WO2017119576A1 - Processus de bioconversion utilisant l'acide oléique hydratase 2 - Google Patents

Processus de bioconversion utilisant l'acide oléique hydratase 2 Download PDF

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WO2017119576A1
WO2017119576A1 PCT/KR2016/011224 KR2016011224W WO2017119576A1 WO 2017119576 A1 WO2017119576 A1 WO 2017119576A1 KR 2016011224 W KR2016011224 W KR 2016011224W WO 2017119576 A1 WO2017119576 A1 WO 2017119576A1
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acid
fatty acid
utr
oleic acid
seq
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Korean (ko)
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박진병
오덕근
송지원
이정후
전은영
강우리
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이화여자대학교 산학협력단
건국대학교 산학협력단
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Priority claimed from KR1020160001809A external-priority patent/KR101769231B1/ko
Priority claimed from KR1020160025454A external-priority patent/KR101856308B1/ko
Application filed by 이화여자대학교 산학협력단, 건국대학교 산학협력단 filed Critical 이화여자대학교 산학협력단
Publication of WO2017119576A1 publication Critical patent/WO2017119576A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a bioconversion process using oleic acid hydratase 2, and more particularly, the present invention relates to a bioconversion process for producing medium and heavy chain omega-hydroxy fatty acids from a long chain unsaturated fatty acid substrate using a novel oleic acid hydratase 2
  • Omega-Hydroxyfatty acid is a type of fatty acid that has one hydroxyl group (hydroxyl group) at the end of fatty acid (HOCH 2 (CH 2 ) n COOH). It is used as a monomer in the production of polyethylene-based plastics. It is widely used in the production and manufacture of cosmetics and pharmaceuticals (J. Am. Chem. Soc., 132: 15451-15455, 2010). It can also be used as a precursor of long-chain dicarboxylic acid synthesis, which is widely used in the production of polyamide, polyester-based plastics, cosmetics and household goods.
  • Medium chain fatty acids are widely used in the synthesis of ester-based perfumes / perfumes, and also have excellent antibacterial activity, and are widely used in the production of cosmetics, food, and household goods.
  • Korean Patent Publication No. 2013-0132254 discloses a series of enzymes consisting of fatty acid double bond hydratase, alcohol dehydrogenase, Baeyer-Villiger monooxygenase (BVMO), and ester hydrolase (esterase). Biotransformation processes have been disclosed that produce heavy and heavy chain omega-hydroxy fatty acids from long chain unsaturated fatty acid substrates using transformants in which the reaction system is expressed.
  • the method has the advantage of using a single transformant to obtain the desired product from the substrate, whereas the expression level of each enzyme expressed in the transformant is not the same, when the expression of one enzyme is inhibited
  • the disadvantage is that the overall bioconversion process does not proceed smoothly. For example, in the case of BVMO, despite being expressed in a large amount in the transformant, the ratio of being expressed in the water-soluble state is not high, and thus shows a low level of the overall biotransformation activity.
  • a method of replacing an enzyme expressed in the transformant with an enzyme showing higher activity or increasing the ratio expressed in a water-soluble state should be developed, but there are still very few research results. It is not reported.
  • the present inventors have made intensive studies to develop a high yield of hydroxyfatty acid from unsaturated fatty acids.
  • a novel oleic hydratase exhibiting superior activity to conventional oleic hydratase from stenotropomonas maltopilaa strains. 2
  • the present invention has been completed.
  • One object of the present invention is to provide a method for producing heavy and heavy chain omega-hydroxy fatty acids from long chain unsaturated fatty acid substrates using the novel oleic acid hydratase 2.
  • Another object of the present invention is to provide a novel oleic acid hydratase 2 used in the above method.
  • Still another object of the present invention is to provide a polynucleotide encoding the oleic acid hydratase 2.
  • Another object of the present invention is to provide a method of increasing the water-soluble expression ratio relative to the total expression level of the protein of interest.
  • FIG. 1 is a graph comparing the production rate of 10-hydroxystearic acid produced from oleic acid (oleic acid) during bioconversion using oleic acid hydrase 2 and the conventional oleic acid hydrase
  • ( ⁇ ) Represents the oleic acid concentration of the reaction solution when using oleic acid hydratase 2
  • ( ⁇ ) represents the 10-hydroxystearic acid concentration of the reaction solution when using oleic acid hydratase 2
  • ( ⁇ ) is a conventional oleic acid hydrase
  • ( ⁇ ) indicates the 10-hydroxystearic acid concentration of the reaction solution when using the conventional oleic acid hydratase.
  • FIG. 2 shows the production of 10-hydroxyoctadec-12-enoic acid produced from linoleic acid during bioconversion using recombinant Escherichia coli expressing oleic acid hydrase 2 and recombinant Escherichia coli expressing conventional oleic hydrase.
  • ⁇ indicates the linoleic acid concentration of the reaction solution when using recombinant E. coli expressing oleic acid hydratase 2.
  • ( ⁇ ) represents the linoleic acid concentration of the reaction solution when using recombinant E. coli with conventional oleic hydrase
  • ( ⁇ ) shows the concentration of 10-hydroxyoctadec-12-enoic acid in the reaction solution when using recombinant E. coli expressing the conventional oleic acid hydratase.
  • FIG. 3 is a graph showing the production rate of 10-hydroxyfatty acid produced from soybean oil at the time of bioconversion using recombinant E. coli expressing lipolytic enzyme (TTL) and oleic acid hydratase 2,
  • TTL lipolytic enzyme
  • ⁇ Represents linoleic acid
  • ⁇ represents 10-hydroxyoctadec-12-enoic acid of the reaction solution
  • ⁇ represents oleic acid of the reaction solution
  • ( ⁇ ) represents 10-hydroxystearic acid of the reaction solution.
  • Figure 4a is an electrophoresis picture of BmoF1-3'UTR CAT mRNA levels in recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT variant
  • lane 1 is recombinant E. coli BL21 (DE3) pET22b-BmoF1-3 ' UTR native
  • lane 2 represents recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT257
  • lane 3 represents recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT357
  • lane 4 Represents recombinant E.
  • coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT557 and lane 5 represents recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT657 .
  • 4B is a photograph showing the results of SDS-PAGE (top panel) and Western blot (bottom panel) analysis of the water soluble fraction of BmoF1 in a variant of recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT .
