US20060204963A1 - Nucleotide sequences of coryneform bacteria coding for proteins involved in l-serine metabolism and method for producing l-serine - Google Patents

Nucleotide sequences of coryneform bacteria coding for proteins involved in l-serine metabolism and method for producing l-serine Download PDF

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US20060204963A1
US20060204963A1 US10/549,262 US54926204A US2006204963A1 US 20060204963 A1 US20060204963 A1 US 20060204963A1 US 54926204 A US54926204 A US 54926204A US 2006204963 A1 US2006204963 A1 US 2006204963A1
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serine
expressed
nucleic acid
microorganism
nucleotide sequence
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Petra Peters-Wendisch
Roman Netzer
Lothar Eggeling
Hermann Sahm
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Forschungszentrum Juelich GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • the invention relates to nucleotide sequences of coryneform bacteria coded for proteins which participate in L-serine metabolism with reduced or omitted L-serine dehydratase and microorganisms for and method of making L-serine.
  • the amino acid L-serine has been found to be useful in the food industry, the animal feed industry and the pharmaceutical industry as well as in human medicine. It serves as a building block for the synthesis of other industrially valuable products like for example L-tryptophan from indole and L-serine.
  • L-serine can be produced by the fermentation of coryneform bacteria strains.
  • a strain of Corynebacterium glycinophilum is capable of forming L-serine from glycine and carbohydrates (Kubota K, Kageyama K, Shiro T and Okumura S (1971) Journal of General Applications in Microbiology, 17: 167-168; Kubota K, Kageyama K, Maeyashiki I, Yamada K and Okumura S (1972) Journal of General Applications in Microbiology 18: 365).
  • the enzyme L-serine-hydroxymethyltransferase here participates in the conversion of glycine to L-serine.
  • L-serine can be produced fermentatively from methanol and glycine with the aid of methylotrophic bacteria like for example Hyphomicrobium strains (Izumi Y, Yoshida T, Miyazaki S S, Mitsunaga T, Ohshiro T, Shiamo M, Miyata A and Tanabe T (1993) Applied Microbiology and Biotechnology, 39: 427-432).
  • the amino acid glycine must be introduced as a precursor for the formation of the amino acid L-serine.
  • coryneform bacteria which can produce the L-serine directly from carbohydrates without further addition of precursors.
  • the objects are achieved, in accordance with the invention with the features given in the characterizing clause of claim 1 . Furthermore, the objects are achieved starting from the preamble of claim 7 with the features given in the characterizing part of claim 7 . The objects are also attained starting from the preamble of claim 8 according to the invention with the features given in the characterizing part of claim 8 . The objects are also achieved starting with the preamble of claim 9 with the features given in the characterizing part of claim 9 . The objects are also achieved starting with the preamble of claim 14 , in accordance with the invention, with the features given in the characterizing part of claim 14 .
  • the objects are also achieved according to the invention by the features given in the characterizing part of claim 20 . Furthermore, the objects are attained according to the invention starting from the preamble of claim 21 by the features of the characterizing part of claim 21 .
  • nucleic acids and polypeptides according to the invention it is possible to produce an L-serine dehydratase such that there is a reduced decomposition of L-serine or no longer any decomposition of L-serine. Furthermore, it is possible to provide microorganisms and a method by which L-serine production can be obtained with higher yield by comparison with hitherto known microbial methods.
  • replicatable and optionally recombinant nucleic acid is provided with nucleotide sequence coding for the L-serine dehydratase, hereinafter referred to also as SDA, which is partially or completely deleted or mutated or is expressed to a reduced extent by comparison with the naturally occurring nucleotide sequence or is not expressed at all.
  • SDA L-serine dehydratase
  • the subject of the invention is, further, the provision of nucleic acids whose sdaA gene sequence is partially or completely deleted or mutated or has, relative to the naturally available nucleotide sequence reduced expression or which does not express at all.
  • nucleic acids with a nucleotide sequence according to SEQ ID No 1 can have its nucleotides from position 506 to position 918, partly or completely deleted or mutated or can be allele, homologue or derivative of this nucleotide sequence or a nucleotide sequence which hybridizes therewith have been found to be advantageous.
  • the wild type L-serine-dehydratase (sdaA) gene sequence is generally known and can be obtained by the artisan from the known data bank (NCBI Accession Nr. AP005279) or from the attached sequence protocol according to SEQ ID No. 1.
