WO2012030195A9 - Micro-organisme mutant ayant une productibilité élevée pour produire de l'éthanol à partir de glycérol ou de glycérol brut, et procédé de production d'éthanol l'utilisant - Google Patents

Micro-organisme mutant ayant une productibilité élevée pour produire de l'éthanol à partir de glycérol ou de glycérol brut, et procédé de production d'éthanol l'utilisant Download PDF

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WO2012030195A9
WO2012030195A9 PCT/KR2011/006536 KR2011006536W WO2012030195A9 WO 2012030195 A9 WO2012030195 A9 WO 2012030195A9 KR 2011006536 W KR2011006536 W KR 2011006536W WO 2012030195 A9 WO2012030195 A9 WO 2012030195A9
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glycerol
genus
microorganism
mutant
enzyme activity
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WO2012030195A2 (fr
WO2012030195A3 (fr
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김철호
서정우
오백록
허선연
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01202—1,3-Propanediol dehydrogenase (1.1.1.202)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/22—Klebsiella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • the present invention relates to a method for producing a mutant microorganism with increased ethanol production ability and ethane using glycerol or waste glycerol as a carbon source, more specifically, alcohol dehydrogenase (Adcohol dehydrogenase, Adhn) Mutant microorganism with high ethanol activity from glycerol or waste glycerol with increased enzymatic activity and reduced enzymatic activity of 1,3-propanediol oxidoreductase (DhaT), An improved mutant microorganism of the mutant microorganism and a method for producing ethanol using the mutant microorganism.
  • Alcohol dehydrogenase Alcohol dehydrogenase, Adhn
  • DhaT 1,3-propanediol oxidoreductase
  • waste glycerol equivalent to about lOT (w / w) of total production (Johnson and Taconi, 2007).
  • waste glycerol not only affects the market price of conventional glycerol, but also can be released directly to the environment, which is becoming an important environmental problem such as treatment cost (da Silva eia / .2009).
  • treatment cost da Silva eia / .2009.
  • Glycerol is genus Lactobac hjs, genus C.
  • the present inventors have made diligent efforts to develop microorganisms that produce industrially useful ethane with high efficiency using glycerol or waste glycerol as a carbon source. To prepare a mutant microorganism having a high performance, and when using the microorganism, it was confirmed that ethane can be produced from glycerol or waste glycerol with high efficiency, to complete the present invention.
  • An object of the present invention is to provide a mutant microorganism having high ethanol from glycerol or waste glycerol and a method for producing the same.
  • Another object of the present invention is to provide a method for preparing ethanol from glycerol or waste glycerol using the mutant microorganisms.
  • the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhll) 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT)
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanediol oxidoreductase
  • the present invention provides a mutant microorganism having high ethanol production from glycerol or waste glycerin with reduced enzymatic activity.
  • the present invention increases the enzyme activity of alcohol dehydrogenase (Adhll) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT).
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanediol oxidoreductase
  • the present invention increases the enzyme activity of alcohol dehydrogenase (Adhll) and the enzyme activity of 1,3-propanediol oxidoreductase (DhaT).
  • Alcohol dehydrogenase Adhll
  • DhaT 1,3-propanediol oxidoreductase
  • Pyruvate dehydrogenase in mutant microorganisms with reduced glycerol or waste glycerol high ethanol production A gene encoding (pyruvate dehydrogenase, Pdc) and a gene encoding aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll) are introduced to provide a mutant microorganism overexpressed.
  • the present invention is an alcohol dehydrogenase (alcohol dehydrogenase, Adhll),
  • Enzyme activity of pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (Adhll) is increased and 1,3-propanediol oxidoreductase (1,3—propanol oxidoreductase, DhaT And mutant microorganisms having high ethanol production from glycerol or waste glycerol with reduced enzymatic activity of Lactate dehydrogenase (LdhA).
  • the present invention (a) by irradiating gamma rays to microorganisms alcohol dihydrogenase
  • the present invention (a) by irradiating gamma rays to microorganisms alcohol dihydrogenase
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanol oxidoreductase
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanol oxidoreductase
  • the present invention ( a ) by irradiating gamma rays to microorganisms alcohol dihydrogenase
  • the present invention (a) by irradiating gamma rays to microorganisms alcohol dihydrogenase
  • Alcohol dehydrogenase (Adhll) to increase the enzyme activity of the 1,3-propanedi mutated oxidase ductase (1,3-propanol oxidoreductase, DhaT) to reduce the enzyme activity;
  • mutated to increase the enzyme activity of the alcohol dehydrogenase (Adhll) and to reduce the enzyme activity of 1,3-propanediol oxidoreductase (DhaT).
