WO2018056794A1 - Microorganism capable of utilizing acetic acid as sole carbon source - Google Patents

Microorganism capable of utilizing acetic acid as sole carbon source Download PDF

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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
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microorganism
gene
acetic acid
coli
seq
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Korean (ko)
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이승구
한귀환
성원재
이대희
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한국생명공학연구원
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • 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
    • C12P7/16—Butanols
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    • 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)
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    • 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)
    • C12Y101/01157—3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
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    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
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    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01038—Trans-2-enoyl-CoA reductase (NADPH) (1.3.1.38)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01055—3-Hydroxybutyryl-CoA dehydratase (4.2.1.55)
    • 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

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.

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Abstract

The present invention relates to a microorganism having improved metabolic capacity of acetic acid and, more particularly, to a mutant microorganism, preferably Escherichia coli, which can utilize acetic acid as a sole carbon source as a result of the mutation of at least one gene selected from the group consisting of patZ, cspC, mukB, lomR, and yhjE in a wild-type microorganism thereof. The mutant microorganism of the present invention not only can grow by use of acetic acid as a sole carbon source, but also can be useful for the production of industrially useful target materials, such as recombinant proteins and butanol, from acetic acid.

Description

아세트산을 유일 탄소원으로 이용할 수 있는 미생물Microorganisms that can use acetic acid as the only carbon source
본 발명은 아세트산에 대한 대사 능력이 향상된 미생물에 관한 것으로서, 보다 상세하게는 아세트산의 세포 내 이용과 관련된 5종의 유전자 중 하나 이상이 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 미생물, 바람직하게는 대장균 균주에 관한 것이다.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.
대장균과 같은 미생물은 아세트산을 단독 혹은 혼합 탄소원으로 사용하여 배양할 경우 그 농도에 따라 대장균의 성장을 저해한다. 또한, 배양액 내 아세트산 농도가 증가할수록 미생물 생육의 유도기(lag phase)가 길어지고, 이로 인해 전체 배양 시간이 길어지는 문제가 있다(Holger Ebbighausen, et al., Arch. Microbiol., 1991, 155(5), 505-5101). 예컨대, 종래 아세트산을 유일 탄소원으로 이용할 수 있는 대장균 균주가 공개되어 있으나, 상기 대장균 균주는 아세트산을 유일 탄소원으로 이용할 경우 성장이 현저하게 느려지는 것으로 알려져 있다(Frank E. Dailey, et al., J. Bacteriol., 1986, 165(2), 453-460).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. In addition, as the acetic acid concentration in the culture medium increases, 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). For example, although 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).
이에, 아세트산의 대사 능력이 향상되어 저가의 아세트산을 유일 탄소원 또는 혼합 탄소원으로 이용하면서도 성장이 느려지지 않고, 또한 유전자 도입을 통해 산업적으로 유용한 바이오연료, 재조합 단백질 등을 생산할 수 있는 신규한 미생물의 개발이 요구되고 있다.As a result, the metabolic ability of acetic acid is improved, and the development of a novel microorganism capable of producing industrially useful biofuels, recombinant proteins, etc., without slowing growth while using low-cost acetic acid as the only carbon source or mixed carbon source. This is required.
본 발명은 야생형 미생물 균주에 특정 유전자가 돌연변이되어 아세트산의 대사 능력이 향상되고 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a mutant microorganism in which a specific gene is mutated to a wild-type microorganism strain, thereby improving the metabolic ability of acetic acid and using acetic acid as the only carbon source.
상기 목적을 달성하기 위하여, 본 발명은 야생형 미생물에 patZ , cspC , mukB, lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제공한다.In order to achieve the above object, 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.
본 발명의 한 구현예에 따르면, 본 발명의 돌연변이 미생물은 patZ 유전자가 필수적으로 돌연변이되고, cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가적으로 돌연변이된 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 본 발명의 돌연변이 미생물은 patZ, cspC , mukB , lomR 및 yhjE의 5종의 유전자가 모두 돌연변이된 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 patZ , cspC, mukB , lomR 및 yhjE 유전자는 각각 서열번호 1 내지 서열번호 5로 이루어지는 염기서열을 갖는 것이 바람직하지만 이에 한정되는 것은 아니다.According to one embodiment of the present invention, 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. According to another embodiment of the invention, 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. According to another embodiment of the present invention, 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.
본 발명의 한 구현예에 따르면, 본 발명의 돌연변이 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 frdA , ldhA , adhE 및 pta 유전자는 각각 서열번호 6 내지 서열번호 9로 이루어지는 염기서열을 갖는 것이 바람직하지만 이에 한정되는 것은 아니다.According to one embodiment of the invention, 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 . According to another embodiment of the present invention, 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.
본 발명의 바람직한 구현예에 따르면, 상기 patZ 유전자의 돌연변이는 서열번호 10의 아미노산 서열로 이루어지는 야생형 효소의 501번째 아미노산인 Trp가 Stop codon으로 돌연변이(Trp501Stop)되고, 상기 cspC 유전자의 돌연변이는 서열번호 11의 아미노산 서열로 이루어지는 야생형 효소의 58번째 아미노산인 Gln이 Stop codon으로 돌연변이(Gln58Stop)되고, 상기 mukB 유전자의 돌연변이는 서열번호 12의 아미노산 서열로 이루어지는 야생형 효소의 54번째 아미노산인 Asp가 Glu로 돌연변이(Asp54Glu)되고, 상기 lomR 유전자의 돌연변이는 서열번호 13의 아미노산 서열로 이루어지는 야생형 효소의 114번째 아미노산인 Pro가 Leu로 돌연변이(Pro114Leu)되고, 상기 yhjE 유전자의 돌연변이는 서열번호 14의 아미노산 서열로 이루어지는 야생형 효소의 210번째 아미노산인 Ile가 Met로 돌연변이(Ile210Met)될 수 있으나 이에 한정되는 것은 아니다.According to a preferred embodiment of the present invention, 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: 14 Ile, the 210th amino acid of the enzyme, is mutated to Met It may be (Ile210Met), but is not limited thereto.
본 발명의 한 구현예에 있어서, 본 발명의 돌연변이 미생물은 부탄올의 생합성 관련 유전자가 추가로 도입되는 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 부탄올의 생합성 관련 유전자는 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제일 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the mutant microorganism of the present invention is preferably, but not limited to, further introduced a biosynthesis related gene of butanol. According to another embodiment of the invention, 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.
본 발명의 한 구현예에 있어서, 상기 미생물은 대장균인 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 바람직한 구현예에 있어서, 상기 미생물은 기탁번호 KCTC13040BP로 기탁된 대장균 SBA01 균주인 것이 바람직하지만 이에 한정되는 것은 아니다.In one embodiment of the present invention, the microorganism is preferably E. coli, but is not limited thereto. In a preferred embodiment of the present invention, the microorganism is preferably E. coli SBA01 strain deposited with accession number KCTC13040BP, but is not limited thereto.
또한, 본 발명은 (1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;In addition, 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.
(2) 상기 돌연변이 미생물에 재조합 단백질을 암호화하는 유전자를 도입한 형질전환 미생물을 제조하는 단계; 및(2) preparing a transformed microorganism in which a gene encoding a recombinant protein is introduced into the mutant microorganism; And
(3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 재조합 단백질의 제조 방법을 제공한다.(3) provides a method for producing a recombinant protein comprising culturing the transformed microorganism in a medium containing a carbon source.
또한, 본 발명은 (1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;In addition, 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.
(2) 상기 돌연변이 미생물에 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제 유전자를 도입한 형질전환 미생물을 제조하는 단계; 및(2) 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogenase Preparing a transformed microorganism into which the aze gene is introduced; And
(3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 부탄올의 제조 방법을 제공한다.(3) It provides a method for producing butanol comprising culturing the transformed microorganism in a medium containing a carbon source.
