CN107083412A

CN107083412A - A kind of application of middle short chain acyl CoA synthase

Info

Publication number: CN107083412A
Application number: CN201710440525.0A
Authority: CN
Inventors: 刘超
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-06-13
Filing date: 2017-06-13
Publication date: 2017-08-22
Anticipated expiration: 2037-06-13
Also published as: CN107083412B

Abstract

The present invention relates to a kind of application of middle short chain acyl CoA synthase.The applying step is as follows：(1) middle short chain acyl CoA synthase of the amino acid sequence as shown in SEQ ID NO.2 is prepared；(2) reaction system is prepared using middle short chain acyl CoA synthase made from step (1)；(3) after reacting, terminating reaction removes zymoprotein, purified, dries, produces.Present invention firstly discloses the technical scheme by middle short chain acyl CoA synthase preparation enoyl CoA in 2 heptan and hexamethylene methylacyl coenzyme A; overcome and be only capable of thinking that malonyl coenzyme A synzyme can only be with 1 in the prior art; 3 dicarboxylic acids or its salt are used as substrate; it is catalyzed them and forms corresponding acyl-CoA product; and the technology prejudice using monocarboxylic acid as the reaction of substrate can not possibly be catalyzed, widen the application of the enzyme.

Description

A kind of application of middle short chain acyl CoA synthase

Technical field

The present invention relates to a kind of application of middle short chain acyl CoA synthase, more particularly to a kind of middle short chain acyl coacetylase Application of the synzyme in 2- enoyl CoAs in heptan, hexamethylene methylacyl coenzyme A is prepared, belongs to technical field of enzyme engineering.

Background technology

Coacetylase (coenzyme A, CoA) contributes to Acyl activating and transfer crucial co-factor during being enzymatic reaction, Aliphatic acid, macrolide (macrolides), aromatic compound biosynthesis, catabolism or oxidizing process, carbohydrate Modification, and played a significant role in the extensive biological pathway such as enzyme regulation and control.It is estimated that thering is 4% to make among known enzyme Co-factor is used as with CoA.

Acyl-CoA (acyl-CoA) is that CoA sulfydryl (- SH) is covalently attached to the carboxyl (- COOH) formation of free carboxy acid Interim compound, as acry radical donor primary and secondary metabolism in be essential.Macrolide, polyethers (polyethers), the compound such as polyenoid (polyenes), flavonoids (flavonoids), stilbene class (stilbenes) is to excavate The important sources of natural products with medical value, and the biosynthesis of above-mentioned polyketone class secondary metabolites all refer to it is all kinds of In, short chain acyl CoA (medium, short-chain acyl-CoA) be used as construction unit.Various acyl-CoA is used as starting Or extension apparatus is integrated into the skeleton of polyketone molecule under polyketide synthase (PKS) catalysis.

Up to the present, researcher still must not independent of various acyl-CoAs,¹³The acyl-CoA of C- marks, radioactivity Mark¹⁴C- acyl-CoAs carry out the research experiment of polyketone class natural drug biosynthesis.Currently, only Sigma-Aldrich etc. Only a few offshore company can provide the acyl-CoA reagent of commercialization, but kind is extremely limited and price is extremely expensive, every gram Price is up to 12.2-53.6 ten thousand yuan, shown in such as table 1 (the commercialization acyl-CoA reagent information of Sigma-Aldrich companies)：

Table 1

Kind just because of the acyl-CoA derivative of existing commercialization is fairly limited, and some species are in economy On make us hanging back or it is commercial can not obtain, be also difficult to synthesize using chemical method, it is domestic each for can independently synthesize The demand of the new acyl-CoA derivative of the form of kind is very urgent.

At present, the substrate broad spectrum activity that has been reported is most wide, synthesizing acyl CoA species most abundant acylCoA synthetase Product is only limitted to malonyl CoA (malonyl-CoA), methylmalonyl CoA (methylmalonyl-CoA), ethyl malonyl CoA (ethylmalonyl-CoA), methoxyl group malonyl CoA (methoxymalonyl-CoA), hydroxyl malonyl CoA (hydroxymalonyl-CoA), report is had no for the enzyme that can synthesize more other acyl-CoAs.

The content of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of application of middle short chain acyl CoA synthase.