  • L-serine dehydratase (sdaA) gene can be achieved for example by directed recombinant DNA techniques. Suitable methods for this purpose are found in Schafer et al. (Gene (1994) 145: 69-73) or also Link et al. (Journal of Bacteriology (1998) 179: 6228-6237). Furthermore, only a part of the gene can be deleted or also mutated fragments of the L-serine dehydratase gene can be formed by replacement. By deletion or replacement it is possible to achieve a loss or a reduction in the L-serine dehydratase activity. An example of such a mutant is the C. glutamicum strain ATCC133032 ⁇ sdaA which has a deletion in the sdaA gene.
  • the promoter and regulatory regions which are located upstream of the structural gene can be mutated.
  • expression regulatory cassettes can be built onto the structural gene, upstream thereof.
  • regulatable promoters it is additionally possible to reduce the expression in the course of fermentative L-serine formation. It is also possible to provide a regulation of the translation in which for the example of stability of the m-RNA is reduced.
  • genes can be used which code for the corresponding enzyme with reduced activity.
  • a reduced expression of the L-serine dehydratase gene can be achieved by varying the medium composition and culture conditions. Guides thereto for the artisan can be found among others in Martin et al.
  • the nucleic acids according to the invention are characterized that they can be isolated from the coryneform bacteria, preferably of corynebacterium or brevibacterium family and especially preferably from Corynebacterium glutamicum.
  • coryneform bacteria wild types from this parental line are for example,
  • Microbacterium ammoniaphilum ATCC 15354 Microbacterium ammoniaphilum ATCC 15354.
  • mutants or production strains suitable for the production of L-serine are organisms from the group of Arthrobacter, Pseudomonas, Nocardia, Methylobacterium, Hyphomycrobium, Alcaligenes or Klebsiella.
  • the present invention is characterized more particularly by the naming of the aformentioned bacterial strains but should not be considered limited thereto.
  • nucleic acid or a “nucleic acid fragment” there is meant, in accordance with the invention, a polymer of RNA or DNA which can be single stranded or double stranded and can have optional natural, chemically synthesized, modified or artificial nucleotides.
  • DNA polymer includes in this case also genomic DNA, cDNA or mixtures thereof.
  • Mutations include substitutions, additions, deletions, replacements or insertions of one or more nucleotide residues. Included here are also sense mutations which in the protein plane can result for example from the replacement of conserved amino acids which however do not lead to any basic alteration in the activity of the protein and thus can be considered functionally neutral. This includes modifications of the nucleotide sequence which involve in the protein plane the N-terminus of a protein without however affecting significantly the function of these proteins.
  • nucleotide sequences are encompassed which, by modification of the nucleotide sequences can result in corresponding derivatives.
  • the target of such modification can, for example, be a restriction of the coding sequence contained therein or for example also the insertion of further restriction enzyme cutting sites.
  • the present invention includes artificial DNA sequences as long as they, as described above, afford the desired characteristics.
  • artificial DNA sequences can, for example, be those obtained by reverse translation from proteins established by means of computer supported programming (molecular modeling) or by in vitro selection.
  • coded DNA sequences which, by reverse translation, can produce a polypeptide sequence which has a specific code on utilization for the host organism.
  • the specific code on utilization can be easily determined by molecular genetic methods common in the art using computer evaluations from other previously known genes of the organism to be transformed.
  • “Homologous sequences” are to be understood in accordance with the invention to be those sequences which are complementary to the nucleotide sequences according to the invention and/or such sequences which can hybridize with them.
  • the hybridizing sequences include, according to the invention, substantially similar nucleotide sequences from the group of DNA or RNA which under stringent conditions known per se undergo a specific interaction (binding) of the aforementioned nucleotide sequences. In this category are to be counted also short nucleotide sequences with a length of for example 10 to 30 and preferably 12 to 15 nucleotides. These include according to the invention among others, also so-called primers or probes.
  • sequence regions are also the coding regions (structure genes) and preceding (5′ or upstream) sequence regions and/or following (3′ or downstream) sequence regions.
  • sequence regions with regulatory functions can influence the transcription, the RNA stability or RNA processing as well as the translation.
  • regulatory sequences are, among others, promoters, enhancers, operators, terminators or translation amplifiers.
  • the subject of the invention is in addition a gene structure containing at least one of the aforedescribed nucleotide sequences and regulatory sequences operatively linked therewith which control expression of the coded sequences in the host cell.
