WO2018056794A1 - Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone - Google Patents

Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone Download PDF

Info

Publication number
WO2018056794A1
WO2018056794A1 PCT/KR2017/010643 KR2017010643W WO2018056794A1 WO 2018056794 A1 WO2018056794 A1 WO 2018056794A1 KR 2017010643 W KR2017010643 W KR 2017010643W WO 2018056794 A1 WO2018056794 A1 WO 2018056794A1
Authority
WO
WIPO (PCT)
Prior art keywords
microorganism
gene
acetic acid
coli
seq
Prior art date
Application number
PCT/KR2017/010643
Other languages
English (en)
Korean (ko)
Inventor
이승구
한귀환
성원재
이대희
Original Assignee
한국생명공학연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국생명공학연구원 filed Critical 한국생명공학연구원
Publication of WO2018056794A1 publication Critical patent/WO2018056794A1/fr

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1025Acyltransferases (2.3)
    • C12N9/1029Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16Butanols
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y101/00Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001Alcohol dehydrogenase (1.1.1.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y101/00Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/011573-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y102/00Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01003Aldehyde dehydrogenase (NAD+) (1.2.1.3)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y103/00Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01038Trans-2-enoyl-CoA reductase (NADPH) (1.3.1.38)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y402/00Carbon-oxygen lyases (4.2)
    • C12Y402/01Hydro-lyases (4.2.1)
    • C12Y402/010553-Hydroxybutyryl-CoA dehydratase (4.2.1.55)
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a microorganism having improved metabolic ability to acetic acid, and more particularly, one or more of the five genes involved in the cellular use of acetic acid is mutated to use acetic acid as the only carbon source, preferably E. coli It is about a strain.
  • Microorganisms such as Escherichia coli inhibit the growth of Escherichia coli depending on their concentration when cultured using acetic acid alone or as a mixed carbon source.
  • the lag phase of microbial growth becomes longer, and thus, the total incubation time is long (Holger Ebbighausen, et al., Arch. Microbiol., 1991, 155 (5). ), 505-5101).
  • E. coli strains that can use acetic acid as the only carbon source is known
  • the E. coli strain is known to significantly slow growth when using acetic acid as the only carbon source (Frank E. Dailey, et al., J. Bacteriol., 1986, 165 (2), 453-460).
  • the present invention is a wild-type microorganism patZ , cspC , mukB, lomR And at least one gene selected from the group consisting of yhjE provides a mutant microorganism capable of utilizing acetic acid as the sole carbon source.
  • the mutant microorganism of the present invention is essentially mutated patZ gene, cspC , mukB , lomR And one or more genes selected from the group consisting of yhjE is preferably mutated, but is not limited thereto.
  • the mutant microorganism of the invention is patZ, cspC , mukB , lomR And all five genes of yhjE are preferably mutated, but are not limited thereto.
  • the patZ , cspC, mukB , lomR And yhjE gene preferably have a nucleotide sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 5, but is not limited thereto.
  • the mutant microorganism of the present invention is preferably, but not limited to, further removed one or more genes selected from the group consisting of frdA , ldhA , adhE and pta .
  • the frdA , ldhA , adhE and pta genes preferably have a nucleotide sequence consisting of SEQ ID NO: 6 to SEQ ID NO: 9, but is not limited thereto.
  • the mutation of the patZ gene is Trp 501th amino acid of the wild type enzyme consisting of the amino acid sequence of SEQ ID NO: 10 is mutated to the stop codon (Trp501Stop), the mutation of the cspC gene is SEQ ID NO: 11 Gln, the 58th amino acid of the wild-type enzyme consisting of the amino acid sequence of Gln58Stop is mutated (Gln58Stop), and the mukB gene is mutated to Alu , the 54th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 12, to Glu ( Asp54Glu), and the mutation of the lomR gene is Pro 114Leu, which is the 114th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 13, is mutated to Leu (Pro114Leu), and the mutation of the yhjE gene is the wild type consisting of the amino acid sequence of SEQ ID NO:
  • the mutant microorganism of the present invention is preferably, but not limited to, further introduced a biosynthesis related gene of butanol.
  • the biosynthesis related gene of butanol is 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA li May be, but are not limited to, ductases and aldehydes and alcohol dehydrogenases.
  • the microorganism is preferably E. coli, but is not limited thereto.
  • the microorganism is preferably E. coli SBA01 strain deposited with accession number KCTC13040BP, but is not limited thereto.
  • the present invention (1) patZ , cspC , mukB , lomR And mutating one or more genes selected from the group consisting of yhjE to use acetic acid as the only carbon source.
  • (3) provides a method for producing a recombinant protein comprising culturing the transformed microorganism in a medium containing a carbon source.
  • the present invention (1) patZ , cspC , mukB , lomR And mutating one or more genes selected from the group consisting of yhjE to use acetic acid as the only carbon source.
  • the transforming microorganism is preferably one or more genes further selected from the group consisting of frdA , ldhA , adhE and pta , but is not limited thereto.
  • the microorganism is preferably E. coli, but is not limited thereto.
  • the microorganism is preferably E. coli SBA01 strain deposited with accession number KCTC13040BP, but is not limited thereto.
  • microorganisms having improved metabolic ability to acetic acid of the present invention can be grown not only using acetic acid as the sole carbon source, but also useful for preparing industrially useful target substances such as butanol from acetic acid.
  • 1 is a diagram showing the types of genes in the process from the carbon source such as glucose or acetic acid to the butanol synthesis pathway via acetyl-CoA.
  • Figure 2 is a diagram showing the synthesis pathway of acetyl-CoA and the genes involved in the acetic acid metabolism of Escherichia coli.
  • Figure 3 is E. coli MG1655 ⁇ frdA ⁇ ldhA ⁇ pta ⁇ adhE
  • G represents the passage of subculture.
  • Figure 5 is a graph showing the results of resistance test against acetic acid of E. coli SBA01 strain of the present invention.
  • Figures 6a and 6b is a table showing a list of the top genes of the E. coli SBA01 strain (experimental group) of the present invention by performing a comparative analysis of the transcripts of each of the increased expression amount, Figure 6c is expressed in the metabolic circuit of E. coli The results obtained by mapping the increased or decreased genes are shown.
  • Figure 7 is a graph showing the results of measuring the intracellular ATP content of spontaneous E. coli MG1655, control DSM01 and SBA01 strain of the present invention.
  • FIG. 8 is a graph and photograph showing growth and expression of fluorescent protein in minimal acetic acid medium of Escherichia coli SBA01 strain transformed with fluorescent protein.
  • FIG. 9 is a graph showing the growth of the E. coli SBA01 strain and acetate, formate use of the present invention, butaneol biosynthesis related genes are introduced.
  • FIG. 10 is a graph showing that butanol was produced as a result of GC and GC / MS analysis of fermentation products after fermentation in acetic acid minimal medium in E. coli SBA01 strain of the present invention into which a butanol biosynthesis related gene was introduced.
  • the present invention relates to wild type microorganisms patZ , cspC , mukB , lomR And at least one gene selected from the group consisting of yhjE provides a mutant microorganism capable of utilizing acetic acid as the sole carbon source.
  • PatZ , cspC , mukB , lomR And yhjE gene may have a base sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 5, but is not limited thereto.