본 발명의 한 구현예에 있어서, 상기 형질전환 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 것이 바람직하지만 이에 한정되는 것은 아니다.In one embodiment of the invention, 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.
본 발명의 한 구현예에 있어서, 상기 미생물은 대장균인 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 바람직한 구현예에 있어서, 상기 미생물은 기탁번호 KCTC13040BP로 기탁된 대장균 SBA01 균주인 것이 바람직하지만 이에 한정되는 것은 아니다.In one embodiment of the present invention, the microorganism is preferably E. coli, but is not limited thereto. In a preferred embodiment of the present invention, the microorganism is preferably E. coli SBA01 strain deposited with accession number KCTC13040BP, but is not limited thereto.
본 발명의 아세트산에 대한 대사 능력이 향상된 미생물은 아세트산을 유일 탄소원으로 이용하여 성장할 수 있을 뿐만 아니라, 아세트산으로부터 부탄올과 같은 산업적으로 유용한 목적 물질의 제조에도 유용하게 사용할 수 있다.The 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.
도 1은 글루코오스 또는 아세트산와 같은 탄소원으로부터 아세틸-CoA를 거쳐 부탄올 합성경로에 이르는 과정의 유전자의 종류를 보여주는 도면이다.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.
도 2는 대장균의 아세트산 대사과정 중에서의 아세틸-CoA의 합성 경로 및 이에 관여하는 유전자의 종류를 보여주는 도면이다.Figure 2 is a diagram showing the synthesis pathway of acetyl-CoA and the genes involved in the acetic acid metabolism of Escherichia coli.
도 3은 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주의 배양 단계별 성장 곡선을 나타내는 그래프로써, "G"는 계대배양 회차를 나타낸다.Figure 3 is E. coli MG1655 Δ frdA Δ ldhA Δ pta Δ adhE As a graph showing the growth curve of each strain of culture, "G" represents the passage of subculture.
도 4는 patZ 유전자가 추가로 제거(knock-out)된 돌연변이 대장균 MG1655 △frdA △ ldhA △pta △ adhE △ patZ 균주 및 본 발명의 대장균 SBA01 균주의 성장을 비교한 그래프이다.4 shows mutant Escherichia coli MG1655 ΔfrdA Δ ldhA Δpta Δ adhE Δ patZ with additional knock-out of patZ gene. It is a graph comparing the growth of the strain and E. coli SBA01 strain of the present invention.
도 5는 본 발명의 대장균 SBA01 균주의 아세트산에 대한 내성 실험 결과를 보여주는 그래프이다.Figure 5 is a graph showing the results of resistance test against acetic acid of E. coli SBA01 strain of the present invention.
도 6a 및 도 6b는 본 발명의 대장균 SBA01 균주(실험군)와 대조군의 전사체 비교 분석을 수행하여 각각 발현양이 증감된 상위 유전자들의 목록을 보여주는 표이고, 도 6c는 대장균의 대사 회로에서 발현이 증감된 유전자를 맵핑하여 비교한 결과를 나타낸다.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.
도 7은 자연연형 대장균 MG1655, 대조군 DSM01 및 본 발명의 SBA01 균주의 세포내 ATP 함량을 측정한 결과를 보여주는 그래프이다.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.
도 8은 형광 단백질로 형질전환된 본 발명의 대장균 SBA01 균주의 아세트산 최소배지에서 성장 및 형광 단백질의 발현을 보여주는 그래프 및 사진이다.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.
도 9는 부탄올 생합성 관련 유전자가 도입된 본 발명의 대장균 SBA01 균주의 성장 및 아세테이트, 포르메이트 이용을 나타내는 그래프이다.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.
도 10은 부탄올 생합성 관련 유전자가 도입된 본 발명의 대장균 SBA01 균주에서의 아세트산 최소배지에서 발효한 후 발효 생성물의 GC 및 GC/MS 분석 결과 부탄올이 생성되었음을 보여주는 그래프이다.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.
본 발명은 야생형 미생물에 patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제공한다. 상기 patZ , cspC , mukB , lomR 및 yhjE 유전자는 각각 서열번호 1 내지 서열번호 5로 이루어지는 염기서열을 가질 수 있으나 이에 한정되는 것은 아니다.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.
종래에는 아세트산을 유일 탄소원으로 이용하는 대장균 균주(Frank E. Dailey, et al., J. Bacteriol., 1986, 165(2), 453-460), 또는 patZ , cspC , mukB 또는 lomR 유전자가 돌연변이된 대장균 균주(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. Bacteriol., 1986, 165(2), 453-460; Zhiqing SONG, et al., J. Radiat. Res., 2012, 53(6), 854-859)가 보고된 바 있으나, 상기 문헌들에는 상기 유전자들이 미생물, 바람직하게는 대장균에서 아세트산을 유일 탄소원으로 이용하는 것과 관련된 유전자임에 대해서는 개시 및 시사하고 있지 않다.Conventionally, 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. Bacteriol., 1986, 165 (2), 453-460; Zhiqing SONG, et al., J. Radiat. Res., 2012, 53 (6), 854-859). The literature does not disclose or suggest that the genes are related to the use of acetic acid as the only carbon source in microorganisms, preferably E. coli.
상기 patZ 유전자는 아세틸트랜스퍼라아제(acetyltransferase) 효소를 발현하고, cspC 유전자는 스트레스 단백질로서 MukB 저온 충격 단백질의 멀티카피 억제제를 발현하며, mukB 유전자는 염색체 파티션 단백질을 발현하고, lomR 유전자는 추정되는 외막 독성 단백질을 발현하며, yhjE 유전자는 내막 대사물 운반 단백질을 발현하는 것으로 알려져 있다.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, and the lomR gene assumes an outer membrane It expresses toxic proteins, and the yhjE gene is known to express endometrial metabolite transport proteins.
본 발명의 한 구현예에 따르면, 상기 돌연변이 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 것이 바람직하지만 이에 한정되는 것은 아니다. 상기 frdA , ldhA , adhE 및 pta 유전자는 각각 서열번호 6 내지 서열번호 9로 이루어지는 염기서열을 가질 수 있으나 이에 한정되는 것은 아니다. 상기 돌연변이의 결과로서 해당 유전자에 의해 발현되는 효소의 활성이 소실될 수 있다.According to one embodiment of the present invention, 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. As a result of the mutation, the activity of the enzyme expressed by the gene of interest may be lost.
본 명세서에 있어서, "돌연변이"란 용어는 유전정보가 기록된 DNA 분자가 여러가지 요인에 의하여 원래의 DNA와 그 서열이 달라지는 것을 의미하는 것으로서, 돌연변이가 일어나면 그 유전자에 의해 생산되는 단백질에 변화가 생기고, 이는 유전형질의 변화를 불러오게 되어, 해당 개체의 생물학적 특징 등에 변형시킬 수 있다.In the present specification, the term "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.
본 발명의 한 구현예에 따르면, 상기 돌연변이는 본 기술분야에서 돌연변이를 유발할 수 있는 것으로서 알려져 있는 임의의 화학적 수단 및/또는 물리적 수단을 미생물에 처리함으로써 도입될 수 있다. 상기 화학적 수단은 돌연변이를 유발하는 물질(돌연변이원)로써 유효한 구아니딘 유도체인 NTG(nitrosoguanidine), MMS(methyl methanesulfonate), EMS(ethyl methanesulfonate), 벤조피렌 등과 같은 화학물질을 들 수 있고, 상기 물리적 수단은 자외선, X-선, γ-선 등과 같은 방사선을 들 수 있으나 이에 한정되는 것은 아니다. 본 발명의 바람직한 구현예에서는 모균주로부터의 아세트산 적응 진화 실험을 통해 돌연변이 균주를 제조하였으나, 이에 한정되는 것은 아니다.According to one embodiment of the invention, 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). And X-rays, γ-rays, and the like, but are not limited thereto. In a preferred embodiment of the present invention, a mutant strain was prepared by acetic acid adaptive evolution experiment from the parent strain, but is not limited thereto.
