CN103725652B - A kind of acyl-CoA synthetase and application thereof - Google Patents
A kind of acyl-CoA synthetase and application thereof Download PDFInfo
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- CN103725652B CN103725652B CN201310728420.7A CN201310728420A CN103725652B CN 103725652 B CN103725652 B CN 103725652B CN 201310728420 A CN201310728420 A CN 201310728420A CN 103725652 B CN103725652 B CN 103725652B
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- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 title claims abstract description 43
- 102000005870 Coenzyme A Ligases Human genes 0.000 title claims abstract description 39
- 239000000758 substrate Substances 0.000 claims abstract description 20
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- JNSSVMGPTZYYIW-UHFFFAOYSA-N 2-chloro-6-methyl-1-oxidopyridin-1-ium Chemical compound CC1=CC=CC(Cl)=[N+]1[O-] JNSSVMGPTZYYIW-UHFFFAOYSA-N 0.000 claims abstract description 7
- OCALSPDXYQHUHA-FNORWQNLSA-N 8-Methyl-6-nonenoic acid Chemical compound CC(C)\C=C\CCCCC(O)=O OCALSPDXYQHUHA-FNORWQNLSA-N 0.000 claims abstract description 7
- 238000010353 genetic engineering Methods 0.000 claims abstract description 7
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
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- 240000008384 Capsicum annuum var. annuum Species 0.000 claims description 13
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- -1 8-methyl nonenyl coenzyme A Chemical compound 0.000 description 5
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- 125000002252 acyl group Chemical group 0.000 description 4
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- 150000002500 ions Chemical class 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- FBUKVWPVBMHYJY-UHFFFAOYSA-N nonanoic acid Chemical compound CCCCCCCCC(O)=O FBUKVWPVBMHYJY-UHFFFAOYSA-N 0.000 description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
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- 229910019142 PO4 Inorganic materials 0.000 description 2
- 235000021355 Stearic acid Nutrition 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
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- 101150047711 acs gene Proteins 0.000 description 2
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
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- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000008117 stearic acid Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- SPSSULHKWOKEEL-UHFFFAOYSA-N 2,4,6-trinitrotoluene Chemical compound CC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O SPSSULHKWOKEEL-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 1
- BZXZFDKIRZBJEP-JMTMCXQRSA-N 3-Oxo-2-(2-entenyl)cyclopentaneoctanoic acid Natural products CC\C=C/C[C@H]1[C@@H](CCCCCCCC(O)=O)CCC1=O BZXZFDKIRZBJEP-JMTMCXQRSA-N 0.000 description 1
- 101710186512 3-ketoacyl-CoA thiolase Proteins 0.000 description 1
- BZXZFDKIRZBJEP-GTOOTHNYSA-N 8-[(1R,2R)-3-oxo-2-{(Z)-pent-2-en-1-yl}cyclopentyl]octanoic acid Chemical compound CC\C=C/C[C@@H]1[C@H](CCCCCCCC(O)=O)CCC1=O BZXZFDKIRZBJEP-GTOOTHNYSA-N 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 101710120269 Acyl-CoA thioester hydrolase YbgC Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 244000056139 Brassica cretica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000511730 Leymus chinensis Species 0.000 description 1
- 235000021360 Myristic acid Nutrition 0.000 description 1
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- BQFCCCIRTOLPEF-UHFFFAOYSA-N chembl1976978 Chemical compound CC1=CC=CC=C1N=NC1=C(O)C=CC2=CC=CC=C12 BQFCCCIRTOLPEF-UHFFFAOYSA-N 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- XJQPQKLURWNAAH-UHFFFAOYSA-N dihydrocapsaicin Chemical compound COC1=CC(CNC(=O)CCCCCCC(C)C)=CC=C1O XJQPQKLURWNAAH-UHFFFAOYSA-N 0.000 description 1
- RBCYRZPENADQGZ-UHFFFAOYSA-N dihydrocapsaicin Natural products COC1=CC(COC(=O)CCCCCCC(C)C)=CC=C1O RBCYRZPENADQGZ-UHFFFAOYSA-N 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- VBCVPMMZEGZULK-NRFANRHFSA-N indoxacarb Chemical compound C([C@@]1(OC2)C(=O)OC)C3=CC(Cl)=CC=C3C1=NN2C(=O)N(C(=O)OC)C1=CC=C(OC(F)(F)F)C=C1 VBCVPMMZEGZULK-NRFANRHFSA-N 0.000 description 1
- LTYOQGRJFJAKNA-IJCONWDESA-N malonyl-coenzyme a Chemical compound O[C@@H]1[C@@H](OP(O)(O)=O)[C@H](CO[P@](O)(=O)O[P@@](O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LTYOQGRJFJAKNA-IJCONWDESA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 125000001402 nonanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 125000003431 oxalo group Chemical group 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000000015 trinitrotoluene Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01086—Fatty-acyl-CoA synthase (2.3.1.86)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of acyl-CoA synthetase and application thereof, its aminoacid sequence is such as shown in SEQ ID NO.1.Build the genetic engineering bacterium that can secrete acyl-CoA synthetase further, utilize recombinant bacterium fermenting and producing acyl-CoA synthetase.Acyl-CoA synthetase provided by the invention be first found up to now to trans 8-methyl-6-nonenoic acid or the activated enzyme of 8-methyl nonanoic acid, can for cloned capsaicin synthase gene provide direct substrate.Acyl-CoA synthetase centering chain fatty acid provided by the invention has activity, it may also be used for field of biodiesel oil.