  • Adhll provides a method for producing a mutant microorganism having high performance in glycerol or waste glycerol comprising overexpressing a gene encoding Adhll).
  • the present invention provides a method for producing ethane comprising the step of culturing the mutant microorganisms in a medium containing glycerol or waste glycerol as a carbon source to produce ethane, and then recovering ethane from the culture solution. to provide.
  • 1 is a schematic diagram showing the metabolic pathway of fermentation of glycerol of crab forla pneumoniae.
  • Figure 2 shows the ethanol production of mutant microorganisms according to the control and the present invention ((a) K. pneumoniae Cui ⁇ , (b) K. pneumoniae GEM1673 ⁇ 4- ⁇ ).
  • Figure 3 shows the production of ethane according to the pH of K. pneumoniae GEM167 variant strains.
  • Figure 4 shows the production of ethane according to the air injection amount of K. pneumoniae GEM167 mutant strain.
  • FIG. 6 is a fed-batch culture using waste glycerol of K. pneumoniae GEM167 mutant strain. Ethanol production by
  • Figure 7 shows the production method (A) of the K. pneumoniae ⁇ IdhA variant strains and Southern hybridization results (B) confirming the ldhA variant.
  • FIG. 8 shows the results of pure glycerol fermentation and ethane production by fed-batch culture of K. pneumoniae GEM167 ldhA mutant strains.
  • Figure 9 shows the results of fermentation and production of ethanol biodiesel waste glycerol by fed-batch culture of K. pneumoniae GEM167 ⁇ ldhA mutant strains.
  • FIG. 10 shows a route for producing improved ethane of K. pneumoniae GEM167 strain.
  • Krebsiela Gamma-irradiation results in mutations that increase the enzyme activity of alcohol dehydrogenase (Adhll) and decrease the enzyme activity of 1,3-propanediol oxidoreductase (DhaT) It was confirmed that the microorganisms prepared and the mutant microorganisms produced were able to produce ethane with high efficiency using glycerol or waste glycerol as a single carbon source.
  • the present invention provides an increase in the enzymatic activity of alcohol dehydrogenase (Adhll) and 1,3-propanedi to oxidoreductase (1,3-propanol oxidoreductase (DhaT).
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanol oxidoreductase
  • the present invention relates to a mutant microorganism having high performance of ethane from glycerol or waste glycerin with reduced enzyme activity.
  • gamma-rays were irradiated with lkGy dose to induce microorganisms, and then cultured in a medium containing glycerol as a single carbon source to isolate mutant microorganisms with improved ethanol production.
  • Metabolite production of mutant microorganisms As a result, compared with the control group, the production of glycerol oxidative metabolite was significantly increased with little production of glycerol reduced metabolite. Especially, ethanol production was increased by 8 times compared with the control group. It was confirmed.
  • the mutant microorganism according to the present invention is cultured in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol with high efficiency.
  • glycerol is used as alcohol dehydrogenase.
  • mutation of a gene encoding (alcohol dehydrogenase, Adh ⁇ ) or a gene encoding 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)) resulting in a change in the enzyme activity encoded by the gene. Changes in some or a substantial part of the biosynthetic pathways that cause changes or enzymes can be attributed to the conversion of glycerol into highly efficient ethanol.
  • the enzyme activity of the mutant microorganism irradiated with gamma rays is the enzyme activity of the mutant microorganism irradiated with gamma rays
  • the enzyme activity of alcohol dehydrogenase (Adhn) did not increase significantly when cultured in a medium containing glucose as a single carbon source, whereas in medium containing glycerol as a single carbon source.
  • the enzyme activity of alcohol dehydrogenase (Adhn) was increased by three times compared to the control group, and 1,3—propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)). It was confirmed that the enzyme activity of the control was reduced.
  • the microorganism is not particularly limited, for example, bacteria,
  • the bacterium is genus Krebsiella (genus j57ebs / e // a), genus Lactobacillus (genus ⁇ ac obac /// i / s), genus Clostridium (genus, genus Enterobacter) Etc., and more preferably, it may be of the genus Krebs.