본 발명의 한 구현예에 따르면, 상기 patZ 유전자의 돌연변이는 서열번호 10의 아미노산 서열로 이루어지는 야생형 효소의 501번째 아미노산인 Trp가 Stop codon으로 돌연변이(Trp501Stop)된 것일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 cspC 유전자의 돌연변이는 서열번호 11의 아미노산 서열로 이루어지는 야생형 효소의 58번째 아미노산인 Gln이 Stop codon으로 돌연변이(Gln58Stop)된 것일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 mukB 유전자의 돌연변이는 서열번호 12의 아미노산 서열로 이루어지는 야생형 효소의 54번째 아미노산인 Asp가 Glu로 돌연변이(Asp54Glu)된 것일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 lomR 유전자의 돌연변이는 서열번호 13의 아미노산 서열로 이루어지는 야생형 효소의 114번째 아미노산인 Pro가 Leu로 돌연변이(Pro114Leu)된 것일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 상기 yhjE 유전자의 돌연변이는 서열번호 14의 아미노산 서열로 이루어지는 야생형 효소의 210번째 아미노산인 Ile가 Met로 돌연변이(Ile210Met)된 것일 수 있으나, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, 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. According to another embodiment of the present invention, 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. According to another embodiment of the present invention, 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. According to another embodiment of the present invention, 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. According to another embodiment of the present invention, 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.
본 발명의 한 구현예에 따르면, 본 발명의 돌연변이 미생물은 patZ 유전자가 필수적으로 돌연변이되고, cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가적으로 돌연변이될 수 있으나 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에 따르면, 본 발명의 돌연변이 미생물은 patZ , cspC, mukB , lomR 및 yhjE의 5종의 유전자가 모두 돌연변이될 수 있으나 이에 한정되는 것은 아니다.According to one embodiment of the present invention, 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. According to another embodiment of the invention, 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.
본 발명의 한 구현예에 따르면, 상기 미생물은 대장균일 수 있으나 이에 한정되는 것은 아니다. 따라서, 본 발명의 바람직한 구현예에 따르면, 본 발명의 돌연변이 미생물은 야생형 대장균 균주에 patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이될 수 있으나 이에 한정되는 것은 아니다. 본 발명의 다른 바람직한 구현예에 따르면, 본 발명의 돌연변이 미생물은 야생형 대장균 균주에 patZ 유전자가 필수적으로 돌연변이되고, cspC , mukB, lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가적으로 돌연변이될 수 있으나 이에 한정되는 것은 아니다. 본 발명의 다른 바람직한 구현예에 따르면, 본 발명의 돌연변이 미생물은 야생형 대장균 균주에 patZ , cspC , mukB, lomR 및 yhjE의 5종의 유전자가 모두 돌연변이될 수 있으나 이에 한정되는 것은 아니다.According to one embodiment of the invention, 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. coli strain, cspC , mukB, lomR And one or more genes selected from the group consisting of yhjE may be additionally mutated, but is not limited thereto. According to another preferred embodiment of the present invention, 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.
본 발명의 바람직한 구현예에 따르면, 본 발명자들은 상기 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 대장균 균주를 "대장균 SBA01" 균주로 명명하고, 2016년 6월 10일자로 한국생명공학연구원 미생물자원센터에 기탁하였다(기탁번호: KCTC13040BP).According to a preferred embodiment of the present invention, 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).
본 발명의 한 구현예에 따르면, 본 발명의 돌연변이 미생물은 부탄올의 생합성 관련 유전자가 추가로 도입되어 부탄올의 제조에 사용될 수 있다. 상기 부탄올의 생합성 관련 유전자는 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제일 수 있으나 이에 한정되는 것은 아니다.According to one embodiment of the present invention, 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.
또한, 본 발명은In addition, the present invention
(1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;(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.
(2) 상기 돌연변이 미생물에 재조합 단백질을 암호화하는 유전자를 도입한 형질전환 미생물을 제조하는 단계; 및(2) preparing a transformed microorganism in which a gene encoding a recombinant protein is introduced into the mutant microorganism; And
(3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 재조합 단백질의 제조 방법을 제공한다.(3) provides a method for producing a recombinant protein comprising culturing the transformed microorganism in a medium containing a carbon source.
또한, 본 발명은In addition, the present invention
(1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;(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.
(2) 상기 돌연변이 미생물에 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제 유전자를 도입한 형질전환 미생물을 제조하는 단계;(2) 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogenase Preparing a transformed microorganism into which the aze gene is introduced;
(3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 부탄올의 제조 방법을 제공한다.(3) It provides a method for producing butanol comprising culturing the transformed microorganism in a medium containing a carbon source.
본 발명의 한 구현예에 있어서, 상기 돌연변이 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거되는 것이 바람직하지만 이에 한정되는 것은 아니다.In one embodiment of the invention, 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.
본 발명의 다른 구현예에 있어서, 상기 재조합 단백질은 특별히 한정되는 것은 아니며, 예컨대, 산업적으로 유용한 임의의 재조합 단백질이 제한없이 사용될 수 있다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 돌연변이 미생물의 아세트산 대사능력 및 외래 단백질의 생산능력을 확인하기 위하여 대표적인 재조합 단백질의 하나로서 GFP(green fluorescent protein) 유전자를 도입한 형질전환 미생물을 제조한 결과, 상기 형질전환 미생물은 아세트산 최소 배지에서 외래 유래의 재조합 단백질인 GFP를 효과적으로 발현함을 확인하였다(도 8 참조).In other embodiments of the present invention, the recombinant protein is not particularly limited, and any recombinant protein useful in the industry may be used without limitation, for example. According to a preferred embodiment of the present invention, 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).
본 발명에 있어서, 상기 배지는 세포 배양용으로 통상적으로 사용되는 임의의 배지가 제한없이 사용될 수 있다. 본 발명의 한 구현예에 따르면, 상기 배지는 질소원, 무기염류 등을 포함할 수 있고, 필요에 따라 생리활성물질을 추가로 포함할 수 있다. 상기 질소원으로는 펩톤 등의 단백질, 아미노산, 요소 등의 유기 질소원과 질산염, 암모늄염 등의 무기 질소원이 사용될 수 있고, 상기 무기염류에는 Na+, K+, Ca2 +, Mg2 +, Cl- 등이 사용될 수 있으며, 상기 생리활성물질에는 비타민 등이 사용될 수 있으나 이에 한정되는 것은 아니다. 또한, 상기 배지에는 효모 추출물, 맥아(malt) 추출물 등이 포함될 수 있고, 세포 배양용으로 시판되는 RPMI(Rosewell Park Memorial Institute), DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimum Essential Medium) 등의 배양 배지들도 사용할 수 있으나 이에 한정되는 것은 아니다. 본 발명의 한 구현예에 따르면, 상기 탄소원으로는 아세트산, 전분, 포도당, 설탕 등이 사용될 수 있으나 이에 한정되는 것은 아니다.In the present invention, the medium may be used without limitation any medium conventionally used for cell culture. According to an embodiment of the present invention, the medium may include a nitrogen source, inorganic salts, and the like, and may further include a bioactive substance as necessary. As 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. In addition, 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. According to one embodiment of the present invention, acetic acid, starch, glucose, sugar, etc. may be used as the carbon source, but is not limited thereto.
