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WO2009066927A1 - Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries - Google Patents

Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries Download PDF

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WO2009066927A1
WO2009066927A1 PCT/KR2008/006807 KR2008006807W WO2009066927A1 WO 2009066927 A1 WO2009066927 A1 WO 2009066927A1 KR 2008006807 W KR2008006807 W KR 2008006807W WO 2009066927 A1 WO2009066927 A1 WO 2009066927A1
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glycerol
corynebacteria
gene
corynebacterium glutamicum
glpdfk
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PCT/KR2008/006807
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English (en)
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Jae-Yeong Ju
Hyun-Ae Bae
Hyo-Jin Kim
Jin-Sook Chang
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Cj Cheiljedang Corporation
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Priority to EP08852445A priority Critical patent/EP2260104A4/fr
Priority to DE112008003111.8T priority patent/DE112008003111B4/de
Publication of WO2009066927A1 publication Critical patent/WO2009066927A1/fr

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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
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • 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
    • 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
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0103—Glycerol kinase (2.7.1.30)

Definitions

  • the present invention relates to Corynebacteria introduced with a foreign gene glpDFK operon involved in the utilization of glycerol. Also, the present invention relates to a method for producing fermentation product from various carbon sources containing glycerol using the Corynebacteria.
  • BioDiesel obtained from plant oil and Bioethanol produced by fermentation are the most attractive candidates.
  • BioDiesel indicates fatty acid methyl ester or fatty acid ethyl ester synthesized by esterification of methanol produced using plant oil as a substrate in the presence of a catalyst.
  • the byproduct, glycerol is necessarily generated by 10% of the total weight.
  • 3 8 3 6 12 6 can provide improved reducing power during the metabolism of a microorganism. Many products produced by fermentation require reducing power in their metabolic pathways. Therefore, if the glycerol could be effectively used as a substrate, the yield and productivity would be improved. Despite the expectation, the studies on glycerol have been limited to reuterin (Talarico et. al., Antimicrob. Agents Chemother., 32:1854-1858 (1988)), 2,3-butanediol (Biebl, et al., Appl Microbiol. Biotechnol. 50:24-29 (1998)), 1,3-propanediol (Menzel, et. al., Enzyme Microb.
  • Glycerol has been produced so far in the industry of soap, fatty acid, wax and surfactants. However, as mentioned above, it has been a new problem to solve how to treat glycerol, the byproduct, produced during the production of BioDiesel that is dramatically increasing. In the meantime, the price of the purified glycerol is also expected to be dropped. Therefore, the production of useful fermentation products using glycerol might bring effects more than expected.
  • E. coli extracellular glycerol infiltrates in cells by using GIpF, one of aquaglyceroporin having permeability for water, glycerol and urea, without energy consumption (Heller et al., J. Bacteriol. 144:274-278, (1980)).
  • the glycerol is converted into glycerol- 3 -phosphate by glycerol kinase, which is converted again into dihydroxyacetonephosphate (DHAP) by glycerol- 3 -phosphate dehydrogenase and then converted into glyceroaldehyde- 3 -phosphate (G-3-P) by triosephosphate isomerase (TpiA), followed by final metabolism.
  • DHAP dihydroxyacetonephosphate
  • TpiA triosephosphate isomerase
  • glycerol kinase activity has no activity
  • glycerol is converted into dihydroxyacetone (DHA) by glycerol dehydrogenase (Gdh), which is converted again into dihydroxyacetone phosphate (DHAP) by glycerol kinase or dihydroxyacetone kianse (DHA kinase), followed by conversion again into glyceraldehydes-3-phosphate (G-3-P) before final metabolism (Paulsen et al., Microbiology, 146:2343-2344, (2000)).
  • DHA dihydroxyacetone
  • DHAP dihydroxyacetone phosphate
  • DHA kinase dihydroxyacetone kianse
  • G-3-P glyceraldehydes-3-phosphate
  • Such glycerol metabolic pathway is regulated by various factors. Particularly, when glycerol and glucose are together, the wild type E. coli reportedly shows diauxic growth in which the wild type E
  • Corynebacterium glutamicum has been used for the production of such amino acids as lysine and monosodium glutamate
