or in which the glb0 gene is amplified.
  • the invention also provides polynucleotides which substantially consist of a polynucleotide sequence, which are obtainable by screening by means of hybridization of a suitable gene library, which contains the complete gene having the polynucleotide sequence according to SEQ ID NO: 1, with a probe which contains the sequence of the stated polynucleotide according to SEQ ID NO: 1, or a fragment thereof, and isolation of the stated DNA sequence.
  • Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA in order to isolate full length cDNA which code for the hemoglobin-like protein (glb0) and to isolate such cDNA or genes, the sequence of which exhibits a high level of similarity with that of the gene for the hemoglobin-like protein.
  • Polynucleotide sequences according to the invention are furthermore suitable as primers for the production of DNA of genes which code for the hemoglobin-like protein by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Such oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides having a length of at least 40 or 50 nucleotides are also suitable.
  • isolated means separated from its natural environment.
  • Polynucleotide generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.
  • Polypeptides are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.
  • polypeptides according to the invention include a polypeptide according to SEQ ID NO: 2, in particular those having the biological activity of the hemoglobin-like protein and also those which are at least 70% identical to the polypeptide according to SEQ ID NO: 2 and which preferably exhibit at least 80% and in particular at least 90% to 95% identity to the polypeptide according to SEQ ID NO: 2 and exhibit the stated activity.
  • the invention furthermore relates to a process for the fermentative production of L-amino acids, in particular L-lysine, using coryneform bacteria, which in particular already produce an L-amino acid and in which the nucleotide sequences which code for the glb0 gene are amplified, in particular overexpressed.
  • the term “amplification” describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme having elevated activity and optionally by combining these measures.
  • the microorganisms may produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
  • the microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, the species Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
  • Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are for example the known wild type strains
  • the inventors have succeeded in isolating the novel glb0 gene which codes for the hemoglobin-like protein from C. glutamicum.
  • the glb0 gene or also other genes from C. glutamicum are isolated by initially constructing a gene library of this microorganism in E. coli.
  • the construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Amsterdam Elnrroundung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) I.B.R. or the manual by Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) I.B.R.
  • One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al.
  • I.B.R. in ⁇ -vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) I.B.R. describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) I.B.R. in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575) I.B.R. Börmann et al.
  • I.B.R. also describe a gene library of C. glutamicum ATCC 13032, using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)) I.B.R.
  • a gene library of C. glutamicum in E. coli may also be produced using plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) I.B.R. or pUC9 (Vieira et al., 1982, Gene, 19:259-268) I.B.R.
  • Suitable hosts are in particular those E. coli strains with restriction and recombination defects.
  • strain DH5 ⁇ MCR which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) I.B.R.
  • the long DNA fragments cloned with the assistance of cosmids may then in turn be sub-cloned in usual vectors suitable for sequencing and then be sequenced, as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977) I.B.R.
  • Coding DNA sequences arising from SEQ ID NO: 1 due to the degeneracy of the genetic code are also provided by the invention.
  • Conservative substitutions of amino acids in proteins for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known in specialist circles as “sense mutations”, which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. It is furthermore known that changes to the N and/or C terminus of a protein do not substantially impair or may even stabilize the function thereof. The person skilled in the art will find information in this connection inter alia in Ben-Bassat et al.
  • DNA sequences which hybridize with SEQ ID NO: 1 or parts of SEQ ID NO: 1 are similarly provided by the invention.
  • DNA sequences produced by the polymerase chain reaction (PCR) using primers obtained from SEQ ID NO: 1 are also provided by the invention.
  • PCR polymerase chain reaction
  • Such oligonucleotides typically have a length of at least 15 nucleotides.
  • coryneform bacteria produce L-amino acids, in particular L-lysine, in an improved manner once the glb0 has been overexpressed.
  • Overexpression may be achieved by increasing the copy number of the corresponding genes or by mutating the promoter and regulation region or the ribosome-binding site located upstream from the structural gene.
  • Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during fermentative L-amino acid production by means of inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover amplified by preventing degradation of the enzyme protein.
  • the genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions.
  • the glb0 gene according to the invention was overexpressed with the assistance of plasmids.
  • Suitable plasmids are those which are replicated in coryneform bacteria.
  • Numerous known plasmid vectors such as for example pZ1 (Menkel et al., Applied and Environmental Microbiology, 64: 549-554(1989)) I.B.R., pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) I.B.R. or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) I.B.R. are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • Plasmid vectors such as for example those based on pCG4 (U.S. Pat. No. 4,489,160) I.B.R., or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) I.B.R., or pAG1 (U.S. Pat. No. 5,158,891) I.B.R. may be used in the same manner.
  • plasmid vectors are those with the assistance of which gene amplification may be performed by integration into the chromosome, as has for example been described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) I.B.R. for the duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli ), but not in C. glutamicum.
  • Vectors which may be considered are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)) I.B.R., pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)) I.B.R., pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994) I.B.R. Journal of Biological Chemistry 269:32678-84 I.B.R.; U.S. Pat. No.
  • I.B.R. pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) I.B.R. or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) I.B.R.
  • the plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation.
  • the conjugation method is described, for example, in Schwarzfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)) I.B.R. Transformation methods are described, for example, in Thierbach et al.
  • the invention accordingly also provides a process for the fermentative production of L-amino acids, in particular L-lysine, in which a strain transformed with a plasmid vector is used and the plasmid vector bears the nucleotide sequence of the gene which codes for the hemoglobin-like protein.
  • L-amino acids in particular L-lysine
  • L-lysine may additionally be advantageous for the production of L-amino acids, in particular L-lysine, to amplify not only the glb0 gene, but also further genes of the biosynthetic pathway of the desired L-amino acid, such that one or more enzymes of the particular biosynthetic pathway, of glycolysis, of anaplerotic metabolism or of amino acid export is/are overexpressed.
  • gap gene which codes for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) I.B.R.,
  • L-amino acids in particular L-lysine, apart from the glb0 gene, simultaneously to attenuate
  • the pck gene which codes for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047) I.B.R. and/or
  • L-amino acids in particular L-lysine
  • L-lysine in addition to overexpressing the glb0 gene, to suppress unwanted secondary reactions
  • the microorganisms produced according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process.
  • a summary of known culture methods is given in the textbook by Chmiel (Bioreatechnik 1. Consum in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) I.B.R. or in the textbook by Storhas (Bioreaktoren und periphere Junior (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) I.B.R.
  • the culture medium to be used must adequately satisfy the requirements of the particular strains.
  • Culture media for various microorganisms are described in “Manual of Methods for General Bacteriology” from the American Society for Bacteriology (Washington D.C., USA, 1981) I.B.R.
  • Carbon sources which may be used are sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose for example, oils and fats, such as soya oil, sunflower oil, peanut oil and coconut oil for example, fatty acids, such as palmitic acid, stearic acid and linoleic acid for example, alcohols, such as glycerol and ethanol for example, and organic acids, such as acetic acid for example. These substances may be used individually or as a mixture.
  • oils and fats such as soya oil, sunflower oil, peanut oil and coconut oil for example
  • fatty acids such as palmitic acid, stearic acid and linoleic acid for example
  • alcohols such as glycerol and ethanol for example
  • organic acids such as acetic acid for example.
  • Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, 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 salts containing sodium.
  • the culture medium has additionally to contain salts of metals, such as magnesium sulfate or iron sulfate for example, which are necessary for growth.
  • essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances.
  • Suitable precursors may furthermore be added to the culture medium.
  • the stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Foaming may be controlled by using antifoaming agents such as fatty acid polyglycol esters for example. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example antibiotics. Oxygen or oxygen-containing gas mixtures, such as air for example, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20° C. to 45° C. and preferably from 25° C. to 40° C. The culture is continued until the maximum quantity of L-lysine has formed. This objective is normally achieved within 10 hours to 160 hours.
  • the invention accordingly provides a process for the fermentative production of L-amino acids, in particular L-lysine, in which the following steps are performed:
  • the purpose of the process according to the invention is the fermentative production of L-amino acids, in particular L-lysine.
  • I.B.R. purchased from Stratagene (La Jolla, USA, product description SuperCos1 Cosmid Vector Kit, code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, code no. 27-0948-02) I.B.R. and also dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04) I.B.R.
  • XbaI Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04
  • Cosmid DNA treated in this manner was mixed with the treated ATCC 13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA Ligase, code no. 27-0870-04) I.B.R.
  • the ligation mixture was then packed in phages using Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, code no. 200217).
  • E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) I.B.R.
  • Cosmid DNA from an individual colony was isolated in accordance with the manufacturer's instructions using the Qiaprep Spin Miniprep Kit (product no. 27106, Qiagen, Hilden, Germany) and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27-0913-02).
  • the DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, product no. 1758250).
  • the cosmid fragments of a size of 1500 to 2000 bp were isolated using the QiaExII Gel Extraction Kit (product no.
  • the DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, product no. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, product no. 27-0868-04). Ligation of the cosmid fragments into the sequencing vector pZero-1 was performed as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) I.B.R., the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated into the E.
  • the resultant nucleotide sequence is stated in SEQ ID NO: 1. Analysis of the nucleotide sequence revealed an open reading frame of 393 base pairs, which was designated the glb0 gene. The glb0 gene codes for a protein of 131 amino acids.