Description
Technical field
The present invention relates to a kind of acyl-CoA synthetase and application thereof, belong to technical field of enzyme engineering.
Background technology
Capsaicin, the trans-8-methy-N-vanillyl-nonenamide of formal name used at school, is a kind of secondary metabolite with zest taste of peppery Fructus Piperis (Capsicum).Capsaicin is synthesized by capsaicin synzyme (CS), and capsaicin synzyme is a kind of acyltransferase.Although the gene of coding CS is not yet decrypted at present, but its Main Function known is to be transferred on Rhizoma et radix valerianae amine by 8-methyl nonene acyl group from 8-methyl enoyl CoA in the ninth of the ten Heavenly Stems, thus forming amide complex.The substrate of CS, 8-methyl nonene acyl is under the effect of acyl-CoA synthetase (ACS), trans-8-methyl-6 nonanoyl acid be transformed.
The process that ACS catalysis carboxylic acid is corresponding acyl-CoA thioesterase experiences two stages altogether.First stage, free fatty acid is converted into a kind of acyl group-AMP intermediate product that can discharge pyrophosphate.Second stage, the carboxyl groups of activation is connected on the sulfydryl of coenzyme A, and discharges AMP and acyl-CoA product (Grootetal., 1976).ACS and some associated protein are considered as have 12 aminoacid sequences of one section of high conservative, and this sequence can form the core (PROSITTEPS00455) connecting AMP connection main body.In the plant model of Arabic mustard, there are about 44 ACS genes identified (Shockeyetal., 2003) supposed.At present; there are about the biochemical function of half identified; including long chain acyl Co A synzyme, acyl-ACP synthase, 4-coumaric acyl CoA ligase; acetyl group CoA synthase; OPC-8:0 CoA ligase, succinylbenzoyl CoA ligase, malonyl coenzyme A synzyme; oxalyl CoA synthase (Shockeyetal., 2003;Koo., 2005;Kooetal., 2006;Kimetal., 2008;LinandOliver, 2008;Chenetal., 2011;Fosteretal., 2012).In paprike, the supposition ACS gene of three kinds of total lengths be cloned (Leeetal., 2001;Mazoureketal., 2009).But, the biochemical function of above-mentioned protein is not yet identified.
Owing to the genome sequence of peppery Fructus Piperis is still unavailable, applicant uses RNA sequencing technologies that the green fruit of India ghost green pepper has been carried out transcript group analysis.India ghost green pepper is a kind of crossbred of Leymus chinensis (Trin.) Tzvel. and Herba Artemisiae Sieversianae chrysanthemum.Applicant, by overlapping assembling of former RNA sequence data is obtained 18987 contigs, wherein has the protein that 33 kinds of codings are similar with acyl-CoA synthetase.In these contigs, Comp2147-1 shows and can be well matched with CaSIG4.CaSIG4 is the supposition acyl-CoA synthetase (Leeetal., 2001) of the cDNA coding of a kind of pathogen-inducible from paprike.In addition, Comp66462 and Comp79520 can mate with Fructus Capsici ACS1 (GenBank:EU616571), Comp167_c0, and Comp167_c1 and Comp46218 can mate with Fructus Capsici ACS2 (GenBank:EU616572).ACS1 and ACS2 is the material standed for (Mazoureketal., 2009) that two kinds of acyl-CoA synthetase transport fatty acid from plastid.
