WO2017034304A1 - Préparation d'un composite enzymatique et procédé de production de matériau cible l'utilisant - Google Patents

Préparation d'un composite enzymatique et procédé de production de matériau cible l'utilisant Download PDF

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WO2017034304A1
WO2017034304A1 PCT/KR2016/009340 KR2016009340W WO2017034304A1 WO 2017034304 A1 WO2017034304 A1 WO 2017034304A1 KR 2016009340 W KR2016009340 W KR 2016009340W WO 2017034304 A1 WO2017034304 A1 WO 2017034304A1
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coa
synthesis
target substance
enzyme
fibrin
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PCT/KR2016/009340
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English (en)
Korean (ko)
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이승구
김하성
성원재
염수진
이대희
정흥채
한귀환
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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 the preparation of an enzyme complex and a method for producing a target substance using the same.
  • a method of directly separating a highly active wild type bacterium (wild wine) from nature a method of using a mutant strain having introduced a target gene necessary for manufacturing a substance into a known microorganism, There is a method of using microorganisms that have introduced mutant strains into highly active wild strains.
  • Patent Literature 1 suppresses the expression or activity of a protein (or enzyme) involved in the pathway of converting acetyl-CoA to acetate, and inhibits acetyl-CoA with butyryl-.
  • a microorganism is disclosed in which the expression or activity of a protein (or enzyme) involved in the pathway to convert to CoA is promoted.
  • HBD 3-hydroxybutyryl-CoA dehydrogenase
  • TER trans-enoyl-CoA reductase
  • AdhE2 aldehyde / alcohol dehydrogenase
  • intermediate products of the butanol synthesis pathway may accumulate in the microorganism through a reaction in which NADH is reduced to NAD + in the butanol synthesis pathway, but is involved in the butanol synthesis pathway inside the cell.
  • various conversion enzymes which actually prevent the accumulation of 3-hydroxybutyryl-CoA, crotonyl-CoA, butyryl-CoA, butyrylaldehyde, intermediates in the butanol synthesis pathway.
  • butyryl-CoA butyrylaldehyde should be converted by aldehyde / alcohol dehydrogenase, but may also be converted to butyryl-P by phosphotrans butyrylase.
  • C4 compounds may accumulate in the microorganism through oxidative phosphorylation of the TCA circuit, but various C4 converting enzymes in addition to the enzymes involved in the TCA circuit inside the cell. Is present, the accumulation of malic acid, fumaric acid and succinic acid is impeded.
  • OAA oxaloacetate
  • it must be converted to malic acid by malic dehydrogenase, but at the same time it is also converted into aspartate by aminotransferase and biosynthesis of fumaric acid from malic acid.
  • the reaction is also considered to be difficult to proceed with the reverse reaction of the dehydrogenation reaction.
  • the present inventors can narrow the physical interval in which the biosynthesis-related enzymes of the target substance are present in order to increase the synthesis efficiency of the target substance, thereby eliminating the problem that the intermediate product immediately participates in the reaction, thereby preventing accumulation by other other enzymes.
  • System was studied, and the cellulose binding domain (CBD), which is known to form insoluble protein constructs in Escherichia coli, was used to fix and accumulate enzymes related to the synthesis of the target substance in cells.
  • the present invention was completed by confirming that the target substance can be produced.
  • An object of the present invention is to provide a complex of enzymes for the synthesis of a target substance, which can improve the production efficiency of the target substance, and a method for producing the target substance using the same.
  • one aspect of the present invention is a cellulose binding domain (CBD); Leucine zippers; And at least two different enzymes involved in the biosynthesis of the target substance from the precursor, wherein each of the at least two different enzymes involved in the synthesis of the target substance is linked to the fibrin binding domain via the leucine zipper.
  • the fibrin binding domains to which the respective enzymes are linked are aggregated with each other to provide an enzyme complex for biosynthesis of a target substance.
  • another aspect of the present invention is a recombinant vector cloned so that the NZ protein constituting the fibrin binding domain and the leucine zipper are linked to each other and expressed at least two different enzymes involved in the synthesis of the target substance.
  • Recombinant vectors cloned such that each of them is linked to and expressed with the CZ protein constituting the leucine zipper;
  • the CZ protein constituting the fibrin-binding domain and the leucine zipper are linked to each other so that the cloned recombinant vector and two or more different enzymes involved in the synthesis of the target substance are linked to the NZ protein constituting the leucine zipper.
  • the cloned recombinant vector provides a transformant for the synthesis of the target material introduced into the host cell.
  • another aspect of the present invention is an in vivo production method of the enzyme complex for the synthesis of the target material comprising the step of expressing the recombinant vector from the transformant, and Expressing and purifying a fusion protein fused with an enzyme involved in the synthesis of a target substance from the transformant and a CZ protein or NZ protein of a leucine zipper and a NZ protein or CZ protein of a fibrin binding domain and a leucine zipper
  • a method for in vitro preparation of an enzyme complex for synthesis of a target substance which comprises the steps of binding and agglutinating the expressed / purified recombinant protein in vitro .
  • another aspect of the present invention comprises the step of reacting the enzyme complex for the synthesis of the target material with a precursor of the target material in vitro, in vitro ( in Provided is a method for producing a target substance in vitro .
  • another aspect of the present invention comprises the step of culturing the transformant in the presence of a precursor of the target material, the target material on the in vivo ( in vivo ) using the enzyme complex To provide a production method.
  • Enzyme complex of the present invention by fixing the synthetic enzymes of the target substance to the fibrin binding domain by narrowing the physical interval of the enzyme acting in a series of processes in the cell from the precursor (eg glucose) of the target substance to the target substance As a result, the intermediate product can be immediately induced to participate in the next reaction, thereby improving the overall production rate or yield of the target substance.
  • the precursor eg glucose
  • the intermediate product can be immediately induced to participate in the next reaction, thereby improving the overall production rate or yield of the target substance.
  • each enzyme to exist in a fixed state in the cell, it is possible to ensure the stability of each enzyme.
  • 1 is a schematic representation of an enzyme complex according to the present invention.
  • FIG. 2 is a diagram illustrating a structure in which fibrin-binding domains and enzymes involved in synthesis of a target substance are linked through a leucine zipper.
  • Figure 6a shows a map of the recombinant vector (pET21a-NZ :: CBD) that is cloned to express the NZ protein and fibrin binding domain forming a part of the leucine zipper.
  • 6B shows a map of a recombinant vector (pET21a-CZ :: CBD) in which a CZ protein constituting a part of a leucine zipper and a fibrin binding domain are cloned to be expressed in connection with each other.
  • FIG. 7A is a control group, 3-hydroxybutyryl-CoA dehydrogenase (HBD), an enzyme involved in butanol synthesis, and 3-hydroxybutyryl-CoA dehydratase (3-hydroxybutyryl-CoA recombinant vector (pACBB-HCTA) designed to express dehydratase (CRT), trans-enoyl-CoA reductase (TER) and aldehyde / alcohol dehydrogenase (AdhE2), respectively Represents a map of.
  • HBD 3-hydroxybutyryl-CoA dehydrogenase
  • pACBB-HCTA 3-hydroxybutyryl-CoA recombinant vector designed to express dehydratase (CRT), trans-enoyl-CoA reductase (TER) and aldehyde / alcohol dehydrogenase (AdhE2)
  • FIG. 7B illustrates a process of constructing the recombinant vector pACBB-HCTA of FIG. 7A.
  • FIG. 8A illustrates a recombinant vector (pACBB-CZ :: HCTA) that is cloned so that CZ proteins constituting a leucine zipper are linked to each other and expressed in HBD, CRT, TER, and AdhE2, which are enzymes involved in butanol synthesis.
  • pACBB-CZ a recombinant vector
  • FIG. 8B shows a process for constructing the recombinant vector pACBB-CZ :: HCTA of FIG. 8A.