  • the genus of the crab sial is preferably crab siella pneumoniae 057ebs / e // a 77 ffl7iw / ae) or glycerol or waste glycerol.
  • Microorganisms capable of using as a carbon source are not limited thereto.
  • the present invention also relates to a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC 11742BP) having high performance in ethane from glycerol or waste glycerol.
  • the alcohol dehydrogenase (alcohol dehydrogenase, Deletion of additional lactate hydrogenase genes in microorganisms in which a mutation occurs in which Adhll) increases the enzyme activity and decreases the enzyme activity of 1,3-propanol oxidoreductase (DhaT). It was confirmed that by improving the ethanol production capacity.
  • the present invention provides an alcohol dehydrogenase (alcohol dehydrogenase,
  • Adhll has increased enzyme activity and reduced enzyme activity of 1,3-propanol oxidoreductase (DhaT).
  • DhaT 1,3-propanol oxidoreductase
  • the present invention relates to a mutant microorganism which has deleted a gene encoding Lactate dehydrogenase (LdhA).
  • Lactate dehydrogenase is used in the mutant microorganisms in order to confirm that excess lactate is produced when glycerol is produced as ethanol using carbon as a carbon source, and to suppress the production of lactate. Reduced the activity of the enzyme.
  • the enzyme activity of alcohol dehydrogenase is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT) is increased.
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanediol oxidoreductase
  • the present invention also relates to lactate dehydrogenase (Lactate dehydrogenase) in a mutant microorganism Klebsiella / weu / »o / 7 / ae GEM167C Accession No. KCTC11742BP having high ethanol production from glycerol or waste glycerol. It relates to a mutant microorganism which has deleted a gene encoding LdhA).
  • the present invention is to increase the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase, DhaT)
  • Adhll alcohol dehydrogenase
  • DhaT 1,3-propanediol oxidoreductase
  • Pdc pyruvate dehydrogenase
  • aldehdyde dehydrogenase aldehdyde dehydrogenase
  • Adhll relates to a mutant microorganism overexpressed by introducing a gene encoding.
  • Pyruvate dehydrogenase (Pdc) and aldehyde dehydrogenase (aldehdyde dehydrogenase) to enhance the pathway of producing ethanol through aldehydes as a carbon source by using microorganisms Adhll) increased the enzyme activity.
  • the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase, DhaT).
  • oxidoreductase (1,3-propanol oxidoreductase, DhaT).
  • Pdc pyruvate dehydrogenase
  • Adhll aldehyde dehydrogenase
  • the amount of ethane produced was confirmed to increase from 21.5g / L to 25g / L.
  • the present invention also relates to pyruvate dehydrogenase (Pdc) in mutant microorganism Klebsiella / eM »o /? / Ae GEM167 (Accession No. KCTC11742BP), which has high ethanol production from glycerol or waste glycerol. It relates to a mutant microorganism overexpressed by introducing a gene encoding a) and a gene encoding an aldehyde dehydrogenase (aldhdyde dehydrogenase, Adhll).
  • Pdc pyruvate dehydrogenase
  • the enzyme activity of alcohol dehydrogenase is increased and 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT)
  • the gene encoding Lactate dehydrogenase is deleted.
  • the present invention relates to a mutant microorganism that is overexpressed by introducing a gene encoding pyruvate dehydrogenase (Pdc) and a gene encoding alpehyde dehydrogenase (Adhll).
  • the activity of the lactate dehydrogenase enzyme is reduced in the mutant microorganisms, and the ethanol production of ethanol via aldehyde as a carbon source is enhanced.
  • Pdc pyruvate dehydrogenase
  • aldehdyde dehydrogenase aldehdyde dehydrogenase
  • the enzyme activity of alcohol dehydrogenase is increased and the enzyme activity of 1,3-propanedi is 1,3-propanol oxidoreductase (DhaT).
  • Lactate dehydrogenase gene was deleted by homologous recombination in the microorganism Klebsiella / «/ TO /? / Ae GEM167, which has undergone this diminished mutation, and pyruvate dehydrogenase (Pdc) was deleted.