본 발명의 한 구현예에 따르면, 상기 배양은 소정의 온도 및 pH 조건하에 행해질 수 있다. 상기 온도는 20 내지 50℃, 바람직하게는 25 내지 40℃, 보다 바람직하게는 28 내지 35℃일 수 있으나 이에 한정되는 것은 아니다. 또한, 본 발명의 다른 구현예에 따르면, 상기 pH는 4 내지 9의 범위, 바람직하게는 5 내지 8의 범위일 수 있으나 이에 한정되는 것은 아니다.According to one embodiment of the invention, 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. In addition, according to another embodiment of the present invention, the pH may be in the range of 4 to 9, preferably in the range of 5 to 8, but is not limited thereto.
본 발명은 아세트산이 유일 탄소원 또는 혼합 탄소원으로 제공되는 경우에 있어서의 아세트산의 효율적인 이용이 가능한 미생물, 바람직하게는 대장균 균주를 제공한다. 이를 위하여, 본 발명의 한 구현예에서는 아세트산 대사 촉진을 위한 진화 실험을 수행하여 아세트산 대사 능력이 향상된 진화 미생물을 분리 동정, 특성을 확인하고, 아세트산 대사 능력이 향상된 미생물, 바람직하게는 대장균을 활용한 재조합 단백질 또는 유용 화합물 생산성을 확인하였다.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. To this end, in one embodiment of the present invention by performing an evolutionary experiment to promote acetic acid metabolism to identify and identify the evolutionary microorganisms with improved acetic acid metabolism ability, confirm the characteristics, using the microorganism with improved acetic acid metabolism ability, preferably E. coli Recombinant protein or useful compound productivity was confirmed.
본 발명의 한 구현예에서는 대장균에서의 아세트산 진화 실험을 통해 아세트산의 대사 능력이 향상된 대장균 균주를 분리 동정하였고(도 3 참조), 이 돌연변이 대장균 균주의 전체 유전자 서열 분석을 통해 유전학적 변화를 조사하였으며, 아미노산 서열에 있어서의 변화가 일어난 5종의 유전자를 확인하였다(표 2 참조). 또한, 분리된 대장균 균주를 이용하여 아세트산 유일 탄소원으로부터 형광 단백질 또는 바이오연료인 부탄올을 생산함으로써 개발한 대장균 균주의 응용 예를 제공하였다(도 8 내지 도 10 참조).In one embodiment of the present invention, 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). In addition, using the isolated 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).
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.
실시예Example 1. 대장균 MG1655  Escherichia coli MG1655 △△ frdAfrdA △ △ ldhAldhA △pta △ △ pta △ adhEadhE 균주의 아세트산 대사능력 개선 Improvement of Acetic Acid Metabolism in Strains
본 발명에서 사용한 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주(Jang-mi Baek et al., Biotechnology and bioengineering, 2013, 110(10), 2790-2794) 는 숙신산, 젖산, 아세트산, 알코올 생산 대사회로 중 각각 frdA, ldhA, pta, adhE 유전자가 제거된 대장균 균주로서, 글루코오스 등과 같은 탄소원으로부터 아세틸-CoA를 다량으로 생산할 수 있도록 개량된 균주이다(도 1). 대장균의 경우 아세트산을 탄소원으로 이용하여 성장하기 위해서는 도 2에 나타낸 두 가지 경로를 통해 아세트산으로부터 아세틸-CoA를 합성한 후 성장에 필요한 에너지를 합성하여 성장하게 된다.E. coli MG1655 △ frdA △ ldhA Δ 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 As an E. coli strain from which the gene has been removed, the strain has been improved to produce a large amount of acetyl-CoA from a carbon source such as glucose (FIG. 1). In the case of 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.
본 발명에서 사용한 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주는 유일 탄소원으로 제공되는 아세트산의 대사 효율이 매우 낮아 외부로부터 제공되는 아세트산의 효율적인 이용이 불가능하였고, 이에 따라 본 발명자들은 상기 대장균 균주의 아세트산 대사 능력을 개선하기 위하여 상기 미생물의 아세트산 적응 진화 실험을 통해 아세트산 대사 능력이 향상된 미생물을 선별하였다.E. coli MG1655 △ frdA △ ldhA Δ pta △ adhE used in the present invention Since the strain has a very low metabolic efficiency of acetic acid provided as the only carbon source, it is impossible to efficiently use acetic acid provided from the outside. Accordingly, the present inventors conducted an acetic acid adaptive evolution experiment of the microorganism to improve the acetic acid metabolism ability of the E. coli strain. Through the selection of microorganisms with improved acetic acid metabolism ability.
실시예Example 2. 대장균 MG1655  2. Escherichia coli MG1655 △△ frdAfrdA △ △ ldhAldhA △pta △ △ pta △ adhEadhE 균주의 아세트산 적응 진화 실험 Acetic acid adaptive evolution experiment of strain
아세트산 적응 진화 실험을 위하여, 표 1에 기재된 아세트산 최소 배지에 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주를 배양하여 성장 속도의 변화를 측정하였다. 이를 위하여, 100㎖의 아세트산 최소 배지에 LB 배지에서 배양된 상기 대장균 균주를 1% 접종하여 37℃, 200rpm의 조건으로 진탕배양기를 이용하여 배양하였다. 또한, 배양 시작 후 미생물의 후기지수성장기(late exponential phase)에서 새로운 배지에 계대배양을 반복 수행하였으며, 각 반복 회차마다 매 12시간 간격으로 600nm에서 흡광도(OD600)를 측정하여 대장균의 성장을 조사하였다.For acetic acid adaptive evolution experiment, the growth rate was measured by culturing E. coli MG1655 Δ frdA Δ ldhA Δpta Δ adhE strain in the acetic acid minimal medium described in Table 1. To this end, 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 ℃, 200rpm conditions. In addition, 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.
아세트산 최소 배지의 조성Composition of Acetic Acid Minimal Medium
성분ingredient 함량content
M9 염 용액(10X)M9 salt solution (10X) 100 ㎖100 ml
Na2HPO4-2H2ONa 2 HPO 4 -2H 2 O 33.7 mM33.7 mM
KH2PO4 KH 2 PO 4 22.0 mM22.0 mM
NaCl NaCl 8.55 mM8.55 mM
NH4ClNH 4 Cl 9.35 mM9.35 mM
1 M1 M MgSOMgSO 44 -- 7H7H 22 OO 1 ㎖1 ml
1 M1 M CaClCaCl 22 0.3 ㎖0.3 ml
미량 원소 용액(100X)Trace Element Solution (100X) 10 ㎖10 ml
EDTA EDTA 13.4 mM13.4 mM
FeCl3-6H2OFeCl 3 -6H 2 O 3.1 mM3.1 mM
ZnCl2 ZnCl 2 0.62 mM0.62 mM
CuCl2-2H2OCuCl 2 -2H 2 O 76 μM76 μM
CoCl2-2H2OCoCl 2 -2H 2 O 42 μM42 μM
H3BO3 H 3 BO 3 162 μM162 μM
MnCl2-4H2OMnCl 2 -4H 2 O 8.1 μM8.1 μM
1 M1 M 나트륨 아세트산 Sodium acetic acid 50 ㎖50 ml
멸균수Sterile water 837.837. 7 ㎖7 ml
합계Sum 1,000 ㎖1,000 ml
대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주의 1차 배양 결과, 배양 시간 192시간 후 0.312의 흡광도를 나타내었다. 1차 배양액 1%를 새로운 아세트산 최소 배지에 접종하여 동일 시간(192시간) 동안 2차 배양을 수행한 결과, 0.455 흡광도를 나타내어 해당 균주의 성장속도가 소폭 증가하였음을 확인하였다. 상기와 동일한 방법으로 각 배양 단계에서 후기지수성장기의 배양액을 새로운 배지에 접종하여 총 10회의 계대배양을 수행하였다.E. coli MG1655 frdA △ △ △ ldhA pta △ 1 subculture result of adhE strain, the culture time after 192 hours showed absorbances of 0.312. Inoculating 1% of the primary culture medium in a new acetic acid minimal medium and performing a second culture for the same time (192 hours) showed 0.455 absorbance, indicating that the growth rate of the strain was slightly increased. In the same manner as above, a total of 10 passages were performed by inoculating the culture medium of the late exponential growth phase in a new medium in each culture step.