  • the present invention relates to a vector containing a nucleotide sequence of the aforedescribed kind with its regulator nucleotide sequence operatively linked thereto as well as additional nucleotide sequences for the selection of host cells capable of effecting transformation, for replication within the host cell or for integration in the corresponding host cell genome.
  • the vector according to the invention can contain a genome structure of the aforedescribed type.
  • Suitable vectors are those which replicate in coryneform bacteria like for example pZ1 (Menkel E, Thierbach G, Eggeling L, Sahm H., 1989, Appl Environ Microbiol 55(3): 684-688), pEKEx2 (Eikmanns et al., Gene 102: 93-98 (1991), or pXMJ19 (Jacoby M., Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors, Biotechnol. Technique 13:437-441).
  • Other plasmid vectors can be used in the same manner. These identifications are however not limiting for the present invention.
  • corresponding probes or primers can be synthesized and used, for example, to amplify and isolate analogous genes from other microorganisms, preferably coryneform bacteria, for example with the aid of the PCR technique.
  • the subject matter of the present invention is thus also a probe for identifying and/or isolating genes coded for proteins participating in the biosynthesis of L-serine, whereby these probes are produced starting from the nucleic acid sequences according to the invention of the aforedescribed type and which contain a suitable marker for detection.
  • a partial segment of the sequences according to the invention for example a conserved region, can be used which for example has a length of 10 to 30 or preferably 12 to 15 nucleotides and under stringent conditions can hybridize with homologous nitride sequences. Numerous suitable markers are known from the literature.
  • the subject matter of the present invention includes, further, an L-serine dehydratase which shows reduced L-serine decomposition by comparison with the wild type L-serine dehydratase and which is coded by a nucleic acid sequence according to the invention or its variants of the aforedescribed type.
  • the present invention thus includes an L-serine dehydratase or an L-serine dehydratase mutant with an amino acid sequence in accordance with sequence ID No. 2 whose amino acids from position 135 to position 274, for example, as a consequence of a directed mutagenesis in the DNA plane, is altered or is a modified form of this polypeptide sequence or an isoform thereof or a mixture thereof.
  • enzymes according to the invention with changes in the sequence, for example, at the N-terminus or C-terminus of the polypeptide or in the regions of the conserved amino acids without however negatively affecting the function of the enzyme. These changes can be in the form of amino acid replacement in accordance with methods known per se.
  • the polypeptides according to the invention are characterized by the fact that they derive from coryneform bacteria and preferably are of the corynebacterium or brevibacterium family and especially of the Corynebacterium glutamicum or Brevibacterium types and especially preferably derive from Corynebacterium glutamicum.
  • Examples of the coryneform bacteria in the strain culture of the wild type are for instance
  • Microbacterium ammoniaphilum ATCC 15354 Microbacterium ammoniaphilum ATCC 15354.
  • mutants or production strands suitable for the production of L-serine are organisms from the group of arthrobacter, pseudomonas, nocardia, methylobacterium, hyphomycrobium, alcaligenes or klebsiella.
  • the present invention has been characterized by listing the aformentioned bacteria strains, but this list should not be considered limiting of the invention.
  • the present invention comprises, further, a genetically altered microorganism characterized in that it contains a nucleotide sequence coding for the L-serine dehydratase which is in part or completely deleted or mutated or expressed to a reduced extent by comparison with the naturally occurring nucleotide sequence or which is not expressed at all.
  • the invention comprises further a microorganism which is characterized in that the sdaA gene is partially or completely deleted or mutated or which is expressed to a reduced extent by comparison with the naturally occurring sdaA gene or which is not expressed at all.
  • the invention encompasses as well a genetically altered microorganism containing in replicatable form a gene structure or a vector of the aforedescribed type.
  • the subject of the present invention is moreover also a genetically modified microorganism containing a polypepetide according to the invention of the aforedescribed type and which in comparison to the corresponding genetically unmodified microorganism has reduced or no L-serine decomposition.
  • a microorganism which, according to the invention has been genetically modified is characterized further in that it is a coryneform bacterium, preferably of the family Corynebacterium or Brevibacterium and especially preferably of the species Corynebacterium glutamicum or Brevibacterium flavum.
  • the genes can, using methods known per se like for example the polymerase chain reaction (PCR), be amplified by the aid of short synthetic nucleotide sequences (primers) and then isolated.
  • the production of the primers used can be effected generally based upon known gene sequences from existing homologies in conserved regions of the gene and/or taking into consideration the GC content of the DNA of the microorganism investigated.