  • aldehyde dehydrogenase aldehdyde dehydrogenase, Adhll
  • the present invention also deletes a gene encoding lactate dehydrogenase (Ld) from a mutant microorganism Klebsiella pneumoniae GEM167 (Accession No. KCTC11742BP) having high ethanol production from glycerol or waste glycerol. and, pyruvate dehydrogenase (pyruvate dehydrogenase, Pdc) of the coding gene and the aldehyde dehydrogenase "relates to a mutant microorganism in which over-expression by introducing a gene encoding a (aldehdyde dehydrogenase, Adhll) to.
  • Ld lactate dehydrogenase
  • the present invention (a) by irradiating gamma rays to microorganisms increases the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidase ductase ( Mutating the enzyme activity of 1,3-propanol oxidoreductase (DhaT) to decrease; And (b) the enzymatic activity of the alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzymatic activity of 1,3-propanol oxidoreductase (DhaT). Beauty It relates to a method for producing a mutant microorganism having high ethanol performance from glycerol or waste glycerol comprising the step of obtaining the organism.
  • the present invention (a) by irradiating gamma rays to the microorganism increases the enzyme activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT); (b) the enzyme activity of the alcohol dehydrogenase (Adhll) is increased and the enzyme activity of 1,3-propanediol oxidoreductase (1,3-propanol oxidoreductase (DhaT) is decreased.
  • the enzymatic activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT);
  • the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT)
  • it relates to a method for producing a mutant microorganis
  • the present invention (a) by irradiating gamma rays to microorganisms increases the enzymatic activity of alcohol dehydrogenase (Adhll) and 1,3-propanediol oxidoreductase (1, Mutating to reduce the enzyme activity of 3-propanol oxidoreductase (DhaT); (b) the enzyme activity of alcohol dehydrogenase (Adhll) is increased and 1,3-propanedi is mutated to decrease the enzyme activity of oxidoreductase (1,3-propanol oxidoreductase (DhaT) Obtaining a microorganism; And (c) a gene encoding lactate hydrogenase in the obtained microorganism, a gene encoding pyruvate dehydrogenase (Pdc) and an aldehyde dehydrogenase (aldehdyde dehydrogenase, Adhll). Over
  • the gamma ray may be irradiated with an appropriate dose depending on the type of the mutant microorganism, for example, 0.1kGy ⁇ 10kGy dose.
  • an appropriate dose for example, 0.1kGy ⁇ 10kGy dose.
  • the present invention includes the step of culturing the mutant microorganism according to the present invention in a medium containing glycerol or waste glycerol as a carbon source to produce ethanol, and then recovering ethane from the culture medium. It relates to a process for producing ethane.
  • the ethanol production yields of the mutant strains of the present invention including K. / weifflOT / ae GEM167 were compared with previously reported results.
  • K. pneumoniae GEM167 ⁇ IdhA strain overexpressing the Pdc-Adhll enzyme gene showed a maximum yield of 31 g / L ethanol, which was 20.7 g / L (the existing maximum yield).
  • the existing maximum yield Compared to recombinant Escherichia coli).
  • the production yield is 40-50 g / L.
  • the culturing and recovery of mutant microorganisms can be carried out using a known culture method, and in addition to the specific medium and the specific culture method used in the embodiment of the present invention, whey , Saccharification fluid such as CSUcorn steep liquor) and other media can be used.
  • the mutant strain produced little metabolites of 1,3-propanediol and 3-hydroxypropionic acid, which are metabolites of glycerol, and metabolites of oxidative metabolic pathways.
  • the production was shown to increase significantly compared to the control.
  • the production of ethanol showed an extremely high yield of ethane (high performance) at 8.6 g / L, an increase of about 8 times from 1.1 g / L (Fig. 2, Table 1).
  • mutant strains showed metabolite production characteristics similar to those of the control.
  • the mutant strain with the improved amount of ethanol specific to glycerol was named GEM167, and was deposited with the KCTC 11742BP accession number to the Korea Biotechnology Research Institute Gene Bank.
  • Example 2 Enzyme activity of ⁇ . / Eiffl? O;? / A ⁇ EM167 mutant strain
  • Enzyme Activity Assay for Determination of Enzyme Activity of Alcohol Dehydrogenase (Adhll)
  • the mixed solution was prepared by glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM).