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US09/813,932 US20020081673A1 (en) 2000-06-02 2001-03-22 Novel nucleotide sequences coding for the glbO gene
EP01940376A EP1287143B1 (de) 2000-06-02 2001-04-27 Für das glbo-gen kodierende nukleotidsequenzen aus corynebacterium glutamicum
DE60127116T DE60127116T2 (de) 2000-06-02 2001-04-27 Für das glbo-gen kodierende nukleotidsequenzen aus corynebacterium glutamicum
CN01810385.5A CN1432066A (zh) 2000-06-02 2001-04-27 编码glb0基因的新核苷酸序列
AU2001273977A AU2001273977A1 (en) 2000-06-02 2001-04-27 Novel nucleotide sequences coding for the glbo gene
PCT/EP2001/004792 WO2001094569A2 (en) 2000-06-02 2001-04-27 Corynebacterium glutamicum nucleotide sequences coding for the glbo gene
AT01940376T ATE356210T1 (de) 2000-06-02 2001-04-27 Für das glbo-gen kodierende nukleotidsequenzen aus corynebacterium glutamicum
US10/139,520 US6759218B2 (en) 2000-06-02 2002-05-07 Nucleotide sequences coding for the glbO gene

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WO2010095642A1 (ja) 2009-02-18 2010-08-26 国立大学法人信州大学 有用物質の製造方法
US9988624B2 (en) 2015-12-07 2018-06-05 Zymergen Inc. Microbial strain improvement by a HTP genomic engineering platform
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EP1287143B1 (de) 2007-03-07
CN1432066A (zh) 2003-07-23
DE60127116T2 (de) 2007-11-15
EP1287143A2 (de) 2003-03-05
ATE356210T1 (de) 2007-03-15
DE60127116D1 (de) 2007-04-19
AU2001273977A1 (en) 2001-12-17

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