  • a further procedure for isolating coding nucleotide sequences is the complementation of so-called defect mutants of the organism to be investigated which at least phenotypically show a function drop in the activity of the gene investigated or the corresponding protein.
  • defect mutants of the organism to be investigated which at least phenotypically show a function drop in the activity of the gene investigated or the corresponding protein.
  • a classical mutagenesis process for producing defect mutants or mutants with a reduced L-serine dehydratase or an L-serine dehydratase which has been shut down is for example the treatment of the bacteria cell with chemicals like for example N-Methyl-N-Nitro-N-Nitrosoguanidine or the use of UV radiation.
  • Such methods of mutation resolution are generally known and can be found among others in Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or the Handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981)).
  • the present invention relates moreover to a method for the microbial production of L-serine whereby the nucleic acids in the microorganisms which code for the L-serine dehydratase in part or completely are deleted or mutated or expressed to a lesser extent or practically not at all by comparison with the naturally available nucleic acids, using these genetically altered microorganisms for the microbial production of L-serine, and isolating the correspondingly formed L-serine from the culture medium.
  • the genetically altered microorganisms produced in accordance with the invention can be used for the purpose of culturing L-serine in continuous cultures or discontinuously in batch processes (set cultivation) or in a fed batch process or a repeated fed batch process.
  • a collection of known cultivation methods can be found in the textbook of Chmiel (Bioreatechnik 1. Einbowung in die Biovonsechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
  • the culture medium used must be suitable to suitably satisfy the requirements of the respective strain.
  • Descriptions of culture media for various microorganisms can be found in the handbook “Manual of Methods for General Bacteriology” der American Society for Bacteriology” der American Society for baceriology (Washington D.C., USA, 1981) as carbon sources, sugars and carbohydrates like for example glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose can be used, oils and fats like for example soy oil, soy flour oil, peanut oil, cocoanut fats can be used, fatty acids like for example palmitic acid, stearic acid and linolaic acids can be used, alcohols like for example glycerine and ethanol can be used and organic acids like for example acetic acid can be used.
  • nitrogen sources organic nitrogen containing compounds like peptones, yeast extract, meat extract, malt extract, corn spring water, soybean meal and urea, or inorganic compounds like ammonium sulfate, ammonium chloride ammonium phosphate, ammonium carbonate and ammonium nitride are used.
  • the nitrogen sources can be used individually or as mixtures.
  • phoshorous sources phosphoric acid, potasiuim dihydrogen phosphate or dipotassium phosphate or the corresponding sodium-containing salts are used.
  • the culture medium must contain further salts of metal like for example magnesium sulfate or iron sulfate which are required for growth.
  • the culture medium can in addition have suitable precursors added to it.
  • the additives can be introduced into the culture in the form of one time addition or can be fed to the culture suitably during cultivation.
  • basic compounds like sodium hydroxide, potassium hydroxide, ammonia or acqueous ammonia can be used or acid compounds like phosphoric acid or sulfuric acid can be used in a suitable way.
  • antifoaming agents like for example fatty acid polyglycol esters can be used.
  • suitable selectively effective substances for example antibiotics can be added to the medium.
  • oxygen or oxygen-containing mixtures like for example air are introduced into the culture.
  • the temperature of the culture is normally between 20° C. and 45° C. and preferably 25° C. to 40° C.
  • the culture is maintained for a duration until L-serine production is a maximum. This duration is normally from 10 hours to 160 hours.
  • the analysis of the L-serine formation can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization as described by Spackman et al. (Analytical Chemistry, 30 (1958), 1190) or the analysis can be effected by reverse phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174.
  • the microorganisms which are the subject of the present invention can produce L-serine from glucose, saccharose, lactose, mannose, fructose, maltose, molasses, starch, cellulose or from glycerine and ethanol.
  • the method can use the coryneforme bacteria representatives which have already been described in detail.
  • a selection of the results of the fermentation has been given in Table 1.
  • the genetically altered microorganisms of the invention show a substantially improved L-serine production by comparison with the corrsponding nontransformed microorganism (wild type) or the micororganisms which contain only the vector without the gene insert. In a special variation of the present invention it has been shown that C.
  • Glutamicum ATCC 13032 ⁇ panBC ⁇ sdaA gives rise to at least a 4-fold increase in the L-serine accomulation in the medium by comparison with the control strain (Table 1).