총 10회의 계대배양 결과, 반복 회차에 따라 대장균의 성장 속도가 증가되는 것을 확인할 수 있었으며, 구체적으로는 계대배양 초기(G1-G3)의 경우 대장균 성장속도가 소폭 증가하였으나, 계대배양 중반(G4-G6)의 경우 배양 50시간 이후부터 지수성장을 시작하여 최종 흡광도 약 0.9까지 대장균의 성장 속도가 증가되었다. 이후, 계대배양 G7-G9 단계를 거치면서 대장균의 성장 속도가 지속적으로 증가되었고, 최종 10회 반복(G10) 단계에서 배양 60시간 후 흡광도가 1.66을 나타내었다. 즉, 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주의 아세트산 적응 진화 실험 결과, 미생물의 성장이 0.312에서 1.66으로 급격히 증가됨을 확인하였다(도 3).As a result of a total of 10 passages, it was confirmed that 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 Δ frdA Δ ldhA Δ pta Δ adhE strain, it was confirmed that the growth of microorganisms rapidly increased from 0.312 to 1.66 (Fig. 3).
실시예Example 3. 전체 게놈 유전자 분석 3. Whole Genome Gene Analysis
상기 실시예 2에서 회수한 대장균(실험군)의 전체 유전자 염기서열을 해독하기 위하여, 상기 균주를 배양용 아세트산 최소 배지에 1% 접종하고, 37℃, 200rpm의 조건으로 60시간 동안 배양하였다. 회수된 균주로부터 분리된 게놈 DNA 전체 유전자 서열을 분석하였고, 최종적으로 아세트산을 유일 탄소원으로 성장할 수 있는 돌연변이 균주를 대장균 SBA01이라 명명하였으며, 2016년 6월 20일자로 한국생명공학연구원 생물자원센터에 기탁하였다(기탁번호: KCTC 13040BP).In order to decode the entire gene sequence of the E. coli (experimental group) recovered in Example 2, the strain was inoculated in culture medium of minimal acetic acid 1%, and incubated for 60 hours at 37 ℃, 200rpm conditions. The entire genome DNA sequence isolated from the recovered strain was analyzed, and finally, 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).
실험에 사용한 대조군 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주와 아세트산 적응 진화를 통해 선별한 본 발명의 대장균 SBA01 균주의 전체 유전자 서열을 비교한 결과, patZ(pka, acetyltransferase), cspC(stress protein, multicopy suppressor of mukB cold shock protein), mukB(Chromosome partition protein), lomR(putative outer membrane virulence protein) 및 yhjE(Inner membrane metabolite transport protein)의 5종의 유전자가 코딩하는 단백질의 아미노산 서열의 변화가 확인되었다(표 2).Control E. coli used in the experiment MG1655 △ frdA △ ldhA △ pta △ adhE strain as a result of comparing the total gene sequence of Escherichia coli SBA01 strain of the present invention selected from the acetate adaptive evolution, patZ (pka, acetyltransferase), cspC (stress protein, Changes in the amino acid sequence of proteins encoded by five genes: multicopy suppressor of mukB cold shock protein, mukB (Chromosome partition protein), lomR (putative outer membrane virulence protein) and yhjE (Inner membrane metabolite transport protein) (Table 2).
대장균 SBA01의 돌연변이 유전자Mutant gene of E. coli SBA01
유전자gene 기능function 서열 변화Sequence change
patZpatZ acetyltransferaseacetyltransferase Trp501Stop (G→A)Trp501Stop (G → A)
cspCcspC stress protein, multicopy suppressor of mukB cold shock proteinstress protein, multicopy suppressor of mukB cold shock protein Gln58Stop (G→A)Gln58Stop (G → A)
mukBmukB Chromosome partition proteinChromosome partition protein Asp54Glu (C→A)Asp54Glu (C → A)
lomRlomR putative outer membrane virulence proteinputative outer membrane virulence protein Pro114Leu (C→T)Pro114Leu (C → T)
yhjEyhjE Inner membrane metabolite transport proteinInner membrane metabolite transport protein Ile210Met (T→G)Ile210Met (T → G)
실시예Example 4.  4. patZpatZ 결실 균주의 제조 및 아세트산 배지에서의 성장 비교 Preparation of Deletion Strains and Comparison of Growth in Acetic Acid Medium
실시예 3에서 나타낸 바와 같이, 본 발명의 대장균 SBA01 균주의 전체 유전자 염기서열 분석 결과, 아세트산 대사과정 중 아세틸-CoA 신타아제(synthase)(ACS)의 아세틸화에 관여하는 patZ(ack) 유전자의 아미노산 서열의 돌연변이를 확인하였다. 이에, 상기 patZ 유전자가 대장균의 아세트산 대사 과정에 미치는 영향을 확인하기 위하여, 대조군 대장균 MG1655 △ frdA △ ldhA △pta △adhE 의 게놈에 존재하는 patZ 유전자를 제거한 후 아세트산 최소 배지에서 배양함으로써 본 발명의 대장균 SBA01 균주와의 성장을 비교하였다.As shown in Example 3, the amino acid of the patZ ( ack ) gene involved in the acetylation of acetyl-CoA synthase (ACS) during acetic acid metabolism, as a result of the entire gene sequence analysis of the E. coli SBA01 strain of the present invention Mutations in the sequence were identified. Thus, in order to confirm the effect of the patZ gene on the acetic acid metabolism process of E. coli, E. coli of the present invention by removing the patZ gene present in the genome of the control E. coli MG1655 △ frdA △ ldhA Δ pta △ adhE Growth with the SBA01 strain was compared.
그 결과, 본 발명의 대장균 SBA01 균주의 경우 대조군 대비 성장 속도가 약 22배 증가된 것과 대조적으로, 대장균 MG1655 △ frdA △ ldhA △pta △ adhE 균주에서 patZ 유전자만을 추가로 제거한 미생물의 경우에는 대조군 대비 성장 속도가 7배 증가되었다(도 4). 상기 결과로부터, 아세트산의 효과적인 이용을 위해서는 patZ 유전자뿐만 아니라 cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자, 바람직하게는 상기 4종의 유전자가 모두 돌연변이되는 복합적인 효과에 의해 아세트산 유일 탄소원 하에서의 SBA01 균주의 성장 속도가 매우 높아진 것임을 확인하였다.As a result, in the case of E. coli SBA01 strain of the present invention, in contrast to that the control group compared to the growth rate by about 22 times, the E. coli MG1655 △ in frdA △ ldhA △ pta △ adhE strain for the microorganism removed by adding only patZ gene has growth control compared to The speed was increased 7 times (FIG. 4). From the above results, for effective use of the gene as well as acetic acid patZ cspC, mukB, lomR And it was confirmed that the growth rate of the SBA01 strain under the only carbon source of acetic acid by the combined effect of one or more genes selected from the group consisting of yhjE , preferably all four genes are mutated.
실시예Example 5. 본 발명의 대장균  5. E. coli of the present invention SBA01SBA01 균주의  Strain 아세트산에 대한 내성 실험Resistance test to acetic acid
본 발명의 대장균 SBA01 균주의 아세트산에 대한 내성 실험을 수행하였다. 이를 위하여, 표 1에 기재된 아세트산 최소 배지의 아세트산 농도를 각각 10, 20, 100, 150, 200 및 250 mM로 조정한 후, 각각의 배지에 대장균 SBA01 균주 1%를 접종하여 37℃, 200 rpm의 조건으로 진탕배양기를 이용하여 배양하였다. 각 반복 회차마다 매 6시간 간격으로 600 nm에서 흡광도(OD600)를 측정하여 대장균의 성장을 조사하였다.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.