  • E. coli strains that use acetic acid as the only carbon source (Frank E. Dailey, et al., J. Bacteriol., 1986, 165 (2), 453-460), or E. coli mutated patZ , cspC , mukB or lomR genes Strains (Sara Castano-Cerezo, et al., Mol. Syst. Biol., 2014, 10, 762; Devashish Rath, et al., J. Bacteriol., 2006, 188 (19), 6780-6785; Hironori Niki, et al., J.
  • the patZ gene expresses an acetyltransferase enzyme
  • the cspC gene expresses a multicopy inhibitor of MukB cold shock protein as a stress protein
  • the mukB gene expresses a chromosomal partition protein
  • the lomR gene assumes an outer membrane It expresses toxic proteins
  • the yhjE gene is known to express endometrial metabolite transport proteins.
  • the mutant microorganism is preferably one or more genes selected from the group consisting of frdA , ldhA , adhE and pta , but is not limited thereto.
  • the frdA , ldhA , adhE and pta genes may have base sequences consisting of SEQ ID NO: 6 to SEQ ID NO: 9, but are not limited thereto.
  • the activity of the enzyme expressed by the gene of interest may be lost.
  • mutation means that the DNA molecule in which genetic information is recorded is changed from the original DNA and its sequence by various factors. When a mutation occurs, a change occurs in a protein produced by the gene. This can lead to changes in the genotype, which can be altered in the biological characteristics of the individual.
  • the mutation can be introduced by treating the microorganism with any chemical and / or physical means known to be capable of causing a mutation in the art.
  • the chemical means may include chemicals such as NTG (nitrosoguanidine), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), benzopyrene, etc., which are effective guanidine derivatives as mutation causing substances (mutants).
  • X-rays, ⁇ -rays, and the like but are not limited thereto.
  • a mutant strain was prepared by acetic acid adaptive evolution experiment from the parent strain, but is not limited thereto.
  • the mutation of the patZ gene may be a mutant (Trp501Stop) Trp, the 501st amino acid of the wild type enzyme consisting of the amino acid sequence of SEQ ID NO: 10, stop codon, but is not limited thereto.
  • the mutation of the cspC gene may be one in which the 58th amino acid Gln of the wild type enzyme consisting of the amino acid sequence of SEQ ID NO: 11 is mutated to the stop codon (Gln58Stop), but is not limited thereto.
  • the mukB gene may be mutated to Glu (Asp54Glu), which is the 54th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 12, but is not limited thereto.
  • the mutation of the lomR gene may be one of the 114th amino acid Pro of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 13 mutated to Leu (Pro114Leu), but is not limited thereto.
  • the yhjE gene may be a mutation in which the 210th amino acid Ile of the wild type enzyme consisting of the amino acid sequence of SEQ ID NO: 14 is mutated to Met (Ile210Met), but is not limited thereto.
  • the mutant microorganism of the present invention is essentially mutated patZ gene, cspC , mukB , lomR And one or more genes selected from the group consisting of yhjE may be additionally mutated, but is not limited thereto.
  • the mutant microorganisms of the invention are patZ , cspC, mukB , lomR And all five genes of yhjE may be mutated, but are not limited thereto.
  • the microorganism may be Escherichia coli, but is not limited thereto. Therefore, according to a preferred embodiment of the present invention, the mutant microorganism of the present invention is a wild type E. coli strain patZ , cspC , mukB , lomR And one or more genes selected from the group consisting of yhjE may be mutated, but is not limited thereto. According to another preferred embodiment of the present invention, the mutant microorganism of the present invention is essentially mutated patZ gene in wild type E.
  • the mutant microorganism of the present invention is wild-type E. coli strains patZ , cspC , mukB, lomR And all five genes of yhjE may be mutated, but are not limited thereto.