  • Figure 10a shows that each of the individual enzymes involved in butanol biosynthesis, soluble fraction and insoluble fraction from the transformant (control group) present in the free state and the transformant (experiment group) forming the enzyme complex according to the present invention (test group)
  • Figure 10b is a schematic diagram showing the process of separating
  • Figure 10b is a graph showing the results of measuring the activity of each enzyme in each of the soluble and insoluble fractions derived from the control group and the experimental group.
  • Figure 11 shows in vitro , each of the individual enzymes involved in butanol biosynthesis, 1 the degree of butanol production when present in the free state and 2 the degree of butanol production when the enzyme complex according to the present invention is formed Is a graph.
  • FIG. 12A and 12B show that each of the individual enzymes involved in butanol biosynthesis is present in the transformant in a free state (control group) and in the presence of an enzyme complex according to the present invention (experimental group).
  • Fig. 12A shows the results of confirming butanol production efficiency in in vivo conditions (aerobic control group, FIG. 12B experimental group).
  • FIG. 13A and 13B show that each of the individual enzymes involved in butanol biosynthesis is present in the transformant in a free state (control group) and in the presence of an enzyme complex according to the present invention (experimental group).
  • Fig. 13A shows the results of confirming butanol generation efficiency in in vivo under anaerobic conditions (FIG. 13A: control, FIG. 13B: experimental group).
  • One aspect of the invention provides an enzyme complex for the synthesis of the desired substance.
  • Figure 1 is a schematic diagram showing the structure of the enzyme complex for the synthesis of the target material as described above.
  • the enzyme complex of the present invention comprises two or more different enzymes and fibrin binding domains involved in the synthesis of the target substance.
  • Enzymes involved in the synthesis of the target substance means all enzymes that participate in a series of metabolic processes in which the target substance is produced from the precursor, and two or more enzymes selected from them.
  • the target substance may be any biosynthetic substance that can be produced by a series of metabolic processes involving two or more enzymes in vivo, such as butanol, 1,4-butanediol, isoprene, succinic acid, ⁇ -caprolactam, and the like. But it is not limited thereto.
  • the enzymes involved in the synthesis of butanol as the target substance are 3-hydroxybutyryl-CoA dehydrogenase (HBD) and 3-hydroxybutyryl-CoA dehydration.
  • HBD 3-hydroxybutyryl-CoA dehydrogenase
  • TER trans-enoyl-CoA reductase
  • AdhE2 aldehyde / alcohol dehydrogenase
  • the enzymes involved in the butanol synthesis are Clostridium acetobutylicum or Treponema It may be an enzyme derived from denticola , but is not particularly limited thereto.
  • the 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydrogenase and aldehyde / alcohol dehydrogenase are derived from C. acetobutylicum
  • trans-enoyl-CoA reductase is T It may be derived from denticola , but is not limited thereto.
  • the 3-hydroxybutyryl-CoA dehydrogenase comprises the amino acid sequence of SEQ ID NO: 1
  • the 3-hydroxybutyryl-CoA dehydrogenase comprises the amino acid sequence of SEQ ID NO: 2
  • the trans-eno One-CoA reductase may include the amino acid sequence of SEQ ID NO: 3
  • the aldehyde / alcohol dehydrogenase may include the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.
  • the enzymes involved in the synthesis of 1,4-butanediol as the target substance are succinyl-CoA synthetase (sucCD) and CoA-dependent succinate semialdehyde.
  • CoA-dependent succinate semialdehyde dehydrogenase (sucD), 4-hydroxybutyrate dehydrogenase (4-HBD), 4-hydroxybutyryl-CoA transferase (4-hydroxybutyryl-CoA transferase, 4) -HBT) and aldehyde / alcohol dehydrogenase (Aldehyde / Alcohol dehydrogenase, AdhE2) may be two or more different from each other selected from the group consisting of.
  • the succinyl-CoA synthetase comprises the amino acid sequence of SEQ ID NO: 11
  • the CoA-dependent succinate semialdehyde dehydrogenase comprises the amino acid sequence of SEQ ID NO: 12
  • the 4-hydroxybutyrate dehydrogenase is SEQ ID NO: 13 wherein the 4-hydroxybutyryl-CoA transferase comprises the amino acid sequence of SEQ ID NO: 14
  • the aldehyde / alcohol dehydrogenase may comprise the amino acid sequence of SEQ ID NO: 4, but It is not limited.
  • the enzymes involved in the synthesis of the target isoprene are hydroxymethylglutaryl-CoA synthase (mvaS), 3-hydroxy-3-methylglutaryl- CoA Reductase (3-hydroxy-3-methylglutaryl-coenzyme A reductase, mvaA), mevalonate kinase (mvaK1), acetyl-CoA acetyl transferase / HMG-CoA reductase (acetyl-CoA acetyltransferase / HMG -CoA reductase (mvaE), phosphomevalonate kinase (mvaK2), diphosphomevalonate decarboxylase (mvaD), isopentyl-diphosphate delta isomerase , idi) and isoprene synthase (IspS) may be two or more different from each other selected from the group consisting of.
  • mvaS hydroxymethylg
  • the hydroxymethylglutaryl CoA synthase comprises an amino acid sequence of SEQ ID NO: 19
  • the mevalonate kinase comprises an amino acid sequence of SEQ ID NO: 20
  • the enzyme comprises the amino acid sequence of SEQ ID NO: 21
  • the phosphomevalonate kinase comprises the amino acid sequence of SEQ ID NO: 22
  • the diphosphosmevalonate decarboxylase comprises the amino acid sequence of SEQ ID NO: 23
  • the isopentyl-diphosphate delta isomerase may include the amino acid sequence of SEQ ID NO: 24, but is not limited thereto.
  • the enzymes involved in the synthesis of the succinic acid as the target substance are phosphoenolpyruvate carboxykinase (pckA), malate dehydrogenase (mdh), and fumarase (fumarase). , fum) and succinate ubiquinone oxidoreductase (sdhABCD).
  • the enzymes involved in the synthesis of the target substance ⁇ -caprolactam are CoA-dependent aldehyde dehydrogenase, an aminotransferase and an amide hydrolase (CoA-dependent aldehyde dehydrogenase).
  • amide hydrolase may be two or more different types selected from the group consisting of.
  • Enzymes involved in the synthesis of the target substance are variants, or fragments of amino acids having a different sequence by deletion, insertion, substitution or combination of amino acid residues within a range that does not affect the function of the protein of each enzyme Can be heard.
  • Amino acid exchange at the protein and peptide levels that does not alter the activity of the enzymes involved in the synthesis of the target substance as a whole is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.
  • the present invention is SEQ ID NO: 1 to SEQ ID NO: 4; SEQ ID NOs: 11 to 14; And a protein having an amino acid sequence substantially identical to a protein comprising the amino acid sequence of SEQ ID NO: 19 to SEQ ID NO: 24 and variants or active fragments thereof.
  • the substantially identical protein means those having homology of at least 80%, preferably at least 90%, most preferably at least 95% of amino acid sequences, but are not limited thereto, and have homology of at least 80% amino acid sequence and the same. If it has enzyme activity, it is included in the scope of the present invention.
  • the 3-hydroxybutyryl-CoA dehydrogenase which is an enzyme involved in butanol synthesis, is encoded by a gene including the nucleotide sequence of SEQ ID NO: 6, and the 3-hydroxybutyryl-CoA dehydratase is SEQ ID NO: Encoded by a gene comprising the nucleotide sequence of 7, wherein the trans-enoyl-CoA reductase is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 8, and the aldehyde / alcohol dehydrogenase is represented by the base of SEQ ID NO: 9. It may be encoded by a gene including a sequence, but is not limited thereto.
  • the succinyl-CoA synthase an enzyme involved in the synthesis of 1,4-butanediol, is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 15, and the CoA-dependent succinate semialdehyde dehydrogenase is the base of SEQ ID NO: 16 Encoded by a gene comprising a sequence, wherein the 4-hydroxybutyrate dehydrogenase is encoded by a gene comprising a nucleotide sequence of SEQ ID NO: 17, and the 4-hydroxybutyryl-CoA transferase is represented by SEQ ID NO: 18 Encoded by a gene comprising a nucleotide sequence, the aldehyde / alcohol dehydrogenase may be encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.