  • C. am- moniagenes has been used for the production of nucleic acid by fermentation industrially. It has reported that Corynebacteria can use various carbon sources such as glucose and raw sugar for fermentation. It has also been reported that Corynebacteria can use xylose by the introduction of a gene such as xylAB (Kawaguchi et al., Appl. Envion. Microbiol. 72(5): 3418-3428 (2006)).
  • Corynebacterium diphtheria is a pathogenic bacterium which is classified as Biosafety level 2.
  • the present inventors continued studies to solve the above problem and found various strains containing a complete gene using glycerol other than Corynebacterium diphtheria.
  • Brevibacterium genus inducing Brevibacterium linens was one containing a complete gene using glycerol.
  • Corynebacterium and Brevibacterium are known to have similar gene specificities. Therefore, there has recently been reported a trend to classify Corynebacterium and Brevibacterium into a single species (Liebl, Ekaman et al., 1991).
  • the present invention provides an operon involved in the use of glycerol to improve glycerol assimilation of Corynebacteria significantly.
  • the present invention provides a mutant of the operon involved in the use of glycerol.
  • the gene involved in the use of glycerol herein indicates a gene encoding glycerol uptake facilitator protein originated from Brevibacterium linens (referred as glpF hereinafter), a gene encoding glycerol kinase which is the enzyme producing glycerol- 3-phosphate by phosphorylation using ATP (referred as glpK hereinafter), and a gene encoding glycerol- 3 -phospho dihydrogenase which is the enzyme producing dihy- droxyacetone-3-phosphate by oxidizing glycerol- 3 -phosphate (referred as glpD hereinafter).
  • the gene includes any gene encoding a polypeptide in Corynebacteria that plays a role in uptake of extracellular glycerol and converting the glycerol into glycerol- 3-phosphate by phosphorylation, and converting glycerol-3-phosphate into dihy- droxyacetone-3-phosphate, and then finally converting dihydroxyacetone- 3 -phosphate into glyceraldehyde-3-phosphate, the intermediate of glycolysis, to metabolize.
  • the gene can be originated from animals, plants, and microorganisms. It is more preferred to select a gene originated from a microorganism, particularly from Brevibacterium linens BL2 (Gene Bank Accession No: NZ_AAGP00000000; SEQ. ID. NO: 7). It is most preferred to select a gene corresponding to SEQ. ID. NO: 3, having a mutation in the nucleotide sequence of glpDFK of Brevibacterium linens BL2
  • the present invention also provides a mutant originated from Corynebacteria using glycerol either alone as a carbon source or together with other carbon sources.
  • the strain used in this invention can be growth consuming glycerol and glucose simultaneously to use glycerol effectively.
  • glucose and glycerol are given simultaneously as a carbon source
  • the wild type E. coli uses glucose exclusively, and after consuming all the glucose the wild type E. coli uses glycerol, which is so called 'diauxy'
  • glycerol which is so called 'diauxy'
  • the present inventors provide a microorganism containing the gene encoding GIpD (Gene Bank Accession No: NP_00380226.1; SEQ. ID. NO: 4), GIpF (Gene bank Accession No: NP_00380225.1; SEQ ID NO: 5) or GIpK (Gene Bank Accession No: NP_00380224.1; SEQ. ID. NO: 6), the protein involved in the use of glycerol, originated from Brevibacterium linens.
  • GIpD Gene Bank Accession No: NP_00380226.1; SEQ. ID. NO: 4
  • GIpF Gene bank Accession No: NP_00380225.1; SEQ ID NO: 5
  • GIpK Gene Bank Accession No: NP_00380224.1; SEQ. ID. NO: 6
  • Corynebacteria with the gene can be performed by the conventional method known to those in the art, and a mutant of Corynebacterium transformed with a vector containing glpDFK operon (SEQ. ID. NO: 3), producing amino acids using glycerol either alone as a carbon source or together with other carbon sources can be provided.
  • glpDFK operon SEQ. ID. NO: 3
  • the vector for the present invention is not limited to a specific one and any informed expression vector can be used. Particularly, E. coli-Corynebacterium shuttle vector pECCG117 (Biotechnology letters vol 13, No.10, p.721-726 (1991)) is preferred.
  • 'transformation' indicates the process of introducing a gene into a host cell and expressing the gene therein.
  • the gene used for the transformation is either inserted into chromosome of the host cell or presented in the outside of chromosome as long as it can be expressed in the cell.
  • the gene is a polynucleotide capable of encoding a polypeptide and containing DNA and RNA.
  • the gene is not limited in its form for introduction, as long as it can be expressed in the host cell.
  • the gene can be introduced into the host cell as an expression cassette, the polynucleotide construct that contains every necessary element for auto-expression.
  • the expression cassette comprising a promoter operably linked to the gene, a transcription terminator, a ribosome binding site, and a translation terminator.