In the present invention, the ACS1 that applicant provides is/long chain acyl Co A synzyme in one, it is possible to by trans-8-methyl-6-nonenoic acid to relevant 8-methyl nonenyl coenzyme A, be intermediate product crucial in a kind of capsaicin biosynthesis pathway.
Summary of the invention
The invention provides a kind of acyl-CoA synthetase, aminoacid sequence is such as shown in SEQIDNO.1.
Present invention also offers a kind of construction method containing acyl-CoA synthetase gene engineering bacteria, comprise the steps of:
(1) the ACS1 gene of ACS1-sumo-F and ACS1-sumo-R primer amplification India ghost green pepper green fruit is utilized；
(2) by after the amplification of step (1) gained gene PCR purification, it is connected on linearizing pETiteN-HisSUMOKan expression vector, obtains recombiant plasmid；
(3) by the recombiant plasmid of step (2) gained, convert to escherichia coli HI-control10G cell, obtain genetic engineering bacterium.
A kind of method that present invention also offers fermenting and producing acyl-CoA synthetase, is acyl-CoA synthetase expressed in microorganism, collects the acyl-CoA synthetase in tunning.
Further, the method for production acyl-CoA synthetase provided by the invention, comprise the steps of:
(1) the ACS1 gene of ACS1-sumo-F and ACS1-sumo-R primer amplification India ghost green pepper green fruit is utilized；
(2) by after the amplification of step (1) gained gene PCR purification, it is connected on linearizing pETiteN-HisSUMOKan expression vector, obtains recombiant plasmid；
(3) by the recombiant plasmid of step (2) gained, convert to escherichia coli HI-control10G cell, obtain genetic engineering bacterium；
(4) engineering bacteria fermentation utilizing step (3) gained produces acyl-CoA synthetase.
Present invention also offers the application of a kind of acyl-CoA synthetase, it is characterised in that be acyl-CoA synthetase expressed in microorganism, collect the acyl-CoA synthetase in tunning, make substrate be converted into acyl-CoA.
Described substrate is middle long-chain fatty acid.
Described substrate is trans 8-methyl-6-nonenoic acid or 8-methyl nonanoic acid.
The invention provides a kind of new acyl-CoA synthetase, substrate can be provided for capsaicin synzyme.Described new enzyme also can for preparing the derivative of fatty acid of medium chain in bio-fuel industry.
ACS1 mono-importance is the fact that, can make it have the effect of capsaicin level in potential adjustment plant by transgenic technology.Realize improving capsaicin content level in capsicum plant by the overexpression of ACS1.By knocking out or strike low ACS1 gene, reduce the capsaicin content level of capsicum plant.
Except accumulation capsaicin in natural capsicum, outside Dihydrocapsaicin, also produce the capsaicin analog (7-11 carbon atom) of other many different side chain lengths.ACS1 can be the acyl-CoA that capsaicin synzyme (CS) provides different chain length, thus controlling the structure of capsaicin analog.In current bio-fuel industry, medium chain acyl CoA synthase is widely used.Therefore, ACS1 has the prospect of application in bio-fuel industry.
Described acyl-CoA synthetase gene can pass through the cell system of antibacterial or yeast expresses.Described ACS gene can come from LCAS4 and the LCAS5 gene of ghost green pepper ACS1, arabidopsis or other sources.
The method have the benefit that acyl-CoA synthetase centering chain fatty acid provided by the present invention has activity to can be used for field of biodiesel oil; be first found up to now to trans 8-methyl-6-nonenoic acid or the activated enzyme of 8-methyl nonanoic acid, can for cloned capsaicin synthase gene provide direct substrate.
Accompanying drawing explanation
Fig. 1 is the His-SUMO-ACS1 SDS-PAGE expressed in BL21 (DE3) cell figure；
(0,20 represent respectively IPTG induction before and induction 20 hours after total protein；C is solubility crude protein extract after IPTG induces 20 hours；E1-E4 is the separation component of Ni-NTA post).
Fig. 2 is the ACS1 activity to different carboxylic acids；
(C2, acetic acid；C4, butanoic acid；C6, caproic acid；C8 is sad；C10, capric acid；C12, lauric acid；C14, myristic acid；C16, Palmic acid；C18, stearic acid).