  • the hydroxymethylglutaryl CoA synthase an enzyme involved in isoprene synthesis, is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 25, and the mevalonate kinase is a gene comprising the nucleotide sequence of SEQ ID NO: 26.
  • Acetyl-CoA acetyltransferase / HMG-CoA reductase is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 27, wherein the phosphomevalonate kinase comprises the nucleotide sequence of SEQ ID NO: 28 Is encoded by a gene comprising the nucleotide sequence of SEQ ID NO: 29, and the isopentyl-diphosphate delta isomerase encodes the nucleotide sequence of SEQ ID NO: 30 It may be encoded by a gene including, but is not limited thereto.
  • Enzymes involved in the synthesis of the target substance of the present invention and genes encoding variants or active fragments thereof are subject to various modifications to the coding region without changing the amino acid sequence of the protein expressed from the coding region.
  • various mutations may be made by substitution, deletion, insertion, or a combination thereof within a range that does not affect the expression of the gene even in a portion except for the coding region, and such a mutation gene is also included in the scope of the present invention.
  • the present invention is SEQ ID NO: 6 to SEQ ID NO: 9; SEQ ID NO: 15 to SEQ ID NO: 18; And a gene consisting of a nucleotide sequence substantially identical to a gene comprising the nucleotide sequence of SEQ ID NO: 25 to SEQ ID NO: 30, and a fragment of the gene.
  • Genes consisting of the same base sequence means those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95%, but are not limited thereto, and at least 80% sequence homology.
  • encoded proteins are included in the present invention if they have the same enzymatic activity.
  • the fibrin-binding domain may be derived from a microorganism of the genus Cellulomonas, and in particular, the amino acid sequence of SEQ ID NO: 5 derived from Cellulomonas fimi is not limited thereto. Any amino acid sequence substantially the same as the amino acid sequence of SEQ ID NO: 5 may be used without limitation.
  • the fibrin binding domain may be encoded by a gene including the nucleotide sequence of SEQ ID NO: 10, but is not limited thereto, and may be used without limitation as long as the nucleotide sequence is substantially the same as the nucleotide sequence of SEQ ID NO.
  • two or more different enzymes involved in the synthesis of the target substance are linked.
  • Two or more different enzymes involved in the synthesis of the fibrin-binding domain and the target substance may be linked through a third mediator.
  • the third mediator may be a leucine zipper.
  • Leucine zippers are heterodimers consisting of CZ and NZ proteins, which can be bound by the electrostatic interaction of glutamic acid and lysine in each protein. have.
  • the leucine zipper is linked by 1) CZ protein fused to two or more different enzymes involved in the synthesis of the target substance and NZ protein fused to the fibrin binding domain to each other (see FIG. 2). Or a state in which the CZ protein and the NZ protein are fused to each other, that is, 2) a NZ protein fused to two or more different enzymes involved in the synthesis of the target substance and a CZ protein fused to the fibrin binding domain. This can be achieved by mutual coupling.
  • the CZ protein comprises the amino acid sequence of SEQ ID NO: 31, is encoded by the nucleotide sequence of SEQ ID NO: 32, and the NZ protein comprises the amino acid sequence of SEQ ID NO: 33, is encoded by the nucleotide sequence of SEQ ID NO: 34, but is not limited thereto. Not.
  • the fibrin binding domain, CZ protein, NZ protein is a variant of the amino acid having a different sequence by deletion, insertion, substitution or combination of amino acid residues within a range that does not affect the function of the protein of each domain Or fragments.
  • Amino acid exchanges at the protein and peptide levels that do not alter the activity of the protein as a whole are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.
  • the present invention includes proteins having an amino acid sequence substantially identical to the protein comprising the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 31 and SEQ ID NO: 33, and variants thereof or active fragments thereof.
  • the substantially identical protein means those having homology of at least 80%, preferably at least 90%, most preferably at least 95% of amino acid sequences, but are not limited thereto, and have homology of at least 80% amino acid sequence and the same. If it has enzyme activity, it is included in the scope of the present invention.
  • the gene encoding the fibrin-binding domain, the leucine zipper proteins, and variants thereof or active fragments thereof of the present invention may be directed to a coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region.
  • Various modifications may be made, and various mutations may be made by substitution, deletion, insertion, or a combination thereof within a range that does not affect the expression of the gene even in a portion except for the coding region, and such a variant gene is also within the scope of the present invention.
  • the present invention includes a gene consisting of the same base sequence as the gene comprising the nucleotide sequences of SEQ ID NO: 10, SEQ ID NO: 32 and SEQ ID NO: 34 and fragments of the gene.
  • Genes consisting of the same base sequence means those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95%, but are not limited thereto, and at least 80% sequence homology.
  • encoded proteins are included in the present invention if they have the same enzymatic activity.
  • the fibrin binding domain to which the enzymes involved in synthesizing the target substance are bound is spontaneously aggregated with each other, the enzymes involved in the synthesis of the target substance bound to the fibrin binding domain can be aggregated and fixed. More specifically, the other ends of the fibrin-binding domain to which the enzymes involved in the synthesis of the target substance are not bound aggregate with each other to form an inclusion body, and the enzymes involved in the synthesis of the target substance have a high density on the surface of the inclusion body thus formed. Can exist in an integrated fashion.
  • the fibrin binding domain serves to bind and fix the enzymes involved in the synthesis of the target substance to the fibrin when fibrin is present.
  • enzymes involved in the synthesis of the target substance and fibrin at the other end of the fibrin binding domain are respectively bound so that the enzymes involved in the synthesis of the target substance are bound and fixed on the fibrin.
  • the other end of the fibrin binding domain may be bound to the fibrin by the fibrin binding property of the domain itself.
  • the fibrin becomes a support of the enzyme complex of the present invention, and all kinds of fibers present in the organism Fibrin. Therefore, the fiber may be derived from an animal or a plant, and in particular, may be cellulose, hemicellulose, lignocellulose, or the like.
  • enzymes sequentially involved in the synthesis of the target substance are densely packed on the encapsulation formed by the fibrin-binding domain or on the fibrin to which the fibrin-binding domain is bound.
  • the resulting intermediate can immediately react with the next enzyme.
  • the enzyme complex of the present invention was applied to the biosynthesis of butanol, representatively, among various target substances, and 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybuty among enzymes involved in butanol biosynthesis
  • An enzyme complex was prepared by linking a reel-CoA dehydratase, a trans-enoyl-CoA reductase, and an aldehyde / alcohol dehydrogenase with a fibrin binding domain through a leucine zipper, and the enzymes such as It was confirmed that it is linked to and expressed in an insoluble state.
  • acetoacetyl-CoA metabolized from glucose on the enzyme complex is 3-hydrated by 3-hydroxybutyryl-CoA dehydrogenase because the enzymes involved in the butanol synthesis are concentrated in the inclusion bodies formed by the fibrin binding domain.
  • the intermediate 3-hydroxybutyryl-CoA can then react directly with the next enzyme, 3-hydroxybutyryl-CoA dehydratase, furthermore 3-hydroxy Crotonyl-CoA, produced by oxybutyryl-CoA dehydratase, is then able to react directly with the next enzyme, trans-enoyl-CoA reductase.
  • Butyryl-CoA produced by trans-enoyl-CoA reductase can directly react with aldehyde / alcohol dehydrogenase, which is also the next enzyme (see FIG. 3). Therefore, the enzyme complex of the present invention is faster than the enzymes involved in the synthesis of butanol, each of which is present in the cytoplasm of the freely produced intermediates to participate in the next enzymatic reaction without delay. Butanol may be produced, and as a result, the yield of butanol may be further improved. In the present invention, the above effects were confirmed through specific examples (see FIGS. 11 to 14b). Based on the results of the above examples, it can be seen that the enzyme complex of the present invention can be sufficiently applied not only to butanol but also to other kinds of target substances biosynthesized by metabolic processes in which a plurality of enzymes work.