  • the expression cassette can be the expression vector capable of auto-replication.
  • the gene can be operably linked to the sequence necessary for the expression in the host cell by introducing into a host cell as in itself or the form of a polynucleotide construct.
  • the microorganism transformed with a gene involved in the use of glycerol to produce amino acids effectively using glycerol can be one of Corynebac- teriaceae, more preferably a microorganism of Corynebacterium genus, and most preferably a microorganism selected from the group consisting of Corynebacterium glutamicum (ex. ATCC 13032), Corynebacterium ammoniagenes (ex. ATCC 6872), Brevibacterium lactofermentum (ex. ATCC13869), Brevibacterium flavum (ex. ATCC14067), Corynebacterium thermoamino genes (ex.
  • FERM-BP 1539 and Corynebacterium efficiens (ex. C. efficiens str. YS-314), but not always limited thereto.
  • the microorganism producing useful materials such as amino acids or nucleic acids, for example Corynebacterium glutamicum SM5 producing glutamic acid and Corynebacterium glutamicum CF 905 (KFCC- 10881) and Corynebacterium glutamicum CgGB 3 producing lysine, can be included, but not always limited thereto.
  • glpDFK gene of Brevibacterium linens was cloned into pECCG117, E. coli- Corynebacterium shuttle vector, followed by transfection of Corynebacterium glutamicum CgGB 3. Corynebacterium glutamicum CgGB 3 was transformed with the vector.
  • the transformed strain was named Corynebacterium glutamicum C003-0011 (KCCM 10886P)
  • the present invention further provides a method for producing fermentation product by fermenting Corynebacteria using glycerol either alone as a carbon source or together with other carbon sources.
  • the present invention provides a method for producing fermentation product using glycerol as a carbon source comprising the steps of : transforming Corynebacteria with the vector containing glpDFK operon represented by SEQ. ID. NO: 3 which is a gene combination facilitating the use of glycerol; culturing the transformed Corynebacteria by inoculating in the culture medium containing glycerol either alone as a carbon source or together with other carbon sources; and separating fermentation product from the culture medium.
  • culture of the microorganism can be performed in a proper medium and under proper culture conditions known to those in the art. These conditions can be regulated according to the selected strain.
  • the cultivation methods are exemplified by batch, continuous and fed-batch cultures, but not always limited thereto. Various culture methods are described in "Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176.
  • the medium has to meet the requirements for the culture of a specific strain.
  • the medium used in this invention contains glycerol alone or glycerol together with other carbon sources, as a carbon source.
  • other carbon sources can be properly added, and at this time glucose is preferred as a carbon source.
  • a nitrogen source such organic nitrogen source as peptone, yeast extract, gravy, malt extract, corn steep liquor and soybean flour, and such inorganic nitrogen source as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be included in the medium.
  • potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium-containing salts can be included in the medium.
  • a metal salt such as magnesium sulfate or iron sulfate can also be included.
  • Amino acids, vitamins and proper precursors can be included as well. These medium or precursors can be added to the culture by batch-type or continuously.
  • PH of the culture can be adjusted during the cultivation by adding such a compound as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid.
  • the generation of air bubbles can be inhibited during the cultivation by using an antifoaming agent such as fatty acid polyglycol ester.
  • oxygen or oxygen-containing gas can be injected into the culture.
  • the temperature of the culture is preferably 20-45 0 C, more preferably 25-4O 0 C.
  • the cultivation can be continued until the production of useful materials reachs a desired level, and the preferable culture time is 10-160 hours.
  • FIG. 1 illustrates a construction of a recombinant plasm ⁇ pECCGl 17-bli glpDFK containing glpDFK operon.
  • Example 1 Screening and cloning of a gene involved in to the use of glycerol which is operable in Corvnebacteria
  • GIpF, GIpK and GIpD are genes of Brevibacterium linens involved in the use of glycerol and adjacent nucleotide sequences was obtained from GeneBank, NIH, USA. Gene Bank Accession No. of GIpF of Brevibacterium linens was ZP_00380225.1, Gene Bank Accession No. of GIpK was ZP_00380224.1, and the Gene Bank Accession No. of GIpD was ZP_00380226.1. The genes were confirmed to be arranged in a series on a genome.
  • PCR could amplify all of the three genes as a single polynucleotide.