  • the present inventors named the mutant Escherichia coli strain that can use the acetic acid as the only carbon source as "E. coli SBA01" strain, and deposited in the Korea Biotechnology Research Institute microbial resource center on June 10, 2016. (Accession Number: KCTC13040BP).
  • the mutant microorganism of the present invention may be introduced to a biosynthesis related gene of butanol and used to prepare butanol.
  • the biosynthesis related genes of butanol include 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogease It may be, but is not limited to, Naase.
  • patZ , cspC , mukB , lomR And mutating one or more genes selected from the group consisting of yhjE to use acetic acid as the only carbon source.
  • (3) provides a method for producing a recombinant protein comprising culturing the transformed microorganism in a medium containing a carbon source.
  • patZ , cspC , mukB , lomR And mutating one or more genes selected from the group consisting of yhjE to use acetic acid as the only carbon source.
  • the mutant microorganism is preferably one or more genes selected from the group consisting of frdA , ldhA , adhE and pta is further removed, but is not limited thereto.
  • the recombinant protein is not particularly limited, and any recombinant protein useful in the industry may be used without limitation, for example.
  • any recombinant protein useful in the industry may be used without limitation, for example.
  • in order to confirm the acetic acid metabolism ability and the production capacity of foreign protein of the mutant microorganism of the present invention to prepare a transgenic microorganism which introduced the GFP (green fluorescent protein) gene as one of the representative recombinant protein As a result, it was confirmed that the transformed microorganism effectively expressed GFP, which is a recombinant protein of foreign origin, in acetic acid minimal medium (see FIG. 8).
  • the medium may be used without limitation any medium conventionally used for cell culture.
  • the medium may include a nitrogen source, inorganic salts, and the like, and may further include a bioactive substance as necessary.
  • the nitrogen source organic nitrogen sources such as proteins, amino acids, urea and the like, and inorganic nitrogen sources such as nitrates and ammonium salts can be used.
  • the inorganic salts include Na + , K + , Ca 2 + , Mg 2 + , Cl ⁇ , and the like. This may be used, but vitamins may be used as the physiologically active substance, but is not limited thereto.
  • the medium may include yeast extract, malt extract, etc., culture such as Rosewell Park Memorial Institute (RPMI), Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), etc., which are commercially available for cell culture. Badges may also be used, but are not limited thereto.
  • RPMI Rosewell Park Memorial Institute
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimum Essential Medium
  • acetic acid, starch, glucose, sugar, etc. may be used as the carbon source, but is not limited thereto.
  • the culturing may be carried out under predetermined temperature and pH conditions.
  • the temperature may be 20 to 50 ° C, preferably 25 to 40 ° C, more preferably 28 to 35 ° C, but is not limited thereto.
  • the pH may be in the range of 4 to 9, preferably in the range of 5 to 8, but is not limited thereto.
  • the present invention provides microorganisms, preferably E. coli strains, which can efficiently use acetic acid when acetic acid is provided as the only carbon source or mixed carbon source.
  • microorganisms preferably E. coli strains
  • E. coli Recombinant protein or useful compound productivity was confirmed.
  • E. coli strains having improved metabolic ability of acetic acid were identified through acetic acid evolution experiments in E. coli (see FIG. 3), and genetic changes were examined through whole gene sequence analysis of the mutant E. coli strains. , Five genes in which a change in the amino acid sequence occurred (see Table 2).
  • E. coli strains provided an application example of the E. coli strains developed by producing butanol, a fluorescent protein or biofuel from the only carbon source of acetate (see FIGS. 8 to 10).