  • Table 1 The control strain
  • Amino acid production strains in accordance with the present invention should be understood to be Corynebacterium glutamicum strains or homologous microrganisms which are altered by classical and/or molecular genetic methods so that metabolic flow is amplified in the direction of the biosynthesis of amino acids or their derivatives (metabolic engineering).
  • these amino acid production strains one or more genes and/or the corresponding enzyme have their regulation altered or are rendered deregulated at different and correspondingly complex regulated key positions in the metabolic pathway.
  • the present invention includes thereby all such already known amino acid production strains preferably of the corynebacterium family or homologous organisms.
  • the Figures show examples of plasmids which can be used as well as experimental results with respect to nucleic acids or microorganisms according to the invention.
  • FIG. 1 The integration plasmid pK19mobsacB-DeltasdaA Markings on the outer edge of the plasmid indicate the respective restriction sites.
  • the portion within the circle indicates the following gene: kan canamycin resistance sacB Sucrase OriT Transfer origin sdA′ 5′ end of the sdaA gene sda′′ 3′ end of the sdaA gene
  • FIG. 2 A graph of the ratio between growth (square symbol ⁇ ) and L-serine breakdown (circle symbol ⁇ ) of C. glutamicum 13032 ⁇ panBC ⁇ sdaA, clone 1 ( ⁇ , ⁇ ) and C. glutamicum 13032 ⁇ panBC ⁇ sdaA, clone 2 ( ⁇ , ⁇ ) compared with C. glutamicum 13032 ⁇ panBC, clone 1 ( ⁇ , ⁇ ) and C. glutamicum 13032 ⁇ panBC,clone 2 ( ⁇ , ⁇ ).
  • the abscissa X represents the fermentation in hours (h).
  • the ordinate y 1 is the growth of the microorganisms measured in terms of optical density at 600 nm.
  • the ordinate Y 2 gives the L-serine concentration in mM.
  • FIG. 3 The expression plasmid pEC-T18mob2-serA fbr CB.
  • the indicia on the outer edge of the plasmid show the resective restriction sites.
  • the indicia within the circle represent the following genes: SerC Phosphoserine Transaminase SerB Phsophoserine Phosphatase Rep Replication origin Per Partition cell partition gene Tet Tetracycline resistance gene RP4-mob Mobilizaiton origin OriV Source of DNA replication SerA-fbr 3-phosphoglycerate dehydrogenase
  • the starting point was Corynebacterium glutamicum with a nuclotide sequence (Genbank-Accession-Number BAB99038; SEQ-ID-No. 1) whose derivative polypeptide sequence showed 40% identity with the described L-serine dehydratase of E.coli (NCBI-Accession-Number P1095).
  • gene protected mutagenesis by the method of Link et al (Link A J, Phillips D, Church G M, Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J. Bacteriol. 1997 October; 179(20):6228-37) and Schafer et al.
  • sdaA-1 5′-TCGTGCAACTTCAGACTC-3′ (AP005279 nucleotide 73635 - 73653)
  • sdaA-2 5′-CCCATCCACTAAACTTAAACACGTCATAATGAACCCACC-3′ (AP005279 complementary to nucleotide 74121 - 74139)
  • sdaA-3 5′-TGTTTAAGTTTAGTGGATGGGCCGACTAATGGTGCTGCG-3′ (AP005279 complementary to nucleotide 74553 - 74571);
  • sdaA-4 5′-CGGGAAGCCCAAGGTGGT-3′ (AP005279 nucleotide 75044 - 75062)
  • the primer combination sdaA-1 and sdaA-2 as well as sdaA-3 and sdaA-4 are used.
  • the PCR reaction is carried out in 30 cycles in the presence of 200 ⁇ m deoxynucleotide triphosphates (dATP, dCTkP, dGTP, dTTP), each with 600 nM of the corresponding oligonucleotide sdaA-1 and sdaA-4 as well as 60 nm of oligonucleotide sdaA-2 and sdaA-3, 100 ng of chromosomal DNA from Corynebacterium glutamicum ATCC13032, 1/10 volumes 10-fold of reagion buffer and 2.6 units of heat stabilized Taq-/Owi-DNA-Polymerase-Mischung mixture (Expand High Fidelity PCR System of Firm of Roche Diagnostics, Mannheim, Germany) in a Sthermocycler (PTC-100, MJ Research, Inc., Watertown, USA) under the following conditions: 94° C.