  • Primers represented by SEQ. ID. NO: 1 and NO: 2 were used for PCR to amplify the genes involved in the use of glycerol of Brevibacterium linens.
  • the primer represented by SEQ. ID. NO: 1 includes a Xbal restriction enzyme site and the primer represented by SEQ. ID. NO: 2 includes a Pstl site.
  • Chromosome of Brevibacterium linens was purchased from American Type Culture
  • ATCQ ATCC ID. No: 9175D
  • PCR was performed using the chromosome of Brevibacterium linens as a template to amplify the gene involved in the use of glycerol.
  • PCR was performed as follows; predenaturation at 94 0 C for 3 minutes, denaturation at 94 0 C for 30 seconds, annealing at 56 0 C for 30 seconds, polymerization at 72 0 C for 4 minutes 30 seconds, 25 cycles from denaturation to polymerization, and final extension at 72 0 C for 5 minutes.
  • the PCR product was transferred on to agarose-gel for electrophoresis. As a result, 4574 bp sized polynucleotide was obtained.
  • the polynucleotide was cloned into pCR2.1 by using TOPO TA cloning kit (Invitrogen).
  • Example 2 Determination of nucleotide sequence of the glpDFK gene [52]
  • the plasmid obtained above was digested with Xbal and Pstl to obtain a DNA fragment containing the gene involved in the use of glycerol.
  • the DNA fragment was cloned into pECCGl 17, E. coli- Corynebacterium shuttle vector, followed by transformation of E. coli TOPlO.
  • the plasmid obtained by the conventional plasmid miniprep was named pECCGl 17-bli glpDFK.
  • DNA nucleotide sequence of the pECCGl 17-bli glpDFK was determined and accordingly it was confirmed that the nucleotide sequence from 252 nd
  • Corynebacterium glutamicum ATCC 13032 wild type by electric pulse method.
  • the cell was cultured on the plate medium containing bacto-peptone 10 g/L, yeast extract 10 g/L, beef extract 5 g/L, NaCl 2.5 g/L and kanamycin 25 ⁇ g/mL.
  • the obtained colonies proceeded to PCR cloning to select the colonies containing the plasmid comprising the gene involved in the use of glycerol.
  • the selected strain was named ATCC13032/pECCGl 17-bli glpDFK.
  • ATCC13032/pECCGl 17-bli glpDFKusing glycerol Corynebacterium glutamicum ATCC 13032 containing the above plasmid and Corynebacterium glutamicum ATCC 13032 not containing the plasmid were respectively cultured in solid LB medium. These were inoculated respectively in 250 ml corner-baffled flask containing 25 ml of the seed medium, followed by shaking-culture (200 rpm) at 3O 0 C for 48 hours. Residues of glycerol and glucose in the medium were measured by HLC analysis and the results are shown in Table 2. The growth of Corynebacterium glutamicum introduced with pECCGl 17-bli glpDFK was confirmed. But, the growth of the strain not introduced with the plasmid was insignificant.
  • Corynebacterium glutamicum ATCC 13032 could not be growing by using glycerol, but Corynebacterium glutamicum ATCC13032/pECCGl 17-bli glpDFK containing glpDFK gene of Brevibacterium linens could be growing by using glycerol as a carbon source.
  • the selected strain was named Corynebacterium glutamicum C003-0011, which was deposited at KCCM (Korean Culture Center of Microorganisms) of KFCC (Korean Federation of Culture Collection), the International Depository Authority located at 361-221, Hongje-1-Dong, Seodaemungu-Gu, Seoul, Korea, on October 19, 2007 (Accession No: KCCM10886P).
  • Example 6 Lysine production of a lysine-producing strain Corynebacterium dutamicum C003-0011 (KCCM10886P) using glycerol
  • Corynebacterium glutamicum mother strain CgGB3 and C003-0011 (KCCM10886P) containing pECCGl 17-bli glpDFK, the strain of the present invention were inoculated respectively in 250 ml corner-baffled flask containing 25 ml of the seed medium, followed by shaking-culture (200 rpm) at 3O 0 C for 20 hours. 1 ml of the seed culture solution was inoculated in 250 ml corner-baffled flask containing 24 ml of the production medium, followed by shaking-culture (200 rpm) at 3O 0 C for 4 days. Upon completion of the culture, L-lysine production was measured with an amino acid analyzer. L-lysine levels in Corynebacterium glutamicum CgGB3 and C003-0011 (KCCM10886P) cultures were investigated and the results are shown in Table 3.
  • Seed medium (pH 7.0 s ): [73] Glucose 40 g, Peptone 10 g, Yeast extract 5 g, Urea 1.5 g, K FPO 8 g, MgSO -7H O
  • the present invention provides a method for producing useful materials with high yield using glycerol effectively which is the byproduct of BioEXesel.
  • the microorganism of the present invention can produce useful materials effectively in the medium containing a complex carbon source comprising glycerol and in the medium containing glycerol alone as a carbon source. Therefore, the microorganism capable of producing useful materials, based on the microorganism of the present invention, can use glycerol as a carbon source effectively.