  • Transformant comprising recombinant vector of enzymes involved in synthesis of target substance
  • a CZ protein comprising a leucine zipper and a recombinant vector cloned such that the NZ protein constituting the fibrin-binding domain and the leucine zipper are linked to each other are expressed.
  • the CZ protein constituting the fibrin-binding domain and the leucine zipper are linked to each other so that the cloned recombinant vector and two or more different enzymes involved in the synthesis of the target substance are linked to the NZ protein constituting the leucine zipper.
  • the cloned recombinant vector provides a transformant for the synthesis of the target material introduced into the host cell.
  • vector a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host cell, said vector being a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector. And the like, and the like.
  • the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.
  • a plasmid vector may be used, wherein the plasmid vector is transformed into (a) a replication initiation point so that replication is efficiently carried out to include several hundred plasmid vectors per host cell, and (b) a plasmid vector.
  • It has a structure comprising a selection marker gene that allows a host cell to be selected and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.
  • the gene of the enzyme involved in the synthesis of the target substance and the gene of the fibrin binding domain can be cloned in the restriction enzyme cleavage site as described above, the gene of the enzyme involved in the synthesis of the target substance and the gene of the fibrin binding domain, respectively It is cloned into a state that can be expressed by fusion with one of the genes of the CZ protein or NZ protein constituting the leucine zipper.
  • fusion protein expressed by the gene of the enzymes involved in the synthesis of the target substance and the gene of the CZ protein constituting the leucine zipper fusion protein design for each enzyme
  • a fusion protein expressed by the gene of the NZ protein constituting the leucine zipper with the gene of the fibrin-binding domain, and a gene construct of the fusion proteins may be prepared first and then inserted into the vector. May be separately inserted into the vector sequentially so that the two genes are fused to each other in the vector.
  • a fusion protein expressed by a gene of NZ proteins constituting a leucine zipper with a gene of enzymes involved in the synthesis of the target substance (design of a fusion protein for each enzyme);
  • a fusion protein expressed by the gene of the CZ protein constituting the leucine zipper with the gene of the fibrin binding domain, and a gene construct of the fusion proteins may be prepared first and then inserted into the vector. May be separately inserted into the vector sequentially so that the two genes are fused to each other in the vector.
  • a recombinant vector pET21a-NZ :: CBD was prepared by inserting a gene encoding a fusion protein of a fibrin binding domain and an NZ protein into a pET21a vector (see FIG. 6A), butanol. Genes encoding individual enzymes involved in synthesis and proteins fused with CZ proteins (ie, CZ: HBD fusion protein, CZ: CRT fusion protein, CZ :: TER fusion protein and CZ :: AdhE2 fusion protein) Inserted into the recombinant vector pACBB-CZ ::: HCTA (see FIGS. 8A and 8B).
  • gene constructs encoding proteins fused individually to the CZ protein constituting the leucine zipper; and genes encoding the fusion protein of the fibrin binding domain and the NZ protein constituting the leucine zipper.
  • Constructs may be separately inserted into different vectors, or two or more gene constructs may be inserted together in a vector.
  • Constructs may be separately inserted into different vectors, or two or more gene constructs may be inserted together in a vector.
  • a fusion protein in which a gene construct of an NZ :: CBD fusion protein in which an NZ protein and a fibrin binding domain are fused is prepared is inserted, and individual enzymes involved in NZ protein and butanol synthesis are fused.
  • a vector was constructed in which each of the gene constructs expressing (CZ: HBD fusion protein, CZ: CRT fusion protein, CZ :: TER fusion protein and CZ :: AdhE2 fusion protein) was inserted into one vector (Example 1).
  • the recombinant vector may include a sequence for facilitating purification of the expressed protein, and specifically, a gene encoding a tag for separation and purification may be linked to be operable to a gene encoding an enzyme involved in synthesis of the target substance.
  • the separation and purification tag may be used alone, or GST, poly-Arg, FLAG, histidine-tag (His-tag) and c-myc, or two or more of them may be sequentially connected.
  • an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen).
  • a pET family vector Novagen
  • a transformant may be prepared by transforming the recombinant expression vector according to the present invention into any one suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells and animal cells according to expression purposes.
  • the host cell may be Escherichia coli ( E. coli BL21 (DE3), DH5 ⁇ , etc.) or yeast cells ( Saccharomyces , Pichia , etc.) and the like.
  • appropriate culture methods, media conditions and the like can be easily selected by those skilled in the art according to the type of host cell.
  • the recombinant expression vector was used as a host cell Escherichia coli MG1655 strain ( ⁇ fed ⁇ ldhA ⁇ adhE ⁇ pta) in which the metabolic circuit to acetyl-CoA was controlled (see Example 2).
  • a known technique that is, a heat shock method, an electric shock method, or the like may be used.
  • Enzyme complex for the synthesis of the target compounds of the present invention wherein the said target substance in the prepared transformant as described in "2.
  • the transformant containing the recombinant vector of the enzymes relating to the synthesis of a target substance” topic Two or more different enzymes and fibrin binding domains that are involved in the synthesis of each of the expression can be prepared in vivo ( in vivo ) by being linked to each other.
  • the enzymes involved in the synthesis of the target substance and the fibrin binding domains are respectively expressed in the transformant, and then linked to each other, the other ends of the fibrin binding domains among these linked proteins spontaneously aggregate to form inclusion bodies on the surface of the inclusion body.
  • Two or more enzymes involved in the synthesis of the target substance on the surface of the fiber by combining two or more enzymes involved in the synthesis of the target substance, or by binding the other end of the fibrin binding domain in the linked protein to the fibers present in the transformant. Can be integrated. Through this process, the protein linked in the transformant is aggregated through the fibrin binding domain so that an enzyme complex may be naturally generated in the transformant.
  • the enzyme complex is generated in the in vivo environment in the transformant as described above, the in vivo environment of the transformant is used as it is for the generation of a target substance, or the transformation
  • the enzyme complex may be separated from the sieve and then used to generate a target substance in an in vitro environment using the separated enzyme complex.
  • the enzyme complex for the synthesis of the target compounds of the present invention in a transformant prepared as described in "2.
  • the transformant containing the recombinant vector of the enzymes relating to the synthesis of a target substance” topic Expresses, isolates, and purifies proteins linked to two or more different enzymes and fibrin binding domains involved in the synthesis of a substance, expresses, isolates, purifies, and links them, respectively, and connects these linked proteins in vitro It can also be prepared in vitro by inducing spontaneous aggregation of binding domains or by binding to fibrin.
  • the enzyme complex prepared as described above can be used for production of a target substance in an in vitro environment.
  • Another aspect of the present invention is a method for producing a target substance in an in vivo environment or in vitro environment using the enzyme complex described in " 1. Enzyme complex for synthesis of the target substance ". To provide.
  • the precursor of the target material may correspond to all intermediate products of the synthesis route of the target material, more specifically, when the target material is butanol, the precursor is glucose, pyruvic acid, acetyl-CoA, acetoacetyl-CoA, 3 At least one selected from the group consisting of -hydroxybutyryl-CoA, crotonyl-CoA, butyryl-CoA, and butyrylaldehyde.
  • the precursor when the target substance is 1,4-butanediol, the precursor is composed of succinic acid, succinyl CoA, succinyl semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl CoA and 4-hydroxybutyraldehyde It may be at least one selected from the group.
  • the precursor when the target substance is isoprene, the precursor is selected from the group consisting of glucose, pyruvic acid, acetyl-CoA, acetoacetyl-CoA, HMG-CoA, mevalonate, phosphomevalonate and diphosphosmevalonate There may be at least one.
  • the precursor when the target substance is succinic acid, the precursor may be at least one selected from the group consisting of glucose, phosphoenolpyruvate, oxaloacetate, maleate and fumarate.
  • the target material when the target material is ⁇ -caprolactam, the precursor may be at least one selected from the group consisting of glucose, pyruvic acid, succinic acid semialdehyde, adipic acid semialdehyde, and 6-aminocaproic acid.