  • Figure 4c is an electrophoresis picture showing the results of SDS-PAGE analysis of the water-insoluble fraction of BmoF1 in the variant of recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT .
  • FIG. 5A is a graph showing the bioconversion of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR native ,
  • ( ⁇ ) represents ricinoleic acid
  • ( ⁇ ) represents 12- Ketooleic acid (12-ketooleic acid)
  • ( ⁇ ) represents the ester of 12-ketooleic acid
  • ( ⁇ ) represents 11-hydroxyundec-9-enoic acid (11-hydroxyundec-9-enoic acid).
  • FIG. 5B is a graph showing the bioconversion of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT257 .
  • FIG. 5B is a graph showing the bioconversion of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT257 .
  • 5C is a graph showing the bioconversion of ricinoleic acid to esters using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT357 .
  • 5D is a graph showing the bioconversion of ricinoleic acid to esters using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT557 .
  • FIG. 5E is a graph showing the bioconversion of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT657 .
  • FIG. 5E is a graph showing the bioconversion of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-BmoF1-3′UTR CAT657 .
  • Figure 6 is a diagram showing the nucleotide sequence of the synthetic 3'UTR (UTRsyn) artificially made to include 18 RNase E cleavage sites, the RNase E cleavage sites in the UTRsyn nucleotides are underlined.
  • the 13th RNase E cleavage site GCATAT indicates that the two RNase E cleavage sites GCAT and ATAT overlap.
  • Figure 7 is the result of a biotransformation in ricinoleate ester of building a pET22b-BmoF1-3'UTR syn having UTRsyn a building in Figure 6, and a recombinant E. coli BL21 (DE3) containing the same pET22b-BmoF1-3'UTR syn
  • ⁇ represents ricinoleic acid
  • ⁇ represents 12-ketooleic acid
  • ⁇ represents ester of 12-ketooleic acid
  • ( ⁇ ) represents 11 -11-hydroxyundec-9-enoic acid.
  • 8A is a graph showing the bioconversion results of ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-MO16-3'UTR native , ( ⁇ ) represents ricinoleic acid, and ( ⁇ ) represents 12 -Represents the ketooleic acid (12-ketooleic acid), ( ⁇ ) represents the ester of 12-ketooleic acid
  • 8B is a graph showing the results of bioconversion from ricinoleic acid to ester using recombinant E. coli BL21 (DE3) pET22b-MO16-3'UTR CAT257 .
  • Figure 9a is a graph showing the results of bio conversion from oleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA, pJOE-E6BVMO, ( ⁇ ) represents oleic acid, ( ⁇ ) is 10-hydroxystearic Acid (10-hydroxystearic acid), ( ⁇ ) represents 10-ketostearic acid, ( ⁇ ) represents the ester of 10-ketostearic acid.
  • FIG. 9B is a graph showing the results of bioconversion from oleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA-FadL, pJOE-E6BVMO, which further introduced FadL in FIG. 9A.
  • FIG. 9C is a graph showing the results of bioconversion from oleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA2-FadL and pJOE-E6BVMO in which the conventional oleic hydrase was replaced with oleic hydrase 2 in FIG. 9B. to be.
  • FIG. 9D shows recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA2-FadL, pJOE-, replacing the conventional pJOE-E6BVMO-3'UTR native with the 3'UTR variant pJOE-E6BVMO-3'UTR syn in FIG. 9C. It is a graph showing the results of bio conversion from oleic acid to ester using E6BVMOsyn.
  • 10A is a graph showing the bioconversion results of linoleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA, pJOE-E6BVMO, where ( ⁇ ) represents linoleic acid, and ( ⁇ ) represents 10-hydroxy Octadec-12-enoic acid (10-hydroxyoctadec-12-enoic acid), ( ⁇ ) represents 10-ketooctadec-12-enoic acid (10-ketooctadec-12-enoic acid), ( Represents a ester of 10-ketooctadec-12-enoic acid.
  • FIG. 10B is a graph showing the results of bioconversion from linoleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA-FadL, pJOE-E6BVMO, which further introduced FadL in FIG. 10A.
  • FIG. 10C shows the results of bioconversion from linoleic acid to ester using recombinant E. coli BL21 (DE3) pACYC-ADH-OhyA2-FadL, pJOE-E6BVMO in which the conventional oleic hydrase was replaced with oleic hydrase 2 in FIG. 10B. It is a graph.
  • Figure 10d is a 10C in the alternative introducing the conventional pJOE-E6BVMO-native 3'UTR with a 3'UTR variants pJOE-E6BVMO-3'UTR syn recombinant E. coli BL21 (DE3) pACYC-ADH- OhyA2-FadL, pJOE- A graph showing the results of bioconversion from linoleic acid to ester using E6BVMOsyn.
  • the present inventors use a transformant expressing a series of enzymatic reaction systems consisting of fatty acid double bond hydratase, alcohol dehydrogenase, BVMO (Baeyer-Villiger monooxygenase) and ester hydrolase (esterase).
  • BVMO Brown-Villiger monooxygenase
  • ester hydrolase ester hydrolase
  • the long chain unsaturated fatty acid is introduced into the transformant, and is converted into a hydroxy fatty acid by a fatty acid double bond hydrase, and the hydroxy fatty acid is produced by an alcohol dehydrogenase. It is converted into a chito fatty acid, which is converted into a fatty acid ester derivative having an ester group introduced into the fatty acid chain by BVMO, and the fatty acid ester derivative is cleaved by ester hydrolase, and thus the heavy chain fatty acid and the heavy chain omega-hydroxy fatty acid.
  • the inventors of the present invention are stenotrophomonas maltopilaa with enhanced activity of oleic acid hydratase itself.
  • maltophilia strain-derived oleic acid hydrase 2 was found to be able to obtain a variety of hydroxy fatty acids in high yield and rate, the oleic acid hydrase 2 is a novel, conventionally used stenotropomonas maltophilia-derived oleic acid hydration
  • the reaction activity and the production rate is remarkably excellent, and when the oleic acid hydrase 2 is applied to the developed transformants, it was confirmed that the production yield of the hydroxy fatty acid can be improved.