  • E. coli MG1655 ⁇ frdA ⁇ ldhA ⁇ pta ⁇ adhE used in the present invention Strains (Jang-mi Baek et al., Biotechnology and bioengineering, 2013, 110 (10), 2790-2794) were used in succinic acid, lactic acid, acetic acid, alcohol production metabolic cycles frdA , ldhA , pta , adhE
  • the strain has been improved to produce a large amount of acetyl-CoA from a carbon source such as glucose (FIG. 1).
  • a carbon source such as glucose (FIG. 1).
  • Escherichia coli in order to grow using acetic acid as a carbon source, it is grown by synthesizing acetyl-CoA from acetic acid through two routes shown in FIG.
  • the growth rate was measured by culturing E. coli MG1655 ⁇ frdA ⁇ ldhA ⁇ pta ⁇ adhE strain in the acetic acid minimal medium described in Table 1.
  • 1% inoculation of the E. coli strain cultured in LB medium in 100ml of acetic acid minimal medium was incubated using a shake incubator at 37 °C, 200rpm conditions.
  • subcultures were repeated in fresh medium at the late exponential phase of microorganisms after the start of the culture, and the absorbance (OD 600 ) was measured at 600 nm every 12 hours at each repeated cycle to investigate the growth of Escherichia coli. It was.
  • M9 salt solution 100 ml Na 2 HPO 4 -2H 2 O 33.7 mM KH 2 PO 4 22.0 mM NaCl 8.55 mM NH 4 Cl 9.35 mM 1 M MgSO 4 - 7H 2 O 1 ml 1 M CaCl 2 0.3 ml Trace Element Solution (100X) 10 ml EDTA 13.4 mM FeCl 3 -6H 2 O 3.1 mM ZnCl 2 0.62 mM CuCl 2 -2H 2 O 76 ⁇ M CoCl 2 -2H 2 O 42 ⁇ M H 3 BO 3 162 ⁇ M MnCl 2 -4H 2 O 8.1 ⁇ M 1 M Sodium acetic acid 50 ml Sterile water 837. 7 ml Sum 1,000 ml
  • the growth rate of E. coli increased according to the repeated rounds. Specifically, the growth rate of E. coli increased slightly in the early stages of passage (G1-G3). In the case of G6), the growth rate of E. coli increased after 50 hours of incubation until the final absorbance was about 0.9. Thereafter, the growth rate of Escherichia coli was continuously increased during the passage of G7-G9 subculture, and the absorbance was 1.66 after 60 hours of culture in the last 10 repetition (G10) stages. That is, as a result of acetic acid adaptive evolution experiment of E. coli MG1655 ⁇ frdA ⁇ ldhA ⁇ pta ⁇ adhE strain, it was confirmed that the growth of microorganisms rapidly increased from 0.312 to 1.66 (Fig. 3).
  • E. coli SBA01 the mutant strain capable of growing acetic acid as the only carbon source was named E. coli SBA01, and deposited on June 20, 2016 at the Korea Research Institute of Bioscience and Biotechnology. (Accession Number: KCTC 13040BP).
  • patZ pka, acetyltransferase
  • cspC stress protein
  • Example 5 E. coli of the present invention SBA01 Strain Resistance test to acetic acid
  • the resistance test against acetic acid of the E. coli SBA01 strain of the present invention was performed. To this end, the acetic acid concentration of the acetic acid minimum medium described in Table 1 was adjusted to 10, 20, 100, 150, 200 and 250 mM, respectively, and then inoculated with 1% E. coli SBA01 strain in each medium to 37 ° C and 200 rpm. The culture was carried out using a shaker incubator. The growth of Escherichia coli was examined by measuring the absorbance (OD 600 ) at 600 nm at each 6-hour interval for each iteration.
  • the E. coli SBA01 strain of the present invention showed the highest growth in the medium containing 50 mM acetic acid, was also found to be excellent growth in the medium containing 100 mM and 150 mM acetic acid, containing more than 200 mM acetic acid Growth was reduced in cultured medium (FIG. 5). From the above results, it was confirmed that the SBA01 strain of the present invention increased the resistance to high concentrations of acetic acid as well as the effective use of acetic acid.
  • RNA-SEQ analysis the expression levels are usually compared by mapping the transcript sequences to the complete genome sequences, but in the present invention, the increase and decrease of the expression level of the experimental group was analyzed by comparing the expression levels of the control group and the experimental group. .