  • Cultivation of such a transformant can be made according to suitable media and culture conditions known in the art. Those skilled in the art can easily use the medium and culture conditions according to the type of host cell of the transformant to be selected. Culture methods can include batch, continuous, fed-batch, or combination cultures thereof.
  • the medium may comprise various carbon sources, nitrogen sources and trace element components.
  • the carbon source may be, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof.
  • the culturing can be performed using glucose as a carbon source.
  • the nitrogen source may be organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and soybean wheat, inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or Combinations thereof.
  • the medium may comprise, as a source of phosphorus, metal salts such as, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts, magnesium sulfate or iron sulfate.
  • amino acids may be included in the medium.
  • the medium or individual components may be added batchwise or continuously to the culture.
  • anti-foaming agents such as fatty acid polyglycol esters can be used during the culture to suppress bubble generation.
  • Cultivation of the transformant as described above may be carried out at 20 ° C to 60 ° C, preferably 25 ° C to 55 ° C, more preferably 30 ° C to 50 ° C.
  • cultivation of the transformant is carried out at a temperature range of less than 20 °C or more than 60 °C does not produce a sufficient amount of intermediate products, despite the formation of the enzyme complex of enzymes involved in the synthesis of the target material, the final product A problem arises that the production amount of phosphorus target substance or not becomes insufficient.
  • the transformant may be cultured in aerobic or anaerobic conditions. Aerobic conditions may be cultured by opening the lid of the culture vessel during the culture process. Anaerobic conditions can be formed, for example, by supplying carbon dioxide or nitrogen at a flow rate of about 0.1 to 0.4 vvm, about 0.2 to 0.3 vvm, or about 0.25 vvm.
  • the culture temperature may be, for example, 20 ° C to 60 ° C or 35 ° C to 55 ° C. The incubation period can last until the desired material is obtained as desired.
  • the culture time in the aerobic condition may be 1 hour to 40 hours, preferably 2 hours to 35 hours, more preferably 3 hours to 30 hours, but is not limited thereto.
  • the culture time in the anaerobic conditions may be 20 hours to 200 hours, preferably 30 hours to 190 hours, more preferably 40 hours to 180 hours, but is not limited thereto. If the incubation time is less than the lower limit, the total amount of microorganisms is not sufficiently increased to synthesize a target substance from the precursor, and thus, the target substance is produced less. If the incubation time exceeds the upper limit, waste and metabolites increase as the growth of the microorganism increases. This accumulation accumulates and the production of the target substance decreases due to pH change.
  • E. coli prepared so that the genes of each of the individual enzymes involved in butanol biosynthesis is expressed and present in a free state as a control, the genes of the enzymes involved in butanol biosynthesis and fibrin binding domain gene E. coli was prepared to form an enzyme complex by being expressed separately and then linked to each other by a leucine zipper, and the two groups of E. coli were respectively aerobic (24 hours culture) or anaerobic condition (168 hours culture) at 37 ° C. When cultured, it was confirmed that butanol production is significantly improved in the experimental group in which the enzyme complex is formed (see FIGS. 12A to 13B).
  • the culturing of the transformant as described above may be performed at pH 4.3 to pH 9.5, preferably at pH 5.0 to pH 9.0, more preferably at pH 6.0 to pH 8.0, but is not limited thereto.
  • the culture pH conditions of the transformant can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium of the transformant.
  • Butanol synthesizing enzymes and fibrin binding domains are not easy to be expressed and linked because the transformants are difficult to grow when the pH condition of the transformants is out of the above range, and these expressed proteins are linked to the enzyme complex. Even if is formed there is a problem that the synthesis of the target material is not made efficiently.
  • the process according to the invention aims at producing the target substance with high efficiency, but intermediate products produced in the process for producing the target substance, for example 3-hydroxybutyryl-CoA, croissant when the target substance is butanol It can also be used for the high efficiency production of tonyl-CoA, butyryl-CoA, butyrylaldehyde.
  • succinyl CoA, succinyl semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl CoA and 4-hydroxybutyric acid It can also be used for high-efficiency production of intermediates such as tyraldehyde.
  • the method according to the present invention can be applied to high efficiency production of intermediate products such as HMG-CoA, mevalonate, phosphomevalonate, diphosphosmevalonate.
  • the target material is succinic acid
  • it can be used for high efficiency production of intermediate products such as oxaloacetate, maleate, and fumarate by applying the method according to the present invention.
  • the target material is ⁇ -caprolactam
  • the method according to the present invention may be applied to high-efficiency production of intermediate products such as adipic acid semialdehyde and 6-aminocaproic acid.
  • butyryl aldehyde in order to produce butyryl aldehyde with high efficiency, it can be achieved by adjusting the incubation time of the transformant as described above to recover butyryl aldehyde before the butanol is produced.
  • the intermediate product can be recovered by adjusting the incubation time to obtain intermediate products produced in the target material manufacturing process.
  • the enzyme complex for the synthesis of a target substance item with the precursor of the target substance, so as to produce a target substance.
  • the precursor of the target substance may correspond to all intermediate products of the synthetic route of the target substance, and more specifically, when the target substance is butanol, glucose, pyruvic acid, acetyl-CoA, acetoacetyl-CoA, 3-hydroxy Butyryl-CoA, crotonyl-CoA, butyryl-CoA, butyrylaldehyde may be at least one selected from the group consisting of.
  • the precursor when the target substance is 1,4-butanediol, the precursor is a group consisting of succinic acid, succinyl CoA, succinyl semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl CoA, and 4-hydroxybutyraldehyde It may be at least one selected from. Further, when the target substance is isoprene, the precursor is at least selected from the group consisting of glucose, pyruvic acid, acetyl-CoA, acetoacetyl-CoA, HMG-CoA, mevalonate, phosphomevalonate and diphosphosmevalonate It can be one.
  • the precursor when the target substance is succinic acid, the precursor may be at least one selected from the group consisting of glucose, phosphoenolpyruvate, oxaloacetate, maleate and fumarate.
  • the target material when the target material is ⁇ -caprolactam, the precursor may be at least one selected from the group consisting of glucose, pyruvic acid, succinic acid semialdehyde, adipic acid semialdehyde, and 6-aminocaproic acid.
  • the reaction of the enzyme complex and the precursor of the target material as described above may be carried out at 30 °C to 60 °C, preferably 40 °C to 57 °C, more preferably 45 °C to 55 °C, at pH 4.3 to pH 9.5, Preferably, the pH may be performed at pH 5.0 to pH 9.0, more preferably at pH 6.0 to pH 8.0, but is not limited thereto.
  • the production method of the target substance in the in vitro environment is also aimed at producing the target substance with high efficiency, but by controlling the type of enzyme constituting the enzyme complex, for example, Butanol may also be used to efficiently produce intermediates produced in the butanol manufacturing process such as 3-hydroxybutyryl-CoA, crotonyl-CoA, butyryl-CoA, butyrylaldehyde and the like.
  • the enzyme complex is composed of only 3-hydroxybutyryl-CoA dehydratase and trans-enoyl-CoA reductase. Can be achieved by reacting with 3-hydroxybutyryl-CoA.
  • 1,4-butanediol such as succinyl CoA, succinyl semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl CoA, 4-hydroxybutyraldehyde and the like It can be used to produce high efficiency intermediate products produced in the process.
  • succinic acid when succinic acid is used as a starting material to produce 4-hydroxybutyryl CoA with high efficiency, succinyl-CoA synthase, CoA-dependent succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4 It can be achieved by constructing the enzyme complex only with -hydroxybutyryl-CoA transferase and reacting it with succinic acid.
  • the target material when the target material is isoprene, it can be used to efficiently produce intermediate products produced in the isoprene manufacturing process such as HMG-CoA, mevalonate, phosphomevalonate and diphosphomevalonate.
  • intermediate products produced in the isoprene manufacturing process such as HMG-CoA, mevalonate, phosphomevalonate and diphosphomevalonate.
  • the intermediate product may be recovered by adjusting the incubation time to obtain intermediate products produced in the target material manufacturing process.