  • dATP deoxynucleotide triphosphates
  • the DNA fragments containing each having a length of 500 bp were isolated with QIAExII Gelextraction kit (Qiagen) in accordance with the requirements of the manufacturer on an 0.8% agarose gel and both fragments were used as templates in the second PCR.
  • Qiagen QIAExII Gelextraction kit
  • primers the primers sdaA-1 and sdaA-4 were used.
  • reaction was carried out in 35 cycles in the presence of 200 ⁇ m deoxynucleotide triphosphates, 600 nM each of the corresponding olegonitrides, 2-mg each of the isolated template DNA from the first PCR, 1/10 volume of 10 fold reaction buffer and 2.6 units of Taq-/Pwo-DNA-Polymerase mixture under the following conditions: 94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 80 seconds. Again the elongation steps after 10 cycles were extended by 5 seconds each.
  • the plasmid is incorporated by electroporation in C. glutamicum 13032 ⁇ panBC (Radmacher E, Vaitsikova A, Burger U, Krumbach K, Sahm H, Eggeling L. Linking central metabolism with increased pathway flux: L-valine accumulated by Corynebacterium glutamicum. Appl Environ Microbiol. 2002 68(5):2246-50) and subject to selection with integration of the vector.
  • This strain is pantothenate auxotropic as a result of the deletion of the pantothenate biosynthesis genes panB and panC and is characterized in that it has an amplified accumulation of pyruvate about 50 mM alanin and 8 mm valine because of the pantothenate limitation.
  • the strain can form about 100 ⁇ M L-serine and is suitable as a starting strain for the construction of L-serine producers. It contains Kanamycin resistant clones of C. Glutamicum 13032 ⁇ panBC by which inactivation vector is integrated in the genome. To allow selection of the excision of the vector, kanamycin-resistant clones are plated out on saccharose containing LB medium (Sambrook et al., Molecular cloning. A laboratory manual (1989) Cold Spring Harbour Laboratory (Press) with 15 g/l Agar, 2% glucose/10% saccharose) and colonies are obtained in which the vector has again been lost as a result of a second recombination event. (Jager et al.
  • strains 13032 ⁇ panBC ⁇ sdaApSerA fbr CB and 13032 ⁇ panBCpSerA fbr CB were obtained.
  • the two strains 13032 ⁇ panBCpSerA fbr CB are cultivated in complex medium (CgIII with 2% glucose and 5 ⁇ g/l tetracycline) and the fermentation medium CGXII (J Bacteriol (1993) 175: 5595-5603), each seeded from the preculture to the medium contained in addition 50 ⁇ g/l kanamycin nd 1 ⁇ M pantothenate.
  • the two starting strains 13032 ⁇ panBC and 13032 ⁇ panBC ⁇ sdaA were cultured in the same manner although the medium did not contain tetracycline. For each at least two independent fermentations were carried out. After culturing for 30 hours at 30° C.
  • the L-serine quantity accumulated in the medium was determined.
  • the determination of the amino acid concentration was carried out by means of high presssure liquid chromatography (J Chromat (1983) 266: 471-482).
  • the results of the fermentation are shown in Table 1 and indicate that the exclusion of L-serine dehydratase led to a 4-fold increase in the L-serine accumulation in the medium independently of whether the L-serine biosynthesis genes serA fbr , serC and serB were overexpressed.
  • the overexpression of the L-serine biosynthesis genes serA fbr , serC and serB however resulted in 16 fold increase in L-serine accumulation in the culture supernatent generally.
  • the cells were cultivated in the presence of 1 mM isopropyl-beta-D-thiogalactopyranoside and in the exponential growth phse at an optical density of 6-8, measured by a Pharmacia Biotech ultrospec 3000 spectral photometer were harvested. They were then centrifuged for 10 minutes at 4500 rpm and 4° C., suspended in 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer (pH 8.0) and centrifuged again.
  • the cells were taken up in 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid buffer (pH 8.0), 1 mM FeSO 4 and 10 mM dithiothreitol.
  • the cell breakdown was effected by means of ultrasonic treatment (Branson sonifier 250; duty cycle 25%, output control 2.5, 10 minutes) on ice.
  • the reaction set contained 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer (pH 8.0), 10 mM dithiothreitol and 10-100 ⁇ l new extract.
  • the detection of the pyruvate formation from the serine was effected as described (Ohmori et al., 1991).