  • the BVMO expressed in the transformant is aggregated in cells when overexpressed to form inclusion bodies, so that the ratio of water-soluble expression to the total expression level of the protein is 50% or less, so that the enzyme reaction efficiency thereof
  • a method of inserting an RNase E cleavage site into 3'UTR affects the stability of mRNA to increase the expression rate of water-soluble protein.
  • a polynucleotide having a form in which a UTR having a nucleotide sequence including an RNase E cleavage site is inserted to reduce the stability of mRNA is obtained, and the polynucleotide is expressed.
  • the expression ratio of the water-soluble protein form to the total expression level of the BVMO is increased, as a result it was confirmed that can improve the enzyme reaction efficiency of BVMO.
  • the transformant uses only the long chain unsaturated fatty acids present on the outside of the cell membrane of the transformant as substrates, the long chain unsaturated fatty acids outside the cell membrane are used in the transformant.
  • the carrier protein to be introduced into the can be improved the efficiency of the biotransformation process of the transformant.
  • FadL is an E. coli-derived long chain fatty acid transport protein, and as a result, confirmed that the efficiency of the bioconversion process was improved.
  • the present inventors have described the use of a transformant expressing a series of enzymatic reaction systems consisting of fatty acid double bond hydratase, alcohol dehydrogenase, BVMO and ester / fat hydrolase to obtain a heavy chain fatty acid from a long chain unsaturated fatty acid substrate.
  • a novel fatty acid double bond hydratase, oleic acid hydratase 2 was discovered, a method of improving the water-soluble expression rate of BVMO, and the influx of substrates into the transformants.
  • a method of promoting this has been developed, which has not been reported at all until now and was first developed by the present inventors.
  • the present invention (a) oleic acid hydrase 2, alcohol dehydrogenase (Baeyer-Villiger monooxygenase) and ester / fat hydrolase (esterase / lipase) Can express; By expressing a polynucleotide conjugated to the UTR (untranslated region) that changes the mRNA stability at the 3 'end of the gene encoding the BVMO, to obtain a transformant for a biotransformation process that can induce water-soluble expression of the BVMO step; And,
  • oleate hydratase 2 of the present invention is one of oleic acid hydrases, which is a type of fatty acid hydrase that catalyzes the reaction of converting oleic acid to 10-hydroxystearic acid, stenotropomonas.
  • hydroxy fatty acids 10-hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12-dienoic acid, 10 10-hydroxy fatty acid, such as hydroxyoctadec-12,15-dienoic acid, 10-hydroxypalmitoleic acid or 10-hydroxystearic acid, 9-hydroxynonanoic acid, etc.
  • the oleic acid hydratase 2 is similar to the conventional oleic hydrase in that hydroxy fatty acids can be produced from long-chain unsaturated fatty acids, but various kinds of hydroxy fatty acids (10-hydroxyoctadec-12-eno Ic acid, 10-hydroxyoctadec-6,12-dienoic acid, 10-hydroxyoctadec-12,15-dienoic acid, 10-hydroxypalmitoleic acid, 10-hydroxystearic acid, etc.) It is distinguished from the conventional oleic hydrase in that it can be produced faster than the conventional oleic hydrase.
  • the oleic acid hydrase 2 is not particularly limited as long as it can exhibit enzymatic activity to produce hydroxy fatty acid, all the amino acid sequence is added to the N- or C- terminal of the amino acid sequence of SEQ ID NO: 1 It may comprise a peptide. In addition, it may further comprise amino acid sequences designed for specific purposes to increase the stability of the targeting sequence, tag, labeled residue, half-life or peptide.
  • alcohol dehydrogenase refers to an enzyme that reversibly catalyzes the reaction of removing hydrogen from alcohol to form aldehydes or ketones.
  • the alcohol dehydrogenase is not particularly limited as long as it can remove the hydrogen from the hydroxy fatty acid produced by the oleic acid hydratase 2 to produce a chito fatty acid.
  • Alcohol dehydrogenase from Micrococcus luteus can be used.
  • Baeyer-Villiger monooxygenase (BVMO) refers to a kind of monooxygenase which is an enzyme capable of catalyzing various oxidation reactions including a Baeyer-Villiger oxidation reaction in which ketones are oxidized to produce lactones or ester compounds. .
  • BVMO Bach-Villiger monooxygenase
  • it is catalyzed by a transformant to produce a fatty acid derivative (10-octyloxy-10-oxodecanoic acid, etc.) in which an ester group is introduced into a chain from chito fatty acids (10-chitostearic acid, etc.).
  • the BVMO is not particularly limited thereto, as long as it exhibits activity, and as an example, Pseudomonas sp. And Rhodococcus sp. Brevibacterium sp., Comanonas sp., Acinetobacter sp., Arthrobacter sp., Brachymonas strain.
  • BVMO derived from a microorganism such as sp.), more preferably Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas putida ( Pseudomonas) putida ), Pseudomonas veronii ), Rhodococcus jostii) or Pseudomonas sp HI-70 (can be the origin BVMO from Pseudomonas sp.
  • strain HI-70 Pseudomonas fluorescein sense (Pseudomonas fluorescens) DSM50106 derived BVMO the BmoF1 or Rhodococcus crude stitcher (Rhodococcus jostii ) may be MO16, a BVMO derived from RHA1.
  • the BVMO may be present at one or more positions of the amino acid sequence constituting the BVMO.
  • the amino acid sequence may include an amino acid sequence in which an amino acid is substituted, deleted, inserted, added or inverted, and as long as it can maintain or enhance the activity of BVMO, at least 80%, preferably 90%, relative to the amino acid sequence of BVMO Or more preferably 95% or more, particularly preferably 97% or more, and may comprise an amino acid sequence having a homology
  • the amino acid sequence of the enzyme exhibiting the activity of the polypeptide may vary depending on the strain, and is not particularly limited thereto.
  • the microorganism containing the activity of the BVMO may be used for substitution, deletion, insertion, addition, or inversion of the amino acid. It may also include naturally occurring mutant sequences or artificially mutated sequences, such as those based on individual or species differences.
  • homology refers to an identity between two different amino acid sequences or nucleotide sequences, which is a BLAST that calculates parameters such as score, identity, similarity, and the like. It can be determined by methods well known to those skilled in the art using 2.0, but is not particularly limited thereto.
  • the BVMO may be expressed from a polynucleotide to which an untranslated region (UTR) which increases mRNA stability at the 3 ′ end of a gene encoding BVMO in the transformant is bound.