Fig. 3 is trans 8-methyl-6-nonenoic acid or the 8-methyl nonanoic acid HPLC spectrogram as the enzyme reaction product of substrate.
The MS/MS of trans 8-methyl-6 nonenyl coenzyme A after purification is analyzed under ion mode by Fig. 4.
The MS/MS of the 8-Nonyl coenzyme A after purification is analyzed under ion mode by Fig. 5.
Fig. 6 is the-different pH buffer impact on ACS1 activity；
(-◆-: Acetate；-■-: Phosphate；-▲-: Tris；-×-: Glycine).
Detailed description of the invention
The invention discloses a kind of acyl-CoA synthetase and application thereof; specific embodiments includes building the genetic engineering bacterium containing acyl-CoA synthetase; the expression of acyl-CoA synthetase in microorganism system; and in reaction system, add zymolyte, utilize recombiant protein to convert a substrate into acyl-CoA.
Substrate be in/long-chain carboxylic acid, wherein long-chain carboxylic acid is generally the carboxylic acid containing 16 or 18 carbon atoms, and Medium chain is then the carboxylic acid containing 10 or 12 carbon atoms.
The construction and expression of embodiment 1 India ghost green pepper ACS1 genetic engineering bacterium
Utilizing the ACS1 gene of the cDNA of ACS1-sumo-F and ACS1-sumo-R primer amplification India ghost green pepper green fruit, the sequence of above-mentioned two primer is respectively as follows: CGCGAACAGATTGGAGGTGCAACAGATAAATTTATTATTG and GTGGCGGCCGCTCTATTATCACTTGGTACCCTTGTACAT.Pcr amplification product, with the agarose gel purification of 1%, and mixes (Lucigen, Middleton, WI) with linearizing pETiteN-HisSUMOKan expression vector.By thermal shock method, DNA mixture is converted to (Lucigen) in escherichia coli HI-control10G cell.Gene after inserting is checked order, it is ensured that its sequence is correct.The aminoacid sequence of its coding is such as shown in SEQIDNO.1.By in pETietN-HisSUMO-ghost green pepper ACS1 gene transformation to HI-ControlBL21 (DE31) cell (Lucigen), His-SUMO-ACS1 expresses the IPTG using 0.5mM at 16 DEG C, induces 20 hours.Mixed protein is purified (see figure 1) by Ni-NTA post.What the molecular weight of ACS1 was about 73.5KDa, His-SUMOtag is about 12KDa.The migration in SDS-PAGE of the ACS1 mixed protein of His-SUMO-ghost green pepper is close to predicting (85KDa is shown in Fig. 1).
Embodiment 2ACS1 determination of activity
HPLC is utilized to measure the activity (Chenetal., 2011) of ghost green pepper ACS1.Specifically, reaction mixture (400 μ L) includes the trinitrotoluene of the DTT, 5mMATP, 10mMMgCl2,0.5mMCoA, 0.1% of Tris-HCl, pH7.5, the 2mM of 0.1M and 200 μMs of carboxylic acids.Reaction starts by adding the purifying enzyme of 20 μ L, adds 20 μ L acetic acid and terminate reaction after reacting 30 minutes.HPLC uses3000LC system (ThermoScientific), adopts120C18 reversed phase chromatographic column (ThermoScientific；3 μ,150x3mm).The composition of mobile phase has solvent orange 2 A (trifluoroacetic acid of 0.1%) and solvent B(acetonitrile).The program of gradient elution is as follows: 0 to 5 minutes, 5% solvent B；5 to 9 minutes, solvent B concentration was linearly increasing from 5% to 80%；9 to 11 minutes, solvent B concentration 80%；11 to 12 minutes, solvent B concentration 5%.Flow velocity is 0.6mL/min.The detection range of diode array detector is 200 to 400nm.The qualitative, quantitative of substrate and product obtains by calculating the area of spectral peak under 257nm.Result shows: ACS1 enzyme Km value when with 8-methyl nonanoic acid for substrate is 0.14 μm of ol/min/mg for 0.57mM, Vmax value;ACS1 enzyme Km value when with (6E)-8-methyl-6-nonenoic acid for substrate is 0.49mM, Vmax value is 0.1214 μm of ol/min/mg.
The selection of embodiment 3ACS substrate specificity
Enzyme reaction system is added 5mM acetic acid, butanoic acid, caproic acid, sad, capric acid, lauric acid, myristic acid, Palmic acid and stearic acid respectively.