  • the top 28 genes with increased expression level of mRNA and the top 26 genes with reduced expression level of mRNA were confirmed (FIGS. 6A and 6B).
  • citrate synthase, aconitase, isocitrate dehydrogenase, ⁇ -ketoglutarate dehydrogenase, succinyl-CoA synthase, fumarase, malate which are genes constituting the TCA circuit
  • the expression levels of dehydrogenase and isocitrate lyase were increased from 2.0 to 6.19 fold.
  • the gene involved in the copper release pump, which releases copper ions into cells was reduced by 5.48 fold.
  • the SBA01 strain of the present invention increased the expression level of genes involved in acetyl-CoA conversion of acetic acid, ATP synthesis, TCA cycle (NADH synthesis) compared to the control strain.
  • E. coli MG1655, control DSM01, and SBA01 strains of the present invention were inoculated with 1% in a minimal medium containing 0.4% glucose, respectively, and absorbed at 37 ° C. and 200 rpm. The strain was recovered at 1.0. The recovered strain was measured by intracellular ATP using Sigma's ATP assay kit.
  • the pUCBB-eGFP vector prepared for expression of fluorescent protein in Escherichia coli is transformed into the E. coli SBA01 strain of the present invention, and then each of the bacteria is incubated in acetic acid minimal medium for 48 hours, and the glycerol concentration is 10 It was made into a storage bacteria solution to% and stored at -80 °C until the culture experiment.
  • 1 ml of the storage solution was inoculated in 50 ml of acetic acid (50 ⁇ g / ml) -added 50 ml of acetic acid minimal medium and incubated for 48 hours, followed by fluorescence spectrophotometer. Fluorescence was measured under conditions of excitation 395 nm and emission 509 nm.
  • E. coli SBA01 strain of the present invention was confirmed that the synthesis of recombinant proteins using acetic acid as a substrate.
  • butanol synthesis was confirmed by introducing a butanol synthesis metabolic circuit to the E. coli SBA01 strain.
  • Butanol synthesis metabolic circuits are the four enzymes involved in the acetyl-CoA and butyryl-CoA synthesis pathways derived from Clostridium acetobutylicum and Treponema denticola , 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl- Recombinant vectors were constructed using CoA dehydratase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogenase.
  • a vector capable of expressing formate dehydrogenase was further configured to supply NADH required in the butanol synthesis pathway.
  • each of the bacteria were incubated for 48 hours in a minimal acetic acid medium, and made into a storage solution so that the concentration of glycerol to 10%, the culture experiment at -80 °C Stored until.
  • Butanol fermentation was performed using a 1 L fermenter with acetic acid minimal medium.
  • 1% of the transformed E. coli SBA01 strain was incubated in a culture medium of minimal acetic acid and incubated at 37 ° C. and 200 rpm for 94 hours.
  • air was injected at a rate of 1vvm to maintain an aerobic state, and then, after 24 hours of culture, the amount of air injected was reduced to 0.02vvm to form a semi-anaerobic condition.
  • the growth curve of the transformed E. coli SBA01 strain is shown in FIG.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un micro-organisme ayant une capacité métabolique améliorée de l'acide acétique et, plus particulièrement, un micro-organisme mutant, préférablement Escherichia coli, qui peut utiliser l'acide acétique comme unique source de carbone en résultat de la mutation d'au moins un gène sélectionné dans le groupe constitué par patZ, cspC, mukB, lomR, et yhjE dans un micro-organisme de type sauvage correspondant. Le micro-organisme mutant de la présente invention non seulement peut se développer par l'utilisation d'acide acétique comme unique source de carbone, mais également peut être utile dans la production de matériaux cibles industriellement utiles, tels que les protéines recombinantes et le butanol, à partir d'acide acétique.
PCT/KR2017/010643 2016-09-26 2017-09-26 Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone WO2018056794A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2016-0123301 2016-09-26
KR20160123301 2016-09-26