  • UTR untranslated region
  • the binding of UTR to the 5 'or 3' end of the polynucleotide encoding the protein improves the stability of mRNA transcribed from the polynucleotide, thereby increasing the level of synthesis of the polypeptide.
  • the polypeptides aggregate with each other to form an inclusion body, and thus there is a problem that the level of the protein expressed in the water-soluble form generated by normal folding is reduced.
  • the RNase E cleavage site is inserted into the UTR and is used as a means for reducing the stability of the mRNA.
  • the RNase E cleavage site is not particularly limited thereto, and includes, for example, 12 to 16 RNase E cleavage sites.
  • hilD nucleotide sequence including 14 RNase E cleavage site (SEQ ID NO: 3), 15 RNase CAT257 base sequence comprising the E cleavage site (SEQ ID NO: 4), CAT357 base sequence comprising the 18 RNase E cleavage sites (SEQ ID NO: 5), CAT557 base sequence comprising the 26 RNase E cleavage sites (SEQ ID NO: 6) , CAT657 nucleotide sequence including 28 RNase E cleavage sites (SEQ ID NO: 7), synthetic base sequence (3'UTR syn , SEQ ID NO: 8) and the like can be used.
  • the technology of controlling the expression level of the protein regulates the induction level of the promoter, thereby reducing the expression level of the aggregate to produce aggregates.
  • Inhibition method 5'UTR or RBS (ribosome binding site) is used to distinguish from the slowdown in the translation level of mRNA.
  • the recombinant protein expression vector may be expressed by combining a target protein with a highly water-soluble amino acid or by connecting it with a signal sequence to secrete it into a periplasm.
  • Methods such as secretion and simultaneously expressing chaperones involved in protein folding are known, and low incubation temperatures are generally known to be advantageous for the water-soluble expression of proteins.
  • the present invention inserts a specific nucleotide sequence including a plurality of RNase E cleavage sites into 3'UTR, thereby reducing mRNA stability and inducing normal folding of the polypeptide, thereby increasing the expression of water-soluble protein. Are distinguished.
  • the term "esterase / lipase” means an enzyme that hydrolyzes an ester bond of a fat / ester compound.
  • the ester / fat hydrolase is used for the purpose of decomposing fatty acid derivatives having ester groups introduced into the chain produced by BVMO into heavy chain fatty acids and omega-hydroxy fatty acids, but is not particularly limited thereto.
  • a lipohydrolase derived from a Thermomyces lanuginosus strain, an ester hydrolase derived from Pseudomonas fluorescens strain, or the like may be used.
  • biotransformation process means a method of converting a substrate into a desired product using an enzyme or a transformant expressing the enzyme.
  • the bioconversion process may be interpreted to mean a method for producing the desired heavy chain fatty acid and heavy chain omega-hydroxy fatty acid from the long chain unsaturated fatty acid substrate using the transformant provided in the present invention.
  • the term “transformer” refers to a gene encoding the fatty acid double bond hydratase of the present invention in a host cell alone or to an alcohol dehydrogenase and Bayer-Villiger monooxygenase (BVMO). It refers to a recombinant microorganism prepared by introducing an expression vector comprising a polynucleotide containing a gene together.
  • BVMO Bayer-Villiger monooxygenase
  • the transformant may be interpreted as a main means for producing the desired heavy chain fatty acid and heavy chain omega-hydroxy fatty acid from the long chain unsaturated fatty acid substrate through the biotransformation process.
  • the host cell used in the preparation of the transformant is not particularly limited, examples of bacterial cells such as E. coli, Corynebacterium, Streptomyces, Salmonella typhimurium; Yeast cells such as Saccharomyces cerevisiae and ski-irradiated Caromyces pombe; Fungal cells such as Pchia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, bow melanoma cells; Or plant cells, and as another example, E. coli.
  • long chain unsaturated fatty acid refers to an unsaturated fatty acid having 16 to 20 carbon atoms among unsaturated fatty acids in which a carbon-to-carbon double bond exists as a chain-like compound having a carbon-carbon unsaturated bond and a carboxyl group in one molecule. do. Due to the nature of the unsaturated fatty acid, the long-chain unsaturated fatty acid is chemically unstable, easy to oxidize, and low in melting point, compared to saturated fatty acid. The position of the double bond included in the long-chain unsaturated fatty acid is indicated by the number of carbons attached from the carboxy group, but the fatty acids present in nature exhibit a certain arrangement. Typical fatty acids of edible oils, oleic acid and linoleic acid, have double bonds at positions 9, 9 and 12, respectively.
  • the long-chain unsaturated fatty acid is not particularly limited thereto, but oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid ( ⁇ -linolenic acid) and the like, and the method for treating the long chain unsaturated fatty acid is also not particularly limited thereto, and the method of culturing the transformant in a medium containing the long chain unsaturated fatty acid may be used.
  • the transformant of the present invention may further express a transporter protein for introducing the long-chain unsaturated fatty acid present in the medium into the transformant.
  • a carrier protein that introduces unsaturated fatty acids into the transformant may be further expressed.
  • the carrier protein to be used is not particularly limited as long as the long chain unsaturated fatty acid in the medium can be introduced into the transformant, but as an example, the protein expressed from the base sequence (SEQ ID NO: 9) encoding FadL is Can be.
  • culture refers to a method of growing a desired microorganism under artificially controlled environmental conditions.
  • the culture is to be interpreted to be carried out to provide a reaction environment for producing the desired heavy chain fatty acid and heavy chain omega-hydroxy fatty acid from the long-chain unsaturated fatty acid substrate using the transformant provided in the present invention Can be.
  • the culture method is not particularly limited thereto, but a batch or continuous culture may be used using a batch process, an injection batch or a repeated fed batch process.
  • the present invention provides oleic acid hydratase 2 expressed in said transformant.
  • the oleic acid hydratase 2 may be composed of an amino acid sequence of the amino acid sequence of SEQ ID NO: 1 or a variety of amino acid sequences added to the N- or C- terminal of the amino acid sequence of SEQ ID NO: 1.
  • the oleic acid hydrase 2 exhibits enzymatic activity against various unsaturated fatty acid substrates (oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid or gamma-linolenic acid), stenotropomonas It was confirmed that the enzyme showed better enzyme activity than the known oleic acid hydratase derived from the maltophilia strain (Fig. 2 and Table 1).