Technical solution of the present invention is as follows：

A kind of application of middle short chain acyl CoA synthase in 2- enoyl CoAs in heptan, hexamethylene methylacyl coenzyme A is prepared.

Above-mentioned application, step is as follows：

(1) middle short chain acyl CoA synthase of the amino acid sequence as shown in SEQ ID NO.2 is prepared；

(2) it is formulated as follows reaction system using middle short chain acyl CoA synthase made from step (1)：

100mM 4- hydroxyethyl piperazineethanesulfonic acids；The glycerine of quality percent by volume 10%, unit g/ml；100mM magnesium chlorides； 15~50mM2- heptenoic acids, the sodium salt of 2- heptenoic acids, the sodium salt of naphthenic acid or naphthenic acid；20mM atriphos； 5mM coacetylases；The malonyl coenzyme A synzyme of 10 μM of purifying；pH 7.5；

(3) above-mentioned reaction system reacts 7.5~10h under the conditions of 20~24 DEG C, and terminating reaction removes zymoprotein, through pure Change, dry, produce.

It is short in preparing amino acid sequence as shown in SEQ ID NO.2 in the step (1) according to currently preferred Chain acyl CoA synthase is adopted with the following method：

The malonyl coenzyme A synthase gene for expanding streptomyces coelicolor is reacted through PCR using primer matBF and matBR MatB, then by construction of expression vector and engineering bacteria, then induced expression closes through Ni-NTA ni-sepharose purifications malonyl coenzyme A Into enzyme, produce；

The nucleotide sequence of the primer matBF and matBR is as follows：

matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’

matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’。

According to currently preferred, in the step (3), remove zymoprotein to remove using ultrafiltration centrifugal filtration.

According to currently preferred, in the step (3), described purifying is to be purified using HPLC chromatogram.

According to currently preferred, in the step (3), the drying is to be carried out using vacuum freeze-drying method.

The encoding gene of above-mentioned middle short chain acyl CoA synthase (malonyl coenzyme A synzyme) derives from sky blue chain Mould ZM12 (being purchased from synthetic biology key lab of the Chinese Academy of Sciences) matB genes, total length 1458bp (base-pair), such as Shown in SEQ ID NO.1,485 residues of its amino acid sequence total length, as shown in SEQ ID NO.2, molecular weight 50.6kDa.Should Enzyme has larger N- ends body (residue 1-387) and less C- end caps (residue 388-485).The enzyme with carboxylic acid or its Salt, ATP, CoA are substrate, are catalyzed the biosynthesis of a variety of acyl-CoAs.

The two-step reaction mechanism of above-mentioned malonyl coenzyme A synzyme is as shown in Figure 1：

In adenylate formation reaction, ATP alpha-phosphate is attacked to form acyl group-adenylate by the carboxyl of carboxylic acid derivates Intermediate and pyrophosphoric acid (PPi)；Thioesters formation reaction in, CoA displacement AMP (AMP) with produce acyl-CoA and AMP。

Inventor has found that the enzyme has widely by furtheing investigate malonyl coenzyme A synzyme of the present invention Carboxylic acid substrate tolerance, this has overturned original cognition to such malonyl coenzyme A synzyme, and the enzyme can not only be with 1,3- bis- Carboxylic acid and its C-2 substitutive derivatives are substrate, moreover it is possible to using straight chain and the monocarboxylic acid with cyclic structure is substrate, such as utilize 2- heptan Olefin(e) acid (2-heptenate), naphthenic acid (cyclohexanecarboxylic acid) generation 2- heptene acyl-CoA and ring Own formyl-CoA.Therefore, malonyl coenzyme A synzyme of the present invention can widely adapt to short chain or middle chain, dicarboxylic acids or Monocarboxylic acid substrate, and it is catalyzed its corresponding acyl-CoA product of formation.

Beneficial effect

Present invention firstly discloses prepare 2- enoyl CoAs in heptan and hexamethylene formyl by middle short chain acyl CoA synthase The technical scheme of coacetylase, overcome be only capable of thinking in the prior art malonyl coenzyme A synzyme can only with 1,3- dicarboxylic acids or its Salt is catalyzed them and forms corresponding acyl-CoA products as substrate, such as malonyl CoA, methylmalonyl CoA, ethyl the third two Acyl CoA, methoxyl group malonyl CoA, hydroxyl malonyl CoA, and can not possibly be catalyzed inclined using monocarboxylic acid as the technology of the reaction of substrate See, widened the application of the enzyme.