  • the reaction mixture was prepared by adding 100 mM ethanol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ° C and then measured the hop intensity at 340 nm (Postma E. et al, Appl Environ Microbiol., 55: 468-477,1989).
  • Enzyme Activity Assay for Measuring Enzyme Activity of Glycerol Dehydrogenase (DhaD) Glycerol was mixed with glycine-KOH buffer (pH 9.0) (50 mM) and NAD + (1 mM) in culture strains. ) was added, and the reaction was started by adding 100 mM glycerol to the prepared mixture. The reaction was carried out for 30 minutes at 37 ° C and then measured for absorbance at 340 nm.
  • Enzyme Activity Assay for Measuring Enzyme Activity of 1,3-propanediol oxidoreductase (DhaT)
  • the mixed solution was formulated with glycine-KOH buffer (pH 9.0) ( 50 mM) and NAD + (1 mM) were added.
  • the reaction was started by adding 100 mM 1,3-propanedi to the prepared mixture. The reaction was measured for 30 minutes at 37 ° C and then measured for absorbance at 340 nm.
  • Alcohol dehydrogenase (Adhll) enzyme of 167 strains was shown to be more than three times higher than that of the control Cu strain.
  • the increase in alcohol dehydrogenase (Adhll) enzyme activity was not significant.
  • Significant increases in alcohol dehydrogenase (Adhll) enzyme activity in glycerol medium are consistent with the metabolite production assays above.
  • 1,3-propanediol oxidoreductase (DhaT) enzyme activity was decreased in GEM 167 mutant strains, which reduced production of 1,3-propanedi and Is a matching result.
  • Enzyme enzyme units of K. pneusioniae Cu and GEM 167 variant strains
  • Enzyme 1 St. units are expressed as unit / g protein.
  • Example 3 Analysis of Ethanol Productivity of Mutant Strains According to pH ⁇ 9i>
  • K. pneumoniae GEM167 In order to determine the effect of pH on the production of ethane of the mutant strain K. pneumoniae GEM167, by using the same method as in Example 1 to change the pH of the culture medium to 5, 6, 7 and 8 The ethanol production of the mutant strain was measured while maintaining the culture. As a result, as shown in Figure 3, it showed a high ethanol productivity in the neutral conditions of pH 6 and 7, especially in the acidic condition pH 5 it was found that the cell growth and ethanol productivity is very poor.
  • GEM 167 was fermented and cultured by a fed-batch method while maintaining the areation at 0.5 wm and the pH at 6.8 to 7.2.
  • the maximum yield of ethane was 21.5 g / L at 23 hours of incubation, and the yield per hour.
  • Lactate deficient GEM167 mutant strain briefly describes the manufacturing process as follows.
  • the top and bottom sequences of the IdhA gene were 0.9 and kb, respectively, PI and P2 primers (upstream). Or amplified using P3, P4 primers (downstream), and then inserted the antibiotic apramycin resistance gene in between to prepare a homologous recombinat ion DNA cassette.
  • Constructed DNA Cassette (/ Q 4 Gene Upstream Sequence-Apramycin Resistance Gene Gene Downstream sequences) were introduced into the K. pneumoniae GEM167 strain by electrolayering, followed by homologous recombinat ion to insert a DNA cassette into chromosomal DNA to isolate transformants that exhibit apramycin resistance.
  • IdhA strain was cultured to analyze the activity of lactate dehydrogenase enzyme protein.
  • the recovered culture cells were washed with 50 mM potassium phosphate buffer (pH 7.5), resuspended, crushed by the sonication method, and used as an enzyme source.