  • the present invention provides oligonucleotides encoding said oleic acid hydratase 2.
  • the nucleotide sequence constituting the polynucleotide encodes a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1 or various amino acid sequences that may be added to the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO:
  • the nucleotide sequence may be in the form of being added to the 5'-terminal or 3 'end of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1, specifically, the sequence number capable of encoding the amino acid sequence of SEQ ID NO: 1
  • the base sequence encoding the various amino acid sequences that may be added to the nucleotide sequence of 2 or the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1 is 5'-terminal or 3'- of the nucleotide sequence of SEQ ID NO: 2 It may be in the form added to the end. Specifically, it may be composed of the nucleotide sequence of SEQ
  • a polynucleotide comprising a nucleotide sequence showing homology with the nucleotide sequence may also be included in the scope of the polynucleotide provided in the present invention.
  • polynucleotide sequence encoding a protein capable of expressing oleic acid hydratase 2 may have, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 2.
  • the expression "having essentially, or consisting of” does not exclude a meaningless sequence addition or a naturally occurring mutation, or a latent mutation thereof, before or after the nucleotide sequence of SEQ ID NO: 2, When expressed as a protein has the same or corresponding activity as the protein encoded by the nucleotide sequence of SEQ ID NO: 2 if the "having, consisting essentially, or consisting of the nucleotide sequence of SEQ ID NO: 2" of the present invention It may correspond to.
  • the polynucleotide may be mutated by one or more bases substituted, deleted, inserted, or a combination thereof.
  • synthetic methods well known in the art may be used, for example, those described in Engels and Uhlmann, Angew Chem Int Ed Eng., 37: 73-127, 1988. , Triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, and the like.
  • the present invention provides a method of increasing the ratio of water soluble expression to the total expression level of a protein of interest.
  • the method of increasing the water-soluble expression ratio relative to the total expression level of the protein of interest provided by the present invention (a) untranslated region (UTR) to increase the mRNA stability at the 3 'end of the gene encoding the desired water-soluble protein Obtaining this bound polynucleotide; And (b) expressing the polynucleotide, wherein a nucleotide sequence including an RNase E cleavage site for lowering mRNA stability is inserted into the nucleotide sequence of the UTR.
  • UTR untranslated region
  • water-soluble protein refers to a protein that expresses both water-insoluble and water-soluble. In particular, when the protein is over-expressed, it means a protein having significantly less water-soluble expression compared to the total expression level.
  • desired water-soluble protein may be used interchangeably with the same meaning as the desired protein.
  • the desired water-soluble protein may be a protein having a water-soluble expression ratio of 50% or less relative to the total expression level of the protein by over-expressing the protein to be aggregated in the cell to form an inclusion body.
  • Pseudomonas fluorescein sense (Pseudomonasfluorescens) DSM50106 derived or Rhodococcus crude stitcher (Rhodococcus josti i) RHA1 derived BVMO (Bayer-Villiger monooxygenase) may be, more specifically, Pseudomonas fluorescein sense (Pseudomonasfluorescens) DSM50106 derived BVMO the BmoF1 or also Rhodococcus jostii ) may be MO16, which is a BVMO derived from RHA1.
  • water soluble protein refers to a protein which is transcribed and translated by expression of a gene of interest, and then shows water solubility by folding.
  • expression level of the water-soluble protein has the same meaning as the water-soluble expression of the protein, can be used interchangeably.
  • UTR untranslated region
  • non-stone portion means a portion of the mRNA chain that is not a template of the protein gene, that is, the portion that is not translated.
  • the UTR includes 5'UTR (5 'non-interpreted portion) and 3'UTR (3' non-interpreted portion).
  • 3'UTR refers to a transcription terminator that contributes to the stability of mRNA in prokaryotes. For example, it has been used to increase the stability of transcriptionally activated mRNA by inserting specific sequences (eg, REP sequences) into the 3'UTR.
  • specific sequences eg, REP sequences
  • RNase E cleavage site refers to a portion where cleavage occurs by RNase E involved in RNA processing and degradation in bacterial cells, and cleavage occurs mainly in a single strand of AU-rich region.
  • the RNase E cleavage site is present in the 3'UTR of BmoF1 or MO16, which is the water-soluble protein of the present invention, including 5 RNase E cleavage sites in the 3'UTR of BmoF1 and 3'UTR of MO16. have.
  • hilD sequence or CAT sequence was used in order to insert the RNase E cleavage site into 3'UTR.
  • the level of expression of water-soluble proteins may increase, and bioconversion activity may be increased.
  • the total expression of the protein of interest is adjusted as the number of RNase E cleavage sites is adjusted.
  • the ratio of water soluble expression to the level can be changed, and specifically, when the BmoF1 expression, as the number of RNase E cleavage sites increases, the ratio of water soluble expression to the total expression level of the protein of interest can be increased.
  • the base sequence including the RNase E cleavage site includes, for example, an hilD sequence including 12 to 16 RNase E cleavage sites, and a CAT base sequence including 13 to 30 RNase E cleavage sites.
  • a hilD sequence comprising 14 RNase E cleavage sites SEQ ID NO: 3
  • a CAT257 base sequence comprising 15 RNase E cleavage sites SEQ ID NO: 4
  • 18 RNase E CAT357 nucleotide sequence comprising the cleavage site SEQ ID NO: 5
  • CAT557 nucleotide sequence comprising the 26 RNase E cleavage sites
  • SEQ ID NO: 7 Synthetic base sequence
  • Example 1-1 Preparation of an Expression Vector and Transformed Recombinant Escherichia Coli Comprising an Oleate Hydrohydrase 2 Gene (ohyA2)
  • Stenotrophomonas maltophilia strain-derived oleic acid hydrase 2 gene was synthesized, and the following experiment was performed using the synthesized gene.
  • a primer (OhyA2A_F (SEQ ID NO: 10): 5'-CATATGAGCCAGCCCACCGCAC-3 ', OhyA2A_R (SEQ ID NO: 11): 5'-CTCGAGTCAGGGCGCGCGCCGCCTG-3') was prepared based on the DNA sequence of the synthesized oleic acid hydratase 2. PCR was performed using the prepared primers to amplify the nucleotide sequence of the gene. The amplified oleic acid hydrase 2 gene was inserted into plasmid vector pET 28 (+) a (manufactured by Novagen) using restriction enzymes Nde I and Xho I to prepare pET 28 (+) a-OhyA2.