  • the target substance produced in an in vivo or in vitro environment may be recovered using separation and purification methods known in the art.
  • the recovery may be by centrifugation, ion exchange chromatography, filtration, precipitation, or a combination thereof.
  • HBD 3-hydroxybutyryl-CoA dehydrogenase
  • Clostridium acetobutylicum and 3-hydroxybutyryl- are enzymes involved in butanol synthesis.
  • TER trans-enoyl-CoA reductase
  • CBD fibrin binding domain
  • CZ protein an antiparallel leucine zipper composed of two stranded heterologous proteins [CZ protein, NZ protein] was used to connect the enzymes involved in the butanol synthesis and the fibrin binding domain.
  • CBD Cellulose monas Pimi ( Cellulomonas fimi Genomic DNA was extracted from the CBD sequence to obtain the antiparallel leucine zipper (NZ protein, CZ protein) is based on the NCBI sequence information NZ :: eGFP (SEQ ID NO: 59 amino acid sequence, SEQ ID NO: 60) ) And CZ :: eGFP gene (amino acid sequence of SEQ ID NO: 61, nucleotide sequence of SEQ ID NO: 62) were synthesized and used.
  • NZ CBD cellulose to Monastir Pimi (Cellulomonas fimi ) genomic DNA as a template
  • PCR using the conditions (1) of Table 2 using primers GS_CBD_F (SEQ ID NO: 37) and CBD_R (SEQ ID NO: 38) to include the nucleotide sequence of SEQ ID NO: 10
  • the gene of fibrin binding domain (CBD) was amplified.
  • PACBB-NZ :: eGFP was used as a template to obtain the NZ sequence, and PCR was carried out using primers NZ_F (SEQ ID NO: 35) and NZ_GS_R (SEQ ID NO: 36) using the conditions (2) in Table 2 below.
  • the PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the CBD sequence and the NZ sequence obtained by PCR so that the molar ratio is 1: 1, PCR was performed using the conditions (3) of Table 2 without the primer. PCR was performed under the conditions (4) of Table 2 by adding primers NZ_F (SEQ ID NO: 35) and CBD_R (SEQ ID NO: 38) to the PCR reaction solution.
  • the gene of fibrin binding domain (CBD) was amplified.
  • pACBB-CZ ::: eGFP was used as a template, and PCR was performed using primers CZ_F (SEQ ID NO: 39) and CZ_GS_R (SEQ ID NO: 40) using the conditions (2) in Table 3 below.
  • the PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the CBD sequence and the CZ sequence obtained by PCR in a molar ratio of 1: 1, PCR was performed using the conditions (3) of Table 3 below without adding a primer. PCR was carried out under the condition (4) of Table 3 by adding primers CZ_F (SEQ ID NO: 39) and CBD_R (SEQ ID NO: 38) to the PCR reaction solution.
  • Clostridium Aceto Beauty Ricum DSM 1731 was used to synthesize the gene (hbd), gene (crt) of the CRT of the HBD based on the sequence information of NCBI of (Clostridium acetobutylicum DSM 1731, Accession no . CP002660), Clostridium Aceto Beauty Ricum Based on the NCBI sequence information of DSM 1731 ( Clostridium acetobutylicum DSM 1731, Accession no. CP002661), gene codons were optimized to synthesize ADHE2 gene (adhE2). Triponema Denticola ( Treponema) denticola ATCC 35405, Accession no. AE017226) optimizes gene codons based on the sequence information of NCBI to synthesize TER genes (ter).
  • Primer pairs of Hbd_F and Hbd_R (SEQ ID NOs: 41 and 42) and primer pairs of Crt_F and Crt_R (SEQ ID NO: 43, respectively) using hbd, crt, ter, and adhE2 synthesized in Example 1-3-1 as templates. 44), PCR was performed with primer pairs of ter_F and ter_R (SEQ ID NOs: 45 and 46) and primer pairs of adhE2_F and adhE2_R (SEQ ID NOs: 47 and 48), respectively, to amplify hbd, crt, ter and adhE2 genes, respectively. .
  • pACBB-hbd was treated with EcoRI, SpeI
  • pACBB-crt was treated with EcoRI, xbaI
  • each treatment was gel-extracted and then ligated using T4 ligase to prepare pACBB-hbd-crt. Screened.
  • the prepared pACBB-hbd-crt was treated with EcoRI and SpeI
  • the pACBB-ter was treated with EcoRI and xbaI.
  • the pACBB-hbd-crt-ter was selected after ligation using T4 ligase. .
  • the prepared pACBB-hbd-crt-ter was treated with EcoRI, SpeI, and the pACBB-adhE2 was treated with EcoRI, xbaI, and each treatment was gel-extracted and then screened after ligation using T4 ligase.
  • the completed pACBB-hbd-crt-ter-adhE2 vector (hereinafter referred to as 'pACBB-HCTA') is shown in FIGS. 7A and 7B.
  • PACBB-CZ eGFP was treated with BamHI (NEB) and XhoI (NEB) for cloning CZ :: hbd.
  • pACBB-hbd plasmid prepared in Example 1-3-2 synthesized to obtain hbd sequence was used as a template, and GS_Hbd_F (SEQ ID NO: 51) and Hbd_R (SEQ ID NO: 42) were used as shown in Table 5 below. PCR was carried out using the conditions.
  • pACBB-CZ :: GFP gene was used as a template, and PCR was performed using CZ_F (SEQ ID NO: 50) and GS_Hbd_R (SEQ ID NO: 52) using the conditions (2) of Table 5 below.
  • the PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the hbd sequence and the CZ sequence obtained by PCR in a molar ratio of 1: 1, PCR was performed using the conditions (3) of Table 5 below without adding a primer.
  • Primer CZ_F (SEQ ID NO: 50) and Hbd_R (SEQ ID NO: 42) were added to the PCR reaction solution, and PCR was performed under the conditions (4) of Table 5 below.
  • BamHI and XhoI were treated with the product which was subjected to two PCRs.
  • the treated solution and the previously prepared pACBB treated with BamHI and XhoI were gel extracted, and then ligated using T4 ligase. Only the ligation was selected and named as pACBB-CZ :: hbd, and was used to prepare a recombinant vector in which enzymes involved in butanol synthesis were linked to and expressed in leucine zipper proteins.
  • PACBB-CZ eGFP was treated with BamHI (NEB) and XhoI (NEB) for cloning CZ :: crt.
  • the pACBB-crt plasmid prepared in Example 1-3-2 synthesized to obtain a crt sequence was used as a template, and GS_Crt_F (SEQ ID NO: 53) and Crt_R (SEQ ID NO: 44) were used as shown in Table 6 below. PCR was carried out using the conditions.
  • pACBB-CZ GFP gene was used as a template, and PCR was performed using CZ_F (SEQ ID NO: 50) and GS_Crt_R (SEQ ID NO: 54) using the conditions (2) of Table 6 below.
  • the PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the crt sequence and the CZ sequence obtained by PCR so that the molar ratio is 1: 1, PCR was performed using the conditions (3) of Table 6 without the primer. Primer CZ_F (SEQ ID NO: 50) and Crt_R (SEQ ID NO: 44) were added to the PCR reaction solution, and PCR was performed under the conditions (4) of Table 6 below.
  • BamHI and XhoI were treated with the product which was subjected to two PCRs.
  • the treated solution and the previously prepared pACBB treated with BamHI and XhoI were gel extracted, and then ligated using T4 ligase. Only the ligation was selected and named as pACBB-CZ :: crt, and was used in the preparation of a recombinant vector in which enzymes involved in butanol synthesis were linked to and expressed in leucine zipper proteins.
  • PACBB-CZ :: eGFP was treated with BamHI (NEB) and XhoI (NEB) for cloning CZ :: ter.
  • pACBB-ter plasmid prepared in Example 1-3-2 synthesized to obtain a ter sequence as a template, and using GS_Ter_F (SEQ ID NO: 55) and Ter_R (SEQ ID NO: 46) in Table 7 (1) PCR was carried out using the conditions.