Publications (1)

Publication Number Publication Date
WO2018056794A1 true WO2018056794A1 (fr) 2018-03-29

Family

ID=61690573

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/010643 WO2018056794A1 (fr) 2016-09-26 2017-09-26 Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone

Country Status (2)

Country Link
KR (1) KR101863239B1 (fr)
WO (1) WO2018056794A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3719121A4 (fr) * 2017-11-30 2021-12-15 Toray Industries, Inc. Micro-organisme à gène modifié destiné à la production d'acide 3-hydroxyadipique, acide -hydromuconique et/ou acide adipique, et procédé de production desdits produits chimiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012099934A2 (fr) * 2011-01-18 2012-07-26 The Regents Of The University Of California Production d'alcool butylique par microorganismes ayant un couplage nadh

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101758910B1 (ko) * 2010-06-10 2017-07-17 지에스칼텍스 주식회사 부탄올 생성능을 가지는 재조합 미생물 및 이를 이용한 부탄올의 제조방법

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012099934A2 (fr) * 2011-01-18 2012-07-26 The Regents Of The University Of California Production d'alcool butylique par microorganismes ayant un couplage nadh

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMAT, M. A.: "The use of acetic acid as a source of carbon by cultured Chondrus crispus (Gigartinales, Rhodophyta) Stackhouse", HYDROBIOLOGIA, vol. 260/261, 1993, pages 451 - 456 *
BAEK, J.-M.: "Butyrate production in engineered Escherichia coli with synthetic scaffolds", BIOTECHNOLOGY AND BIOENGINEERING, vol. 110, no. 10, 22 April 2013 (2013-04-22), pages 2790 - 2794, XP002761845, DOI: doi:10.1002/BIT.24925 *
CASTANO-CEREZO, S.: "Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli", MOLECULAR SYSTEMS BIOLOGY, vol. 10, no. 762, 27 November 2014 (2014-11-27), pages 1 - 12, XP055603229, ISSN: 1744-4292, DOI: 10.15252/msb.20145227 *
DATABASE NUCLEOTIDE 30 October 2014 (2014-10-30), GRENIER F. ET AL.: "Escherichia coli BW25113, complete genome", XP055603232, retrieved from NCBI Database accession no. CP009273 *

Also Published As

Publication number Publication date
KR101863239B1 (ko) 2018-06-01
KR20180034280A (ko) 2018-04-04

Similar Documents

Publication Publication Date Title
Leigh et al. Model organisms for genetics in the domain Archaea: methanogens, halophiles, Thermococcales and Sulfolobales
CN110551671B (zh) 一株产surfactin基因工程菌及其构建方法和应用
Spector et al. Identification and characterization of starvation-regulated genetic loci in Salmonella typhimurium by using Mu d-directed lacZ operon fusions
WO2019203436A1 (fr) Levure résistante aux acides avec voie de production d'éthanol supprimée et procédé de production d'acide lactique l'utilisant
Schmid et al. Genetic analysis of temperature-sensitive lethal mutants of Salmonella typhimurium.
JP7507897B2 (ja) 生産効率を向上させる線毛無し大腸菌の構築及び使用
US20220282293A1 (en) Enterobacter chengduensis for producing nicotinamide mononucleotide and application thereof
CN108026546A (zh) 微杆菌属菌株用于生产抗菌剂的用途
KR20190026851A (ko) 당 포스포트랜스퍼라제 시스템 (pts)을 코딩하는 유전자를 포함하는 미생물에 의한 메티오닌 또는 그의 히드록시 유사체 형태의 발효적 생산을 위한 방법
CN112251456B (zh) 一种通过林可链霉菌调控基因组合改造提高林可霉素产量的方法
Kumari et al. Vibrio panuliri sp. nov., a marine bacterium isolated from spiny lobster, Panulirus penicillatus and transfer of Vibrio ponticus from Scophthalmi clade to the newly proposed Ponticus clade
Lukáčová et al. Euglena gracilis can grow in the mixed culture containing Cladosporium westerdijkiae, Lysinibacillus boronitolerans and Pseudobacillus badius without the addition of vitamins B1 and B12
WO2018056794A1 (fr) Micro-organisme apte à utiliser l'acide acétique comme unique source de carbone
CN111154705B (zh) 热葡萄糖苷酶地芽孢杆菌工程菌及其构建方法及应用
Hughes et al. Structural gene for NAD synthetase in Salmonella typhimurium
US20140199372A1 (en) Microbial growth factors
Murat et al. Deletion of the Escherichia coli uup gene encoding a protein of the ATP binding cassette superfamily affects bacterial competitiveness
CN109929853B (zh) 嗜热菌来源的热激蛋白基因的应用
WO2020116941A2 (fr) Microorganisme pour produire de l'acide dicarboxylique, et procédé de production d'acide dicarboxylique l'utilisant
KR102245274B1 (ko) 최소 유전체를 갖는 신규 미생물 및 이의 제조 방법
KR101402659B1 (ko) 악티노바실러스 숙시노게네스 및 이를 이용한 숙신산의 제조 방법
WO2018212627A1 (fr) Micro-organisme mutant à capacité de production d'acide succinique améliorée et méthode de production d'acide succinique l'utilisant
WO2015046978A1 (fr) Micro-organisme recombiné ayant une aptitude accrue à produire du 2,3-butanediol et procédé de production de 2,3-butanediol l'utilisant
JP6222647B2 (ja) 1,3−βガラクトシル−N−アセチルヘキソサミンホスホリラーゼの製造方法
Chen et al. Pelagibacterium flavum sp. nov., isolated from soil sample

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17853508

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17853508

Country of ref document: EP

Kind code of ref document: A1