  • the recombinant expression vector thus obtained was transformed into E. coli ER 2566 or BL21 (DE3) strain.
  • the sample containing the oleic acid hydrase 2 was prepared after culturing E. coli and purifying oleic acid hydratase 2.
  • Recombinant Escherichia coli ER 2566 of Example 1-1 was incubated at 37 ° C. under aeration conditions of 200 rpm in a flask to which 500 mL of LB medium and 50 ⁇ g / ml of kanamycin were added.
  • IPTG was added at a final concentration of 0.1 mmol / L to induce oleic hydrase expression, and the culture was incubated with stirring at 150 rpm at 16 ° C. for 12 hours. .
  • the culture was washed twice with 0.85% sodium chloride (NaCl) after centrifugation at 6,000 xg at 4 ° C. for 30 minutes, 50 mM sodium phosphate (NaH 2 PO 4 ), 300 mM sodium chloride, 10 mM immidazole. After the addition of 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride), the cell solution was disrupted with a sonicator. The cell lysate was centrifuged at 13,000 xg at 4 ° C. for 20 minutes to separate only the cell supernatant.
  • NaCl sodium phosphate
  • 10 mM immidazole 10 mM immidazole.
  • protease inhibitor phenylmethylsulfonyl fluoride
  • the cell supernatant was subjected to a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA) to obtain an active fraction, and the active fraction was subjected to a histidine tag (Hisidine tag, His-tag).
  • the oleic acid hydrase 2 was purified by applying to a Hisrap HP adsorption column, and a sample containing the purified oleic acid hydrase 2 was prepared.
  • Example 1-3 Comparison of Inactivation by Substrate of Oleic Acid Hydrolase 2
  • oleic acid oleic acid
  • palmitoleic acid palmitoleic acid
  • linoleic acid linoleic acid
  • alpha-linolenic acid ⁇ -linolenic acid
  • gamma- Hydration was performed by adding linolenic acid ( ⁇ -linolenic acid) as a substrate.
  • the oleic acid hydration reaction was performed for 10 minutes in 50 mM citrate / phosphate buffer solution (pH 6.0) containing 0.14 g / L oleic acid and 0.005 g / L oleic acid hydratase 2, respectively.
  • the reaction was terminated by adding acetate (ethyl acetate), the reaction activity was compared by measuring the production of 10-hydroxystearic acid (Table 1).
  • oleic acid hydrase 2 As shown in Table 1, the hydration activity of oleic acid hydrase 2 was measured using various unsaturated fatty acids as a substrate, and the highest activity was observed in oleic acid, followed by palmitoleic acid, linoleic acid, alpha-linolenic acid, and gamma-linolenic acid. It was confirmed that the order.
  • the inactivation of the oleic acid hydratase 2 (the amount of the product according to the amount of enzyme in 7 reaction time) is J. Biotechnol. 158: 17-23, 45% for oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid, or gamma-linolenic acid, respectively, compared to the substrate-specific inactivity of oleic acid hydratase derived from stenotropomonas maltophilia disclosed in 2012 , 10%, 80%, 90%, 130% was confirmed to be high.
  • reaction activity of oleic acid hydratase 2 was highest when oleic acid was used as a substrate, and higher than that of homologous oleic hydrase when the substrate was used.
  • Example 1-4 Production of 10 -Hydroxystearic Acid Using Oleic Acid Hydrolase 2
  • oleic acid hydrase 2 produced 10 g / L of 10-hydroxystearic acid, and showed a production rate of 3.4 g / L per hour and a conversion yield of 90% or more relative to the reduced substrate.
  • the oleic acid hydrase 2 shows a production rate of about 20% or more higher It was confirmed (FIG. 1).
  • oleic acid hydratase 2 is an oleic acid hydrase superior to oleic hydrase derived from the same strain, and it is possible to produce 10-hydroxystearic acid in high yield using the enzyme.
  • Example 1-5-1 Production of 10-hydroxyoctadec-12-enoic acid using recombinant E. coli expressing oleic acid hydratase 2
  • the recombinant Escherichia coli was cultured in Regenberg medium, and when the recombinant Escherichia coli reached the stationary phase, linoleic acid was added to the culture solution at 30 mM (8.4 g / L) for 6 hours.
  • oleic acid hydratase 2 is superior to the oleic acid hydratase derived from the same strain through the above results, and using the enzyme can produce 10-hydroxyoctadec-12-enoic acid in high yield.
  • using the enzyme can produce 10-hydroxyoctadec-12-enoic acid in high yield.
  • Example 1-5-2 Recombinant Escherichia coli and lipohydrolase expressing oleic acid hydratase 2 ( TLL 10- using) Hydroxyoctadec -12- Enoick Acid and 10- Hydroxystea Production of arsenic
  • E. coli and TLL were used to measure the hourly production of the reaction product during bioconversion and compared with conventional oleic hydratase.
  • oleic acid hydrase 2 is an oleic acid hydrase that is superior to oleic acid hydrase derived from the same strain, and in particular, the 10-hydroxyoctadec-12-enoic acid from soybean oil using the enzyme is higher It was found that the yield can be produced.
  • Pseudomonas fluorescein sense Pseudomonas fluorescens . It is known that various oxidases, including BmoF1, BVMO (Bayer-Villiger monooxygenase) derived from DSM50106, are difficult to express in water-soluble form in E. coli strains (Appl. Microbiol. Biotechnol. 73: 1065-1072, 2012).
  • BmoF1 BmoF1
  • BVMO Bach-Villiger monooxygenase
  • the stability of Salmonella enterica hilD mRNA in E. coli is significantly influenced by the presence or absence of 3'untranslated reigion (310'UTR) consisting of 310 nucleotides containing 14 RNase E cleavage sites.
  • 310'UTR 3'untranslated reigion
  • increased to 14 RNase E is significantly hilD mRNA stability in E. coli when removing hilD 3'UTR in hilD gene containing the cleavage site by hilD mRNA concentration is markedly increased, which results markedly increased amount hilD expressed in E. coli It was.