  • pACBB-CZ :: GFP gene was used as a template, and PCR was performed using CZ_F (SEQ ID NO: 50) and GS_Ter_R (SEQ ID NO: 56) using the conditions (2) of Table 7 below.
  • the PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the ter sequence and the CZ sequence obtained by PCR in a molar ratio of 1: 1, PCR was carried out using the conditions (3) of Table 7 without the primer. PCR was performed under the condition (4) of Table 7 by adding primers CZ_F (SEQ ID NO: 50) and Ter_R (SEQ ID NO: 46) to the PCR reaction solution.
  • BamHI and XhoI were treated with the product which was subjected to two PCRs.
  • the treated solution and the previously prepared pACBB treated with BamHI and XhoI were gel extracted, and then ligated using T4 ligase. Only the ligation was selected and named as pACBB-CZ :: ter, and was used in the preparation of a recombinant vector in which enzymes involved in butanol synthesis were linked to and expressed in a leucine zipper protein.
  • PACBB-CZ eGFP was treated with BamHI (NEB) and XhoI (NEB) for cloning CZ :: adhE2.
  • the pACBB-adhE2 plasmid prepared in Example 1-3-2 synthesized to obtain an adhE2 sequence was used as a template, and GS_AdhE2_F (SEQ ID NO: 57) and AdhE2_R (SEQ ID NO: 48) were used as shown in Table 8 below. PCR was carried out using the conditions.
  • pACBB-CZ GFP gene was used as a template, and PCR was performed using CZ_F (SEQ ID NO: 50) and GS_AdhE2_R (SEQ ID NO: 58) using the conditions (2) of Table 8 below.
  • PCR result obtained above was subjected to overlap PCR as a template. Specifically, after mixing the adhE2 sequence and the CZ sequence obtained by PCR in a molar ratio of 1: 1, PCR was performed using the conditions (3) of Table 8 below without adding a primer. PCR was carried out under the condition (4) of Table 8 by adding primers CZ_F (SEQ ID NO: 50) and AdhE2_R (SEQ ID NO: 48) to the PCR reaction solution.
  • BamHI and XhoI were treated with the product which was subjected to two PCRs.
  • the treated solution and the previously prepared pACBB treated with BamHI and XhoI were gel extracted, and then ligated using T4 ligase. Only the ligation was selected and named as pACBB-CZ :: adhE2, and used in the preparation of a recombinant vector in which enzymes involved in butanol synthesis were linked to the leucine zipper protein.
  • pACBB-CZ :: hbd was treated with EcoRI, SpeI
  • pACBB-CZ :: crt was treated with EcoRI, xbaI
  • each treatment was gel-extracted and then ligated with T4 ligase and then pACBB-CZ :: hbd-CZ :: crt was selected.
  • the prepared pACBB-CZ :: hbd-CZ :: crt was treated with EcoRI and SpeI
  • the pACBB-CZ :: ter was treated with EcoRI and XbaI.
  • pACBB -CZ :: hbd-CZ :: crt-CZ :: ter was selected.
  • the prepared pACBB-CZ :: hbd-CZ :: crt-CZ :: ter was treated with EcoRI and SpeI, and the pACBB-CZ :: adhE2 was treated with EcoRI and xbaI, and each treated product was gel-extracted using T4 ligase. Screening after ligation.
  • 'pACBB-HCTA' The completed pACBB-CZ :: hbd-CZ :: crt-CZ :: ter-CZ :: adhE2 vector (hereinafter referred to as 'pACBB-HCTA') is shown in Figs. 8A and 8B.
  • pET21a - NZ CBD, pACBB - HCTA (Control), pACBB -CZ :: HCTA (experimental group) Expression and Purification
  • PET21a-NZ CBD prepared in Example 1-1 and pACBB-CZ :: HCTA prepared in Example 1-4-5, E. coli MG1655 strain ( ⁇ fed ⁇ ldhA in which the metabolic circuit to acetyl-CoA was controlled) ⁇ adhE ⁇ pta) to prepare an experimental group in which enzymes involved in butanol biosynthesis by binding of CZ protein and NZ protein are bound to the fibrin binding domain and the fibrin binding domain aggregates with each other to form an enzyme complex.
  • pET21a-NZ CBD prepared in Example 1-1 and pACBB-HCTA prepared in Examples 1-3-3 were also transformed with the MG1655 strain ( ⁇ fed ⁇ ldhA ⁇ adhE ⁇ pta), and CZ protein and NZ protein. Was unable to form an enzyme complex, so that a control group was prepared in which individual enzymes existed in a free state.
  • the transformants of the experimental group and the control group were inoculated in LB medium containing 50 ⁇ g / ml of empicillin and chloramphenicol 34 mg / ml, respectively, and pre-incubated at 37 ° C. and 200 rpm for 16 hours, and then the culture medium of 1% glucose was used.
  • the culture medium of the overexpressed transformant was centrifuged at 6,000 ⁇ g for 30 minutes at 4 ° C., and pH 7.4 10 mM PBS buffer solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH) was used. 2 PO 4 ), followed by adding 0.1 mM of phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, to disrupt the cell solution with a sonicator.
  • PMSF phenylmethylsulfonyl fluoride
  • the cell lysate was again centrifuged at 13000 ⁇ g for 20 minutes at 4 ° C., and the proteins expressed in the cell supernatant were separated into soluble fractions and insoluble fractions expressed in pellets, and proteins (CRT, HBD, TER, AdhE2, NZ :: CBD and CZ :: CRT binding protein, NZ :: CBD and CZ :: HBD binding protein, NZ :: CBD and CZ ::: TER binding protein, NZ ::: CBD and CZ :: AdhE2 binding protein).
  • fibrin binding domain (16kda) was found in both insoluble protein layers in both experimental and control groups.
  • HBD (30kda), CRT (28kda), TER (44kda) and AdhE2 (94kda) were not found in SDS-PAGE of the insoluble protein of the control group, but CZ: : HBD (32kda), CZ :: CRT (30kda), CZ :: TER (46kda) and CZ :: AdhE2 (96kda) were all identified (FIG. 9).
  • enzymes involved in biosynthesis of a target substance are linked to cellulose binding domains through leucine zippers, respectively.
  • the present inventors confirmed whether the enzymes are linked to the fibrin binding domain through the leucine zipper, or whether the enzymes and the fibrin binding domains show the enzyme activity even if they are linked through the leucine zipper. Since fibrin-binding domains form insoluble protein constructs in E. coli, the cell lysates are divided into soluble fractions and insoluble fractions, respectively, and then the enzyme activity of the insoluble fractions is confirmed. It can be confirmed (Fig. 10a).
  • Example 2 After culturing the control and experimental group transformants of Example 2, the cultured cells were lysed, and each lysate was partitioned into soluble and insoluble fractions to confirm the activity of enzymes or enzyme complexes contained in each fraction.
  • E. coli was secured by centrifugation after incubating the control and experimental group transformants of Example 2 for 24 hours in LB medium.
  • the cultured control group and the experimental group Escherichia coli were disrupted and then partitioned into soluble and insoluble so that the enzyme activity of the control group (enzyme present in the free state) and the experimental group (enzyme complexes linked to fibrin-binding domain through leucine zipper) It was confirmed.
  • acetoacetyl-CoA (Acetoacetyl-CoA) 0.1 mM, NADH 0.1mM containing MOPS buffer (pH 7.2) was prepared as the enzyme reaction solution. 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the control group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at a spectrophotometer 340 nm at 10 minute intervals to measure the amount of NADH reduction. The enzyme activity of HBD was measured to compare the relative enzyme activity of the soluble fraction and the insoluble fraction.
  • the enzyme activities of soluble fraction and insoluble fraction were 0.403 umol / mg / min and 0.124 umol / mg / min, respectively, and their relative enzyme activities were 76.4% in soluble fraction and 23.6% in insoluble fraction.
  • MOPS buffer containing pH 7.2 of acetoacetyl-CoA and 0.1 mM of NADH was prepared as an enzyme reaction solution. 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the experimental group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at the spectrophotometer 340 nm at 10 minute intervals to measure the NADH reduction amount.