  • the reaction was started by adding 50 mM L-serine and after 10 minutes was stopped by the addition of 1,2-diamino-4,5-dimethoxybenzene reagent in a ratio of 1:1.
  • the reagent, as described in Ohmori et al 1991 was comprised of 4 mg 1,2-diamino-4,5-dimethoxybenzol dissolved in 42.4 ml H 2 O, 3.5 ml ⁇ -mercaptoethanol and 4.1 ml HCl (37% ig) then incubation was carried out for 2 hours at 102° dry heat.

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US20090181435A1 (en) * 2005-10-17 2009-07-16 Lothar Eggeling Method for Determining L-Serine, Gene Sequene, Vectors and Micro-Organisms
US20150223504A1 (en) * 2013-08-07 2015-08-13 Cj Cheiljedang Corporation Method for Preparing IMP Fermented Broth or Glutamic Acid Fermented Broth as Raw Material for Preparation of Natural Flavor
US20150272186A1 (en) * 2013-07-23 2015-10-01 Cj Cheiljedang Corporation Method For Preparing Natural Neutral Flavor
US20150272187A1 (en) * 2013-07-23 2015-10-01 Cj Cheiljedang Corporation Method for Preparing Natural Beef Flavor
US20150296848A1 (en) * 2013-07-23 2015-10-22 Cj Cheiljedang Corporation Method for Preparing Natural Kokumi Flavor
US20180016546A1 (en) * 2015-01-27 2018-01-18 Danmarks Tekniske Universitet Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

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PL2342103T3 (pl) 2008-10-22 2014-11-28 Johnson Controls Tech Co Zamek siedzenia pojazdu
BR112012016908B1 (pt) 2009-12-30 2020-03-31 Evonik Degussa Gmbh Linhagens e métodos para a produção de metionina
KR102279696B1 (ko) * 2021-04-20 2021-07-20 씨제이제일제당 주식회사 신규한 l-세린 암모니아 분해 효소 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법
WO2025244452A1 (ko) * 2024-05-24 2025-11-27 씨제이제일제당 (주) 글리신 절단 시스템의 활성이 강화된 미생물 및 이의 용도

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US3623952A (en) * 1967-11-29 1971-11-30 Ajinomoto Kk Method of producing l-serine by fermentation
US4528273A (en) * 1983-11-22 1985-07-09 W. R. Grace & Co. Microorganism strains for the fermentative preparation of L-serine
US6258573B1 (en) * 1998-01-12 2001-07-10 Ajinomoto Co., Inc. Method of producing L-serine by fermentation

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JP4066543B2 (ja) * 1998-01-12 2008-03-26 味の素株式会社 発酵法によるｌ−セリンの製造法
EP1257649B1 (en) * 1999-06-25 2010-01-20 Paik Kwang Industrial Co., Ltd. Corynebacterium glutamicum genes encoding metabolic pathway proteins
JP4623825B2 (ja) * 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド

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US3623952A (en) * 1967-11-29 1971-11-30 Ajinomoto Kk Method of producing l-serine by fermentation
US4528273A (en) * 1983-11-22 1985-07-09 W. R. Grace & Co. Microorganism strains for the fermentative preparation of L-serine
US6258573B1 (en) * 1998-01-12 2001-07-10 Ajinomoto Co., Inc. Method of producing L-serine by fermentation

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090181435A1 (en) * 2005-10-17 2009-07-16 Lothar Eggeling Method for Determining L-Serine, Gene Sequene, Vectors and Micro-Organisms
US20150272186A1 (en) * 2013-07-23 2015-10-01 Cj Cheiljedang Corporation Method For Preparing Natural Neutral Flavor
US20150272187A1 (en) * 2013-07-23 2015-10-01 Cj Cheiljedang Corporation Method for Preparing Natural Beef Flavor
US20150296848A1 (en) * 2013-07-23 2015-10-22 Cj Cheiljedang Corporation Method for Preparing Natural Kokumi Flavor
US20150223504A1 (en) * 2013-08-07 2015-08-13 Cj Cheiljedang Corporation Method for Preparing IMP Fermented Broth or Glutamic Acid Fermented Broth as Raw Material for Preparation of Natural Flavor
US20180016546A1 (en) * 2015-01-27 2018-01-18 Danmarks Tekniske Universitet Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways
US10513682B2 (en) * 2015-01-27 2019-12-24 Cysbio Aps Method for the production of L-serine using genetically engineered microorganisms deficient in serine degradation pathways

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