  • the present invention by introducing the RNase E cleavage site in the 3'UTR of the target gene to reduce the mRNA stability of the target gene by lowering the protein expression level per mRNA of the target gene to reduce the insoluble expression of the target gene and improve the water-soluble expression.
  • the following experiment was performed using CAT (Chloramphenicol acetyltransferase) sequence containing 28 estimated RNase E cleavage sites to increase the water-soluble expression of BmoF1 in E. coli.
  • Example 2-1 Water Soluble Expression of Protein BmoF1 Using CAT Sequence as 3′UTR
  • the amplified CAT sequence fragment is a CAT657 nucleotide sequence including 657 nucleotides, and is inserted into the HindIII-XhoI restriction enzyme region of pET22b-BmoF1 into the 3'UTR of the BmoF1 gene, thereby recombining Escherichia coli BL21 (DE3) pET22b-BmoF1-3 'UTR CAT657 was produced.
  • Example 2-1-2 Reverse transcription PCR reverse transcription PCR )In accordance mRNA Level analysis
  • Reverse transcription PCR was performed to control BmoF1-3'UTR CAT657 mRNA levels in recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT657 and to control BmoF1-3 'in recombinant E. coli BL21 (DE3) pET-BmoF1-3'UTR native . UTR native mRNA levels were compared. RNA extraction and reverse transcription PCR were performed according to the experimental method reported in the previous study (Sci. Reports 6: 29406 (doi: 10.1038 / srep29406), 2016).
  • Example 2-1- 3 3'UTR Protein expression level analysis by insertion of CAT sequence
  • variants were constructed that included partial deletion of the CAT sequence (3'UTR CAT ) inserted into the 3'UTR.
  • Fragments with partial deletion of CAT sequences inserted into the 3'UTR are forward primer CAT_F and other reverse primers CAT257_R (5'-CTCGAGCGCCCCGCCCTGCCACTCATCG-3 ', SEQ ID NO: 14), CAT357_R (5'- Amplified by PCR using CTCGAGTCCCATATCACCAGCTCA-3 ', SEQ ID NO: 15) and CAT557_R (5'-CTCGAGTATGTGTAGAAACTGCCG-3', SEQ ID NO: 16) and inserted into pET22b-BmoF1.
  • the number of RNase E cleavage sites in CAT 257, CAT357 and CAT557 was 15, 18 and 26, respectively.
  • Example 2-2-2 mRNA Level analysis
  • BmoF1 of recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT variants containing CAT sequences having different RNase E cleavage sites -3'UTR CAT mRNA levels were compared using reverse transcription PCR.
  • BmoF1-3'UTR CAT mRNA levels of recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT variants gradually decreased ( 4a)
  • the total expression level of BmoF1 of recombinant E. coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT variants decreased as the number of RNase E cleavage sites present in the CAT sequence increased (FIGS. 4B and 4C).
  • the level of water soluble expression of BmoF1 was significantly increased in Escherichia coli BL21 (DE3) pET22b-BmoF1-3'UTR CAT variants.
  • PCR was performed with Syn_F (5'-AAGCTTATAAAGGCTTAAATCACTGG-3 ', SEQ ID NO: 17) and Syn_R (5'-CTCGAGAGTGTAGGAGAATTCTTCTAACACGG-3', SEQ ID NO: 18) as forward and reverse primers. And amplified. The fragment obtained through the amplification was subcloned into the HindIII-XhoI restriction enzyme site of pET22b-BmoF1 to prepare pET22b-BmoF1-3'UTR syn .
  • Example 2-4 using CAT sequencing Rhodococcus jostii RHA1 origin BVMO Water-soluble expression
  • BVMO derived from Pseudomonas fluorescens DSM50106, Rhodococcus jostii
  • RHA1-derived BVMO ie, sequence homology with MO16, BmoF1: 23.3%, FIG. 8A
  • E. coli ChemBioChem 10: 1208-1217, 2009. Therefore, in order to improve the expression of soluble protein of MO16 in E. coli, a study was performed to increase the number of RNase E cleavage sites in 3'UTR.
  • MO16_CAT257_F 5'-AAGCTTATGGAGAAAAAAATCACTGGATAT-3 ', SEQ ID NO: 19
  • MO16_CAT257_R 5'-CTCGAGCGCCCCCTGCCACTCATCG-3', SEQ ID NO: 20
  • the MO16_CAT sequence fragment was subcloned into the HindIII-XhoI restriction enzyme site of pET- MO16 to construct pET22b-MO16-3'UTR CAT257 (ChemBioChem 10: 1208-1217, 2009).
  • Example 2-4-2 Whole cell Biotransformation
  • the recombinant E. coli BL21 (DE3) expressing pET-MO16-3'UTR CAT257 produced an ester product at a 35% higher rate than the recombinant E. coli BL21 (DE3) expressing the control pET-MO16-3'UTR native . Formation was confirmed to improve the catalytic activity of MO16 (Fig. 8a and 8b).
  • the results showed that the MO16 soluble protein was increased by adjusting the number of RNase E cleavage sites present in the 3'UTR to improve the water-soluble expression and bioconversion activity of the MO16 enzyme in the microorganism.
  • Example 3-1 Ester Production from Ele Coli Using E. Coli Expressing Oleic Acid Hydrolase, Alcohol Dehydrogenase (ADH) and Bayer-Villiger Monooxygenase (BVMO)
  • Oleic Acid Hydrolase (OhyA) and Micrococcus, which have been used to produce esters from oleic acid using recombinant E. coli long chain secondary alcohol dehydrogenase (ADH) derived from luteus NCTC2665, Pseudomonas Recombinant E. coli BL21 transformed with pACYC-ADH-OhyA (Angew. Chem. Int. Ed. 52: 2534-2537, 2013) and pJOE-E6BVMO (Sci. Reports 6: 29406, 2016) expressing BVMO from putida Whole cell bioconversion was performed using (DE3). Whole cell bioconversion was performed according to the experimental method reported in the previous study (Adv Synth Catal. DOI: 10.1002 / adsc. 201600216, 2016).
  • ADH E. coli long chain secondary alcohol dehydrogenase
  • pACYC-ADH-OhyA Angew. Chem. Int. Ed. 52
  • Example 3-2 Ester Production from Oleic Acid Using Recombinant E. Coli Expressing FadL
  • Example 3-2-1 FadL Expression vector comprising and the production of transformed recombinant E. coli
  • genomic DNA was extracted from E. coli and the following method was performed.