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Abstract

La présente invention porte sur des corynébactéries qui peuvent utiliser diverses sources de carbone contenant du glycérol et sur un procédé de fabrication d'un produit de fermentation à partir de diverses sources de carbone contenant du glycérol à l'aide de ces corynébactéries. Plus précisément, la présente invention porte sur un procédé de fabrication d'un produit de fermentation avec un rendement élevé et une productivité élevée, par fermentation de corynébactéries dans lesquelles est introduit le gène étranger glpDFK facilitant l'utilisation de glycérol et accumulant des acides aminés industriellement utiles dans le milieu de culture.
PCT/KR2008/006807 2007-11-20 2008-11-19 Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries WO2009066927A1 (fr)

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EP08852445A EP2260104A4 (fr) 2007-11-20 2008-11-19 Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries
DE112008003111.8T DE112008003111B4 (de) 2007-11-20 2008-11-19 Corynebakterien, die Glyzerin enthaltende Kohlenstoffquellen verwerten, und Verfahren zur Herstellung eines Fermentationsprodukts unter Verwendung derselben

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KR1020070118455A KR100924904B1 (ko) 2007-11-20 2007-11-20 글리세롤을 포함한 탄소원을 이용할 수 있는코리네박테리아 및 이를 이용하여 발효산물을 생산하는방법
KR10-2007-0118455 2007-11-20

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DE112008003111B4 (de) 2023-05-25
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EP2260104A4 (fr) 2012-03-14
EP2260104A1 (fr) 2010-12-15

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