  • Enzyme activity assay mixture to measure LdhA enzyme activity was prepared by adding glycine-KOH buffer (pH 9.0) (50mM) and NAD + (ImM) to the culture strain pulverized solution, and 100 raM lactate in the prepared mixture. The reaction was started by addition. The reaction was carried out for 30 minutes at 37 ° C and then measured for absorbance at 340 nm. As a result, as shown in Table 6, GEM167! Lactein / Q1 ⁇ 24 strain Ytt dehydrogenanaze enzyme activity was significantly reduced. Table 6
  • Example 9 Improvement of eM ⁇ 2 / aeGEM167 Mutant Strain: Overexpression of Pdc-adhll Gene
  • Pdc the genes and gene Adhll of 1.15kb of 1.8kb, respectively
  • P P dc-F (5'- 7C7 fi4atgagttatactgtcggtacctatttagc-3 '( SEQ ID NO: 5); italic - bal site)
  • Ppdc- R (5'- (: fi 6CT (X: AGctagaggagcttgttaacaggct: tac-3 '(SEQ ID NO: 6); italic site, underscore-/ 3 ⁇ 4l site)
  • PaldB-F (5'- 3 ⁇ 4tggcttcttcaacttttttatattcc-3' (SEQ ID NO: 7); italic- ⁇ gJII Site)
  • PaldB-R (5'-C7C 6TCTAGAttagaaagcgctcaggaagagt
  • the gene expression promoter / ac promoter sequence ( ⁇ -aldB) was expressed as PlacZ-aldB-F (5'— 477 agcgggcagtgagcgcaa-3 '(SEQ ID NO: 9); Italic-Ec (R ⁇ ⁇ ⁇ Q ]) and P lacZ- aldB-R (5 '-CTCAGAkGklZl agctgt 11 cctgtgtgaaat tg-3 (SEQ ID NO:
  • the Adhll and Pdc genes are inserted in turn to insert pGEM-P / ac ⁇ a 1 clB, pG-? lacZ -a 1 cIB-pdc .
  • p / ⁇ -a / o-/ c: cassette was reinserted into the pBR322 vector to make pBR—akiB-jxk, followed by K. pneumoniae Cu, GEM16.
  • Each of the GEM167 JdhA strains was introduced.
  • Pdc Pyruvate decarboxylase
  • Adhll aldehyde dihydrate of recombinant cells
  • Pdc enzymatic activity assays to determine the activity common hapaek is imidazole hydrochloride buffer (pH. 6.5) to the culture strain crushing solution (40mM),
  • K was overexpressed with the Pdc-Adhll enzyme gene while maintaining a culture temperature of 37 ° C., agitation speed of 200 rpm, an air injection rate of 0.5wm, and a pH of 6.8 to 7 ⁇ 2 using 2 L of the medium described in Example 1.
  • Pneumoniae mutant strains were fermented and cultured in a fed-batch method to analyze the fermentation and ethanol production of glyce, and the results are shown in FIGS. 11 and 12.
  • Pdc-Adhll enzyme gene roll overexpression K.
  • Pneumoniae GEM167 ⁇ IdhA strains showed a maximum ethane- production yield of 31 g / L, which is far superior to the maximum production of Kichon 20.7 g / L (recombinant Escherichia coli).
  • the production yield is 4 . If it is about 0-50 g / L, it is expected to have economic feasibility.
  • the production yield of the mutant strains of the present invention if the optimization of the culture process is made, it is considered that the commercial value is warm.
  • accession number KCTC11742BP
  • the present invention has the effect of providing a mutant microorganism that produces ethane with high efficiency by irradiating gamma rays to microorganisms using glycerol or waste glycerol as a carbon source.
  • the mutant microorganism according to the present invention has little production of metabolites of glycerol as a reduced metabolic pathway, and produces ethane, a high value-added product, by using waste glycerol, a by-product of the biodiesel industry, with high efficiency. It is useful industrially.

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Abstract

Cette invention concerne un micro-organisme mutant, dont l'aptitude à produire de l'éthanol, en utilisant du glycérol ou du glycérol brut à titre de source de carbone, est améliorée, et un procédé de production d'éthanol l'utilisant. Plus particulièrement, cette invention concerne un micro-organisme mutant ayant une productibilité élevée pour produire de l'éthanol à partir de glycérol ou de glycérol brut, l'activité enzymatique alcool déshydrogénase (AdhII) étant augmentée et l'activité enzymatique 1,3-propanediol oxydoréductase (DhaT) étant diminuée chez ledit organisme mutant. Cette invention concerne également un micro-organisme amélioré à l'aide du micro-organisme mutant précité, et un procédé de production d'éthanol utilisant ledit micro-organisme mutant.
PCT/KR2011/006536 2010-09-03 2011-09-02 Micro-organisme mutant ayant une productibilité élevée pour produire de l'éthanol à partir de glycérol ou de glycérol brut, et procédé de production d'éthanol l'utilisant WO2012030195A2 (fr)

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KR10-2010-0086550 2010-09-03

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