  • FadL_F (SEQ ID NO: 21): 5'-GCCACGCGATCGCTGCTTAAGTCGAAAGAAAGTA-3 '
  • FadL_R (SEQ ID NO: 22): 5'-GTCAGCGATCGCTCAGTCCTCCTGCACCAGC-3'
  • PCR PCR was performed to amplify the nucleotide sequence of the gene.
  • the amplified FadL gene was inserted into pACYC-ADH-OhyA using restriction enzyme PvuI to prepare pACYC-ADH-OhyA-FadL.
  • the recombinant expression vector and pJOE-E6BVMO obtained as described above were transformed into E. coli BL21 (DE3) strain to construct a whole cell catalyst.
  • FadL expression level can significantly increase the oleic acid bioconversion rate of the recombinant E. coli catalyst.
  • Example 3-3 Ester Production from Oleic Acid Using Recombinant Escherichia Coli Expressing Oleic Acid Hydrolase 2
  • OhyA2 cloned in Example 1-1 was introduced in place of the conventional fatty acid hydrase OhyA to construct a recombinant E. coli biocatalyst.
  • Example 3-3-1 oleic acid hydrase 2 ( OhyA2 Expression vector and the production of transformed recombinant E. coli
  • primers (OhyA2B_F (SEQ ID NO: 23): 5'-GCCACGCGATCGCTGCTTAAGTCGAACAGAAAGTAATCGTA-3 ', OhyA2B_R (SEQ ID NO: 24): 5'-CGTCAGCGATCGCTAGGGCGCGCCGCC), produced a polymerase chain reaction, and produced a PCR primer. (PCR) was performed to amplify the nucleotide sequence of the gene. The amplified oleic acid hydrase 2 gene was inserted into pACYC-ADH-FadL using restriction enzyme PvuI to prepare pACYC-ADH-OhyA2-FadL.
  • the recombinant expression vector and pJOE-E6BVMO obtained as described above were transformed into E. coli BL21 (DE3) strain to construct a whole cell catalyst.
  • Recombinant Escherichia coli BL21 (DE3) pACYC-ADH-OhyA2-FadL and pJOE-E6BVM were constructed using pACYC-ADH-OhyA2-FadL constructed in Example 3-3-1, and the whole cell bioconversion reaction was performed. . 10 mM oleic acid was added to the reaction solution as a substrate to initiate bioconversion and the hourly production of the reaction product was measured.
  • Example 3-4 Ester Production from Oleic Acid Using Recombinant Escherichia Coli Expressing BVMO Incorporating Synthetic UTR
  • Synthetic 3'UTR (SEQ ID NO: 8) containing 18 RNAse E cleavage sites was used to improve the water-soluble expression of BVMO to increase the production yield by increasing the ester concentration of the final product.
  • Example 3-4-1 Preparation of an Expression Vector Comprising BVMO Incorporating Synthetic UTR and Transformed Recombinant Escherichia Coli
  • RNAse E cleavage site was arranged to synthesize a nucleotide sequence having 18 RNAse E cleavage sites (SEQ ID NO: 8).
  • the synthesized UTR sequences were amplified by PCR with CAT657B_F (5'-GGATCCATAAAGGCTTAAATCACTG-3 ', SEQ ID NO: 25) and CAT657B_R (5'-GGATCCAGTGTAGGAGAATTCTTCTA-3', SEQ ID NO: 26) primers as forward and reverse primers.
  • the amplified nucleotide fragment was inserted into the BamHI restriction enzyme site of pJOE-E6BVMO into 3'UTR of the E6BVMO gene to prepare pJOE-E6BVMOUTR, and the prepared pJOE-E6BVMOUTR and pACYC-ADH-OhyA2-FadL were recombinant E. coli BL21. (DE3) was transformed to construct whole cell catalyst.
  • Example 3-4-2 Whole cell Biotransformation
  • Recombinant Escherichia coli BL21 (DE3) pACYC-ADH-OhyA2-FadL and pJOE-E6BVMOUTR were constructed using pJOE-E6BVMOUTR constructed in Example 3.4.1, and the whole cell bioconversion reaction was performed. 10 mM oleic acid was added to the reaction solution as a substrate to initiate bioconversion and the hourly production of the reaction product was measured.
  • Example 4 oleic hydrase, Alcohol Dehydrogenase (Alcohol dehydrogenase ) And Bayer-Billiger oxidase ( Bayer - Villiger monooxygenase , BVMO Ester Production from Linoleic Acid Using Escherichia Coli
  • Biotransformation reaction for ester production from linoleic acid was performed using the whole-cell catalysts constructed in Example 3. In order to confirm the increase in the bioconversion efficiency of each whole cell catalyst, the whole cell bioconversion reaction was performed. Whole cell bioconversion was performed in the same manner as in Example 3-1.
  • FadL is very important for linoleic acid bioconversion.

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Abstract

La présente invention concerne un processus de bioconversion permettant de produire un acide gras à chaîne lourde et un acide gras oméga-hydroxylé à chaîne lourde à partir d'un substrat d'acide gras insaturé à longue chaîne en utilisant une nouvelle acide oléique hydratase 2 ; une nouvelle acide oléique hydratase 2 utilisée pour ledit processus ; un polynucléotide destiné à coder pour l'acide oléique hydratase 2 ; et un procédé permettant d'augmenter la proportion d'expression d'une protéine hydrosoluble vers un niveau d'expression total d'une protéine cible. Lors de l'utilisation de l'acide oléique hydratase 2 et du procédé permettant d'augmenter la proportion d'expression d'une protéine hydrosoluble fournie par la présente invention, il est possible d'augmenter l'efficacité d'un processus de bioconversion classique pour produire un acide gras à chaîne lourde et un acide gras oméga-hydroxylé à chaîne lourde à partir d'un acide gras insaturé à longue chaîne, et, par conséquent, la présente invention peut être largement utilisée pour le développement d'un processus de bioconversion plus efficace.
PCT/KR2016/011224 2016-01-07 2016-10-07 Processus de bioconversion utilisant l'acide oléique hydratase 2 WO2017119576A1 (fr)

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