그 결과, 본 발명의 대장균 SBA01 균주는 50 mM 아세트산이 포함된 배지에서 가장 높은 성장도를 나타내었고, 100 mM 및 150 mM 아세트산이 포함된 배지에서도 성장이 우수한 것으로 측정되었으며, 200 mM 이상의 아세트산이 포함된 배지에서는 성장이 감소되었다(도 5). 상기 결과로부터, 본 발명의 SBA01 균주는 아세트산의 효과적인 이용뿐만 아니라 고농도의 아세트산에 대한 내성이 증가되었음을 확인하였다.As a result, 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.
실시예Example 6. 본 발명의 대장균  6. Escherichia coli of the present invention SBA01SBA01 균주의  Strain 전사체Transcript 비교 분석 comparison analysis
본 발명의 대장균 SBA01 균주(실험군)와 대조군의 전사체 비교 분석을 수행하였다. 이를 위하여 본 발명의 SBA01(실험군) 및 대조군 균주를 글루코오스 0.4%가 포함된 최소 배지에서 각각 1%를 접종한 후, 37℃, 200 rpm의 조건으로 배양한 흡광도가 1.0 일 때 균주를 회수하였다. 회수한 실험군과 대조군 균주로부터 각각 mRNA를 추출하였고, 추출한 각각의 mRNA를 이용하여 RNA-SEQ 분석을 수행하였다(TruSeqStranded mRNA Sample Preparation Protocol). 상기 RNA-SEQ 분석의 경우 통상적으로 완전한 게놈 염기서열에 전사체 염기서열을 맵핑하여 발현양을 비교하지만, 본 발명에서는 대조군과 실험군의 발현양을 비교하여 아세트산에 대한 실험군의 발현양 증감을 분석하였다. 그 결과, mRNA의 발현량이 증가된 상위 28개의 유전자와 mRNA의 발현량이 감소된 상위 26개의 유전자 정보를 확인하였다(도 6a 및 도 6b).Comparative analysis of the transcripts of the E. coli SBA01 strain (experimental group) and the control group of the present invention was performed. To this end, the SBA01 (experimental group) and the control strains of the present invention were inoculated with 1% each in a minimal medium containing 0.4% glucose, and the strains were recovered when the absorbance was 1.0 at 37 ° C and 200 rpm. MRNA was extracted from each of the recovered experimental and control strains, and RNA-SEQ analysis was performed using each extracted mRNA (TruSeqStranded mRNA Sample Preparation Protocol). In the case of 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. . As a result, 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).
또한, 대장균의 대사 회로에서 증감된 유전자를 맵핑하여 비교한 결과를 도 6c에 나타내었다. 그 결과, 발현량이 증가된 대사 회로의 유전자로는 아세테이트를 아세틸-CoA롤 전환하는 아세틸-CoA 신타아제가 약 7.05배 증가하였고, 세포내 ATP 합성에 관여하는 F1F0 ATP 신타아제 5.62배, 전자 전달계에 관여하는 시토크롬 bo3 5.79배가 증가되었다. 또한, TCA 회로를 구성하는 유전자인 시트레이트 신타아제, 아코니트라아제, 이소시트레이트 디히드로게나아제, α-케토글루타레이트 디히드로게나아제, 숙시닐-CoA 신타아제, 푸마라아제, 말레이트 디히드로게나아제, 이소시트레이트 라이아제의 발현량이 2.0에서 6.19배 증가되었다. 이와 대조적으로, 구리 이온의 세포내로 배출하는 구리 방출 펌프에 관여하는 유전자는 5.48배 감소되었다.In addition, the result of mapping and comparing the genes increased and decreased in the metabolic circuit of E. coli is shown in Figure 6c. As a result, the acetyl-CoA synthase that converts acetate to acetyl-CoA was increased by about 7.05 fold, and the gene of the metabolic circuit with increased expression was 5.62 fold for F1F0 ATP synthase, which is involved in intracellular ATP synthesis. Involved cytochrome bo3 5.79 times increased. In addition, citrate synthase, aconitase, isocitrate dehydrogenase, α-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. In contrast, the gene involved in the copper release pump, which releases copper ions into cells, was reduced by 5.48 fold.
상기 결과로부터, 본 발명의 SBA01 균주는 대조군 균주 대비 아세트산의 아세틸-CoA 전환, ATP 합성, TCA 회로(NADH 합성)에 관여하는 유전자의 발현량이 증가되었음을 확인하였다.From the above results, it was confirmed that 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.
실시예Example 7.  7. 세포내Intracellular ATP 함량 측정 ATP content measurement
세포 내 ATP 함량을 측정하기 위하여 자연형 대장균 MG1655, 대조군 DSM01 및 본 발명의 SBA01 균주를 글루코오스 0.4%가 포함된 최소 배지에서 각각 1%를 접종하였고, 37℃, 200 rpm의 조건으로 배양한 흡광도가 1.0 일 때 균주를 회수하였다. 회수한 균주를 시그마 사의 ATP 분석 키트를 활용하여 세포내 ATP 양을 측정하였다.In order to determine the intracellular ATP content, 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.
그 결과, 자연형 균주는 0.3264±0.0776 nmol/㎕, 대조군 DSM01 균주는 0.3031±0.0346 nmol/㎕, 본 발명의 SBA01 균주는 0.4814±0.0544 nmol/㎕의 ATP가 측정되어, 본 발명의 SBA01 균주의 경우 대조군 대비 세포내 ATP 함량이 약 40% 증가되었음을 확인하였다(도 7). 실시예 6의 전사체 분석 결과 및 상기의 ATP 함량 측정의 결과로부터, 본 발명의 SBA01 균주는 세포내 ATP 합성 효율이 대조군에 비해 증가되었음을 확인하였다.As a result, ATP of 0.3264 ± 0.0776 nmol / μl of natural strain, 0.3031 ± 0.0346 nmol / μl of control DSM01 strain, 0.4814 ± 0.0544 nmol / μl of SBA01 strain of the present invention was measured, and in case of SBA01 strain of the present invention It was confirmed that the intracellular ATP content was increased by about 40% compared to the control (Fig. 7). From the results of the transcript analysis and the ATP content measurement of Example 6, it was confirmed that the SBA01 strain of the present invention increased the intracellular ATP synthesis efficiency compared to the control.
실시예Example 8.  8. GFPGfp 단백질의 발현 Expression of protein
대장균 SBA01 균주의 아세트산 대사능력 및 외래 단백질의 생산능력을 확인하기 위하여 대표적인 재조합 단백질의 하나인 GFP의 발현 여부를 관찰하였다. 이를 위하여, 대장균 내에서 형광 단백질의 발현이 가능하도록 제작된 pUCBB-eGFP 벡터를 본 발명의 대장균 SBA01 균주에 형질전환시킨 후, 각각의 균들을 아세트산 최소 배지에 48시간 동안 배양시키고, 글리세롤 농도가 10%가 되게 보관용 균액으로 만든 다음 -80℃에서 배양실험 때까지 저장하였다. 상기 보관용 균액 1㎖을 250㎖ 플라스크에 들어있는 앰피실린(50㎍/㎖)이 첨가된 50㎖의 아세트산 최소 배지에 접종하여 48시간 동안 배양한 후, 형광광학측정기(fluorescence spectrophotometer)를 이용하여 조사(excitation) 395 ㎚, 방출(emission) 509 ㎚의 조건에서 형광을 측정하였다.In order to confirm the acetic acid metabolism of E. coli SBA01 strain and the ability to produce foreign protein, expression of GFP, one of the representative recombinant proteins, was observed. To this end, 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 ℃ until the culture experiment. 1 ml of the storage solution was inoculated in 50 ml of acetic acid (50 µg / 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.