  • the enzyme activity of CZ :: HBD was measured to compare the relative enzyme activity of the soluble and insoluble fractions.
  • MOPS buffer solution containing 3-hydroxybutyryl-CoA (0.1 mM, NADH 0.1 mM) (pH 7.2) was prepared as an enzyme reaction solution.
  • 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the control group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at a spectrophotometer 340 nm at 10 minute intervals to measure the amount of NADH reduction.
  • the enzyme activity of the CRT was measured to compare the relative enzyme activity of the soluble fraction and the insoluble fraction.
  • CZ In order to measure the enzyme activity of CZ :: CRT, MOPS buffer (pH 7.2) containing 3-hydroxybutyryl-CoA 0.1 mM and NADH 0.1 mM was prepared as an enzyme reaction solution. 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the experimental group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at the spectrophotometer 340 nm at 10 minute intervals to measure the NADH reduction amount. The enzyme activity of CZ :: CRT was measured to compare the relative enzyme activity of the soluble and insoluble fractions.
  • MOPS buffer pH 7.2
  • 0.1 mM of crotonyl-CoA 0.1 mM of NADH was prepared as an enzyme reaction solution.
  • 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the control group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at a spectrophotometer 340 nm at 10 minute intervals to measure the amount of NADH reduction.
  • the enzyme activity of TER was measured to compare the relative enzyme activity of soluble and insoluble fractions.
  • the enzyme activities of soluble fraction and insoluble fraction were 0.192 umol / mg / min and 0.212 umol / mg / min, respectively, and their relative enzyme activity was 47.5% in soluble fraction and 52.5% in insoluble fraction.
  • CZ :: TER MOPS buffer containing pH 7.2 of crotonyl-CoA and 0.1 mM of NADH was prepared as an enzyme reaction solution. 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the experimental group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at the spectrophotometer 340 nm at 10 minute intervals to measure the NADH reduction amount. The enzyme activity of CZ :: TER was measured to compare the relative enzyme activity of the soluble fraction and the insoluble fraction.
  • the enzyme activity of the TER was determined to be higher than the insoluble fraction in the soluble fraction, and in the experimental group (enzyme complex), the activity of each enzyme was measured to be higher than the soluble fraction in the insoluble fraction (FIG. 10B).
  • MOPS buffer solution pH 7.2
  • MOPS buffer solution pH 7.2
  • 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the control group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at a spectrophotometer 340 nm at 10 minute intervals to measure the amount of NADH reduction.
  • the enzyme activity of AdhE2 was measured to compare the relative enzyme activities of the soluble and insoluble fractions.
  • the enzyme activities of the soluble fraction and the insoluble fraction were 0.077 umol / mg / min and 0.026 umol / mg / min, respectively. Their relative enzyme activities were 74.7% in the soluble fraction and 25.3% in the insoluble fraction.
  • CZ :: AdhE2 MOPS buffer (pH 7.2) containing butyryl-CoA 0.1 mM, NADH 0.1 mM, and DTT 1 mM was prepared as an enzyme reaction solution. 25 ⁇ l of the soluble fraction and 25 ⁇ l of the insoluble fraction of the experimental group obtained above were added to each enzyme reaction solution, and the absorption spectrum was measured for 90 minutes at the spectrophotometer 340 nm at 10 minute intervals to measure the NADH reduction amount. The enzyme activity of CZ :: AdhE2 was measured to compare the relative enzyme activities of the soluble and insoluble fractions.
  • the activity of each enzyme in the insoluble fraction of the experimental group was measured to be associated with the fibrin-binding domain by leucine zipper and precipitated together with the fibrin-binding domain during centrifugation, and enzymes involved in butanol biosynthesis. It can be seen that the activity of enzymes involved in butanol biosynthesis is maintained even when the fibrin-binding domain is linked with a leucine zipper.
  • the degree of enzymatic activity varies depending on the enzymes involved in butanol synthesis and the fibrin-binding domains linked by leucine zippers, especially in the case of HBD, CRT, and TER, rather than free enzymes. It was found that the enzyme activity increased when.
  • a conversion reaction solution containing acetoacetyl-CoA 20 mM, NADH 50 mM, and DDT 1 mM was prepared in a MOPS buffer of pH 7.4.
  • 50 ⁇ l of the free enzyme of the control group and 50 ⁇ l of the enzyme complex of the experimental group prepared in 150 ⁇ l of the reaction solution were added to the reaction solution, respectively, and the total amount was 200 ⁇ l.
  • 50 ⁇ l of the sample was taken from the reaction solution every 3 hours or 6 hours, centrifuged at 12000 rpm for 5 minutes, the supernatant was taken through a 0.21 ⁇ m filter, and the insoluble protein and impurities were removed for the butanol analysis.
  • Butanol was measured using Agilent's gas chromatography 7890B model and flame ion detector (FID), and analytical column Agilent's DB-WAX capillary column (30 m, 0.32 mm i.d., 0.50 um film thickness) was used. 1 ⁇ l of the sample prepared above was injected in splitless mode (15 ml / min at 0.75 min). The gas chromatography oven temperature was allowed to stand at 60 ° C. for 4 minutes and then raised to 120 ° C. at 15 ° C. per minute. Thereafter, the temperature was raised to 230 ° C. at 50 ° C. per minute. The carrier gas was helium and the gas inlet pressure was maintained at 9.3 psi. The gas inlet temperature of the gas chromatography was maintained at 250 ° C and the detector at 300 ° C.
  • the transformants were inoculated into LB medium containing 50 ⁇ g / ml of empicillin and 34 mg / ml of chloramphenicol, and pre-incubated at 37 ° C. and 200 rpm for 16 hours, and then, LB medium containing 2% glucose as the main culture medium.
  • LB medium containing 2% glucose was inoculated at 1% (v / v).
  • Incubation was performed by injecting air at a rate of 0.5vvm at 37 ° C and 200rpm. After the culture, the absorbance value (OD600) was 0.4, and 0.1 mM IPTG was added thereto, followed by incubation for 24 hours in aerobic conditions to induce the expression of butanol synthase HBD, CRT, TER, ADHE2.
  • the transformants were inoculated into LB medium containing 50 ⁇ g / ml of empicillin and 34 mg / ml of chloramphenicol, and pre-incubated at 37 ° C. and 200 rpm for 16 hours, and then, LB medium containing 2% glucose as the main culture medium.
  • LB medium containing 2% glucose as the main culture medium.
  • OD600 absorbance value
  • 0.1mM IPTG was added and 0.5vvm of air was injected for 12 hours, followed by incubation under aerobic conditions, to produce butanol synthase HBD, CRT, Expression of TER, ADHE2 was induced.
  • Example 4-2 and Example 4-3 From the results of Example 4-2 and Example 4-3, when the enzymes involved in butanol synthesis are aggregated with each other through the fibrin binding domain to form an enzyme complex, at least about 30% in both aerobic and anaerobic cultures. It can be seen that butanol production efficiency is significantly increased.

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Abstract

La présente invention concerne un composite enzymatique d'enzymes synthétiques impliquées dans la synthèse d'un matériau cible et un procédé de fabrication d'un matériau cible l'utilisant. L'invention concerne un composite enzymatique qui contient des domaines de liaison de fibres et deux types ou plus de différentes enzymes impliquées dans la synthèse d'un matériau cible, les deux types ou plus de différentes enzymes impliquées dans la synthèse du matériau cible étant liés aux domaines de liaison de fibres, respectivement. Le composite enzymatique diminue l'intervalle physique des enzymes qui agissent sur une série de processus de la conversion d'un précurseur du matériau cible en matériau cible, et peut ainsi induire un produit intermédiaire qui participera immédiatement à une réaction suivante, permettant ainsi d'améliorer la vitesse ou le rendement de la production globale du matériau cible.
PCT/KR2016/009340 2015-08-25 2016-08-23 Préparation d'un composite enzymatique et procédé de production de matériau cible l'utilisant WO2017034304A1 (fr)

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