As in figure 2 it is shown, in different substrates, the most high activity of India ghost green pepper ACS1 is capric acid.On the contrary, acetic acid and butanoic acid are not had any activity by ACS1.
The production of embodiment 4 acyl-CoA
Utilize intermediate product trans-8-methyl-6 n-nonanoic acid (6E) raw in capsaicin biosynthesis pathway and 8-methyl nonanoic acid (8M), as substrate, produce acyl-CoA.
As it is shown on figure 3, ACS1 is with above-mentioned two materials for the measurement of substrate, the activity of 6E is higher.Have collected corresponding HPLC detached peaks, utilize SpeedVac concentrator dried, be further analyzed with MS/MS.
Each drying sample uses 40 μ L methanol: water: acetonitrile ratio is the buffer solution of 1:1:2.Use TriVersa(Advion, Ithaca, NY) is directly injected into 10 μ L.Mass spectrograph (LTQ-QrbitrapVelo (ThermoFisherScientific, Waltham, MA)) adopts ion mode to carry out.The scope of mass scanning is 300-2000m/z.Resolution is set to 60000400m/z.The broken MS/MS that adopts of CID, detection adopts the separation trap of separator window 1.5m/z.Broken with the 35% of normalization collision energy.As shown in Figure 4 and Figure 5, mass spectrometric data matches with the molecular weight of trans-8-methyl-6-nonenyl coenzyme A and 8-Nonyl coenzyme A.
The pH stability of embodiment 5 acyl-CoA synthetase
Adopt acetate, phosphate, Tris and glycine/NaOH buffer solution, regulate pH4.0 to 10.5, measure the pH stability of the acyl-CoA synthetase produced.As shown in Figure 6, the optimum pH of ACS1 is 9.5 to result.
Claims (7)
1. an acyl-CoA synthetase, it is characterised in that aminoacid sequence is such as shown in SEQIDNO.1.
2. the construction method containing acyl-CoA synthetase gene engineering bacteria described in claim 1, it is characterised in that step is as follows:
(1) the ACS1 gene of ACS1-sumo-F and ACS1-sumo-R primer amplification India ghost green pepper green fruit is utilized；
(2) by after the amplification of step (1) gained gene PCR purification, it is connected on linearizing pETiteN-HisSUMOKan expression vector, obtains recombiant plasmid；
(3) by the recombiant plasmid of step (2) gained, convert to escherichia coli HI-control10G cell, obtain genetic engineering bacterium.
3. the production method of acyl-CoA synthetase described in a claim 1, it is characterised in that be that such as the acyl-CoA synthetase shown in SEQIDNO.1 is expressed in microorganism by aminoacid sequence, collects the acyl-CoA synthetase in tunning.
4. method described in claim 3, it is characterised in that step is as follows:
(1) the ACS1 gene of ACS1-sumo-F and ACS1-sumo-R primer amplification India ghost green pepper green fruit is utilized；
(2) by after the amplification of step (1) gained gene PCR purification, it is connected on linearizing pETiteN-HisSUMOKan expression vector, obtains recombiant plasmid；
(3) by the recombiant plasmid of step (2) gained, convert to escherichia coli HI-control10G cell, obtain genetic engineering bacterium；
(4) engineering bacteria fermentation utilizing step (3) gained produces acyl-CoA synthetase.
5. the application of acyl-CoA synthetase described in claim 1, it is characterised in that be acyl-CoA synthetase expressed in microorganism, collects the acyl-CoA synthetase in tunning, makes substrate be converted into acyl-CoA.
6. apply described in claim 5, it is characterised in that described substrate is middle long-chain fatty acid.
7. apply described in claim 5, it is characterised in that described substrate is trans 8-methyl-6-nonenoic acid or 8-methyl nonanoic acid.
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| PL3094320T3 (en) | 2014-01-17 | 2023-12-04 | Conagen Inc. | Methods of using capsaicin synthase for the microbial production of capsaicinoids |
| US10655150B2 (en) | 2016-01-07 | 2020-05-19 | Conagen Inc. | Methods of making capsinoids by biosynthetic processes |
| AU2017299615A1 (en) | 2016-07-19 | 2019-01-17 | Conagen Inc. | Method for the microbial production of specific natural capsaicinoids |
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| CN102618556A (en) * | 2012-04-12 | 2012-08-01 | 重庆大学 | Pepper CaCOI1.2 gene, recombinant expression vector and application thereof |
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