그 결과, 대조군의 경우 균체의 성장이 거의 이루어지지 않은 반면, 본 발명의 SBA01 균주는 600nm에서 2.0의 흡광도를 나타내는 것으로 보아 형광 단백질이 발현됨을 확인하였다(도 8).As a result, in the case of the control group, the growth of the cells was hardly achieved, whereas the SBA01 strain of the present invention showed an absorbance of 2.0 at 600 nm, confirming that the fluorescent protein was expressed (FIG. 8).
실시예Example 9. 바이오 부탄올의 생산 9. Production of Bio Butanol
실시예 8에서 확인한 바와 같이, 본 발명의 대장균 SBA01 균주는 아세트산를 기질로 이용하여 재조합 단백질의 합성이 가능함을 확인하였다. 이와 더불어 본 발명의 대장균 SBA01 균주를 이용한 고부가 화학물질의 생산 가능성을 확인하기 위하여, 상기 대장균 SBA01 균주에 부탄올 합성 대사회로를 도입하여 부탄올 합성 여부를 확인하였다. 부탄올 합성 대사회로는 Clostridium acetobutylicum 및 Treponema denticola 유래의 아세틸-CoA 및 부티릴-CoA 합성 경로에 관여하는 4종의 효소인 3-히드록시부티릴-CoA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제를 이용하여 재조합 벡터를 구성하였다. 또한, 부탄올 합성경로에서 필요한 NADH의 공급을 위하여 포르메이트 디하이드로게나아제의 발현이 가능한 벡터를 추가로 구성하였다. 상기 두 가지 벡터를 본 발명의 SBA01 균주에 형질전환시킨 후, 각각의 균들을 아세트산 최소 배지에 48시간 동안 배양하였고, 글리세롤 농도가 10%가 되게 보관용 균액으로 만든 후, -80℃에서 배양실험 때까지 저장하였다.As confirmed in Example 8, E. coli SBA01 strain of the present invention was confirmed that the synthesis of recombinant proteins using acetic acid as a substrate. In addition, in order to confirm the production potential of the high value-added chemicals using the E. coli SBA01 strain of the present invention, 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. In addition, a vector capable of expressing formate dehydrogenase was further configured to supply NADH required in the butanol synthesis pathway. After transforming the two vectors into the SBA01 strain of the present invention, 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 ℃ Stored until.
부탄올 발효는 아세트산 최소 배지가 포함된 1 L 발효기를 이용하였다. 배양용 아세트산 최소 배지에 형질전환된 대장균 SBA01 균주를 1% 접종하여 37℃, 200 rpm의 조건으로 94시간 동안 배양하였다. 배양 초기 24시간은 호기성 상태를 유지하기 위하여 1vvm의 속도로 공기를 주입한 후, 배양 24시간 이후 0.02vvm으로 공기 주입량을 줄여 반혐기성 조건을 조성하였다. 형질전환된 대장균 SBA01 균주의 생장 곡선을 도 9에 나타내었다.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. In the first 24 hours of culture, 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.
배양 종료 후 배양액 내 부탄올의 농도를 측정하기 위하여 가스크로마토그래피(GC) 분석을 수행한 결과, 11.1 ㎎/L의 부탄올을 생산하였으며, 동일 시료를 GC/MS 분석한 결과 생산된 물질이 부탄올임을 확인하였다(도 10).Gas chromatography (GC) analysis was carried out to measure the concentration of butanol in the culture medium after the end of the culture, producing 11.1 mg / L butanol, and GC / MS analysis of the same sample confirmed that the produced material was butanol. (FIG. 10).
[수탁번호][Accession number]
기탁기관명: 한국생명공학연구원Depositary Name: Korea Research Institute of Bioscience and Biotechnology
수탁번호: KCTC13040BPAccession number: KCTC13040BP
수탁일자: 2016년 6월 10일Deposited Date: June 10, 2016
본 발명을 지원한 국가연구개발사업 (1)National R & D project supporting the present invention (1)
과제고유번호: 20133030000300Assignment unique number: 20133030000300
부처명: 산업통상자원부Department name: Ministry of Trade, Industry and Energy
연구관리전문기관: 한국에너지기술평가원Specialized research management organization: Korea Institute of Energy Research
연구사업명: 에너지기술개발사업Project Name: Energy Technology Development Project
연구과제명: 전기활성 미생물을 이용한 저급탄소원의 바이오연료 전환기술Project title: Biofuel conversion technology for lower carbon sources using electroactive microorganisms
기여율: 50/100Contribution rate: 50/100
주관기관: 한국생명공학연구원Organizer: Korea Research Institute of Bioscience and Biotechnology
연구기간: 2015.10.01 ~ 2016.09.30Research period: 2015.10.01 ~ 2016.09.30
본 발명을 지원한 국가연구개발사업 (2)National R & D project supporting the present invention (2)
과제고유번호: 2016004919Assignment number: 2016004919
부처명: 미래창조과학부Department name: Ministry of Science, ICT and Future Planning
연구관리전문기관: 한국연구재단(대전)Specialized research management organization: Korea Research Foundation (Daejeon)
연구사업명: 원천기술개발사업(C1가스리파이너리)Project name: Original Technology Development Project (C1 Gas Refinery)
연구과제명: 고속 C1 전환 효소 스크리닝 기술 및 바이오촉매 개발Project title: Development of high speed C1 converting enzyme screening technology and biocatalyst
기여율: 20/100Contribution rate: 20/100
주관기관: 한국생명공학연구원Organizer: Korea Research Institute of Bioscience and Biotechnology
연구기간: 2016.03.01 ~ 2017.02.28Research period: 2016.03.01 ~ 2017.02.28
본 발명을 지원한 국가연구개발사업 (3)National R & D project supporting the present invention (3)
과제고유번호: 20163030091540Assignment Number: 20163030091540
부처명: 산업통상자원부Department name: Ministry of Trade, Industry and Energy
연구관리전문기관: 한국에너지기술평가원Specialized research management organization: Korea Institute of Energy Research
연구사업명: 에너지기술개발사업Project Name: Energy Technology Development Project
연구과제명: 슬러지 에너지화 경제성 향상을 위한 에너지전환율 25% 향상 슬러지 가용화용 효소 재설계 및 bio-augmentation 기술 개발Project name: 25% energy conversion to improve the economic efficiency of sludge Redesign of enzyme for sludge solubilization and development of bio-augmentation technology
기여율: 20/100Contribution rate: 20/100
주관기관: 한국생명공학연구원Organizer: Korea Research Institute of Bioscience and Biotechnology
연구기간: 2016.05.01 ~ 2016.12.31Research period: 2016.05.01 ~ 2016.12.31
본 발명을 지원한 국가연구개발사업 (4)National R & D project supporting the present invention (4)
과제고유번호: KGM2121622Assignment No .: KGM2121622
부처명: 미래창조과학부Department name: Ministry of Science, ICT and Future Planning
연구관리전문기관: 국가과학기술연구회Specialized research management organization: National Institute of Science and Technology
연구사업명: 주요사업(2015-2018)Project Name: Major Projects (2015-2018)
연구과제명: 바이오부품/회로 및 합성생물학 기술 개발사업Project title: Bio components / circuit and synthetic biology technology development project
기여율: 10/100Contribution rate: 10/100
주관기관: 한국생명공학연구원Organizer: Korea Research Institute of Bioscience and Biotechnology
연구기간: 2016.01.01 ~ 2016.12.31.Research period: 2016.01.01 ~ 2016.12.31.
Figure PCTKR2017010643-appb-I000001
Figure PCTKR2017010643-appb-I000001

Claims (19)

  1. 야생형 미생물에 patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물.In wild-type microorganisms patZ , cspC , mukB , lomR And one or more genes selected from the group consisting of yhjE can use acetic acid as the only carbon source.
  2. 청구항 1에 있어서,The method according to claim 1,
    patZ 유전자가 필수적으로 돌연변이되고, cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가적으로 돌연변이된 돌연변이 미생물. patZ gene is essentially mutated, cspC , mukB , lomR And a mutant microorganism further mutated with at least one gene selected from the group consisting of yhjE .
  3. 청구항 1에 있어서,The method according to claim 1,
    patZ , cspC , mukB , lomR 및 yhjE의 5종의 유전자가 모두 돌연변이된 돌연변이 미생물. patZ , cspC , mukB , lomR And a mutant microorganism in which all five genes of yhjE are mutated.
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 patZ , cspC , mukB , lomR 및 yhjE 유전자는 각각 서열번호 1 내지 서열번호 5로 이루어지는 염기서열을 갖는 돌연변이 미생물. PatZ , cspC , mukB , lomR And yhjE gene has a nucleotide sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 5, respectively.
  5. 청구항 1에 있어서,The method according to claim 1,
    frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 돌연변이 미생물.A mutant microorganism further comprising one or more genes selected from the group consisting of frdA , ldhA , adhE and pta .
  6. 청구항 5에 있어서,The method according to claim 5,
    상기 frdA , ldhA , adhE 및 pta 유전자는 각각 서열번호 6 내지 서열번호 9로 이루어지는 염기서열을 갖는 돌연변이 미생물. Wherein said frdA , ldhA , adhE and pta genes each have a nucleotide sequence consisting of SEQ ID NO: 6 to SEQ ID NO: 9.
  7. 청구항 1에 있어서,The method according to claim 1,
    상기 patZ 유전자의 돌연변이는 서열번호 10의 아미노산 서열로 이루어지는 야생형 효소의 501번째 아미노산인 Trp가 Stop codon으로 돌연변이(Trp501Stop)되고, 상기 cspC 유전자의 돌연변이는 서열번호 11의 아미노산 서열로 이루어지는 야생형 효소의 58번째 아미노산인 Gln이 Stop codon으로 돌연변이(Gln58Stop)되고, 상기 mukB 유전자의 돌연변이는 서열번호 12의 아미노산 서열로 이루어지는 야생형 효소의 54번째 아미노산인 Asp가 Glu로 돌연변이(Asp54Glu)되고, 상기 lomR 유전자의 돌연변이는 서열번호 13의 아미노산 서열로 이루어지는 야생형 효소의 114번째 아미노산인 Pro가 Leu로 돌연변이(Pro114Leu)되고, 상기 yhjE 유전자의 돌연변이는 서열번호 14의 아미노산 서열로 이루어지는 야생형 효소의 210번째 아미노산인 Ile가 Met로 돌연변이(Ile210Met)되는 돌연변이 미생물.The mutation of the patZ gene is Trp, the 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 58 of the wild type enzyme consisting of the amino acid sequence of SEQ ID NO: 58 The first amino acid Gln is mutated to the stop codon (Gln58Stop), the mukB gene mutation is the 54th amino acid Asp of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 12 is mutated to Glu (Asp54Glu), the mutation of the lomR gene Pro is the 114th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 13 Pro is mutated to Leu (Pro114Leu), the mutation of the yhjE gene is 210 amino acids of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 14 Ile is Met Mutant microorganisms mutated (Ile210Met).
  8. 청구항 1에 있어서,The method according to claim 1,
    부탄올의 생합성 관련 유전자가 추가로 도입된 돌연변이 미생물.A mutant microorganism further introduced with a biosynthesis related gene of butanol.
  9. 청구항 8에 있어서,The method according to claim 8,
    상기 부탄올의 생합성 관련 유전자는 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제인 돌연변이 미생물.The biosynthesis related genes of butanol include 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogena Azein mutant microorganisms.
  10. 청구항 1 내지 청구항 9 중 어느 한 항에 있어서,The method according to any one of claims 1 to 9,
    상기 미생물은 대장균인 돌연변이 미생물.The microorganism is E. coli mutant microorganisms.
  11. 청구항 1 내지 청구항 7 중 어느 한 항에 있어서,The method according to any one of claims 1 to 7,
    상기 미생물은 기탁번호 KCTC13040BP로 기탁된 대장균 SBA01 균주인 돌연변이 미생물.The microorganism is a mutant microorganism is Escherichia coli SBA01 strain deposited with accession number KCTC13040BP.
  12. (1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;(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.
    (2) 상기 돌연변이 미생물에 3-히드록시부티릴-COA 디하이드로게나아제, 3-히드록시부티릴-CoA 디하이드라타아제, 트랜스-에노일-CoA 리덕타아제 및 알데히드 및 알코올 디하이드로게나아제 유전자를 도입한 형질전환 미생물을 제조하는 단계;(2) 3-hydroxybutyryl-COA dehydrogenase, 3-hydroxybutyryl-CoA dehydrolase, trans-enoyl-CoA reductase and aldehyde and alcohol dehydrogenase Preparing a transformed microorganism into which the aze gene is introduced;
    (3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 부탄올의 제조 방법.(3) A method for producing butanol comprising culturing the transformed microorganism in a medium containing a carbon source.
  13. 청구항 12에 있어서,The method according to claim 12,
    상기 형질전환 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 부탄올의 제조 방법.The transforming microorganism is a method for producing butanol, wherein one or more genes selected from the group consisting of frdA , ldhA , adhE and pta are further removed.
  14. 청구항 12 또는 청구항 13에 있어서,The method according to claim 12 or 13,
    상기 미생물은 대장균인 부탄올의 제조 방법.The microorganism is E. coli butanol production method.
  15. 청구항 12에 있어서,The method according to claim 12,
    상기 돌연변이 미생물은 기탁번호 KCTC13040BP로 기탁된 대장균 SBA01 균주인 부탄올의 제조 방법.The mutant microorganism is Escherichia coli SBA01 strain deposited with accession number KCTC13040BP method of producing butanol.
  16. (1) patZ , cspC , mukB , lomR 및 yhjE로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 돌연변이되어 아세트산을 유일 탄소원으로 이용할 수 있는 돌연변이 미생물을 제조하는 단계;(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.
    (2) 상기 돌연변이 미생물에 재조합 단백질을 암호화하는 유전자를 도입한 형질전환 미생물을 제조하는 단계; 및(2) preparing a transformed microorganism in which a gene encoding a recombinant protein is introduced into the mutant microorganism; And
    (3) 상기 형질전환 미생물을 탄소원을 포함하는 배지에서 배양하는 단계를 포함하는 재조합 단백질의 제조 방법.(3) a method for producing a recombinant protein comprising culturing the transformed microorganism in a medium containing a carbon source.
  17. 청구항 16에 있어서,The method according to claim 16,
    상기 형질전환 미생물은 frdA , ldhA , adhE 및 pta로 이루어진 군으로부터 선택되는 하나 이상의 유전자가 추가로 제거된 재조합 단백질의 제조 방법.The transforming microorganism is a method for producing a recombinant protein further removed one or more genes selected from the group consisting of frdA , ldhA , adhE and pta .
  18. 청구항 16 또는 청구항 17에 있어서,The method according to claim 16 or 17,
    상기 미생물은 대장균인 재조합 단백질의 제조 방법.The microorganism is Escherichia coli method for producing a recombinant protein.
  19. 청구항 16에 있어서,The method according to claim 16,
    상기 돌연변이 미생물은 기탁번호 KCTC13040BP로 기탁된 대장균 SBA01 균주인 재조합 단백질의 제조 방법.The mutant microorganism is E. coli SBA01 strain deposited with accession number KCTC13040BP method of producing a recombinant protein.
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