Brief description of the drawings

Fig. 1 is the two-step reaction mechanism of malonyl coenzyme A synzyme；

Fig. 2 is N- ends His₆The SDS-PAGE detection figures of the malonyl coenzyme A synzyme of-tag fusion；

In figure：T, S are LB culture mediums, 15 DEG C, the total protein of expressive host bacterium under 0.5mM IPTG inductive conditions respectively With supernatant (control)；

Ft is Flow-through effluxes；

15 be the eluent under 15mM imidazole concentrations；

150 be the eluent under 150mM imidazole concentrations；

500 be the eluent under 500mM imidazole concentrations；

M is protein molecular weight standard (PageRuler Broad Range Unstained Protein Ladeder)；

Fig. 3 is test group and the HPLC-MS testing results of control group；

In figure：1) control group, 2) test group.

Embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.

Biological sample is originated

Streptomyces coelicolor ZM12 is existing known bacterial strain, purchased from synthetic biology key lab of the Chinese Academy of Sciences；

Coacetylase is purchased from Sigma-Aldrich (China) company.

Equipment source

Ultrafiltration in embodiment is usedUltra-0.5 ultrafiltration centrifugal filter devices；

Detection couples MS (ESI mass spectrometry, electrospray ionization mass spectrometry) using HPLC, by analyzing ultrafiltration A small amount of sample after removing protein, it is determined that and checking the retention time (retention time) of acyl-CoA product, ultraviolet absorption peak Shape (UV absorption peak shape) and molecular weight (molecular weight)；

Purifying uses preparation HPLC chromatographic column；

Dry using MAXI dry lyo vacuum concentrations/freeze-drying system (Heto-Holten, Denmark).

Embodiment 1

1. the structure of expression vector and engineering bacteria

Use primer matBF and matBR, using streptomyces coelicolor genomic DNA as template, PCR amplifying target genes matB.PCR primer after purified is connected to by identical digestion processing simultaneously using being purified again after NdeI and XhoI double digestions And dephosphorylized contain N- ends His₆The expression vector pET-28a (+) of-label.Plasmid pET-28a (+)-matB of formation Host Strains E.coli BL21 (DE3) are transformed into after being verified through DNA sequencing is used for protein expression.

matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’

matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’

2. the expression and purifying of malonyl coenzyme A synzyme

It is used for the LB culture mediums for being inoculated with 37 DEG C of pre-temperatures of addition 50mg/l kanamycins after starting culture growth overnight.When OD₆₀₀When=0.4,15 DEG C of cooling culture mediums are expressed with 0.5mM isopropyl-β-D-thiogalactosides (IPTG) inducible protein. After 16h, by the way that cell is harvested by centrifugation, being resuspended in lysis buffer, (quality percent by volume is 10% glycerine, g/ml, 0.5M NaCl, 100mMHEPESpH7.5) in, it is ultrasonically treated, and 16,000g centrifugation 45min are to remove cell fragment.Acellular cracking Nickel-NTA the posts that liquid is balanced by using lysis buffer.Wash the post with the lysis buffer containing 15mM imidazoles, and with containing The lysis buffer elution albumen of 150mM imidazoles.

Polyacrylamide gel electrophoresis (SDS-PAGE) is detected, as a result as shown in Fig. 2 destination protein is N- ends His₆The malonyl coenzyme A synzyme of-tag fusion, molecular weight 52.8kDa.Use Thermo Scientific Nanodrop 1000 determine final protein concentration for 62.7 μM.

The in vitro test of 2- enoyl CoA in the heptan biosynthesis of embodiment 2

The reaction system of malonyl coenzyme A synzyme as shown in table 2, adds each component successively in order, in 22 DEG C of reactions Under the conditions of constant temperature stand overnight.

Table 2

The reaction system of control group such as test group, difference is that malonyl coenzyme A synzyme first passes through 96 DEG C of heat in advance Inactivation processing 10min.Response sample passes throughUltra-0.5 Centrifugal Filter Devices ultrafiltration is removed It is used for HPLC-MS (high-resolution positive-ESI mass spectrometry, high-resolution positive electricity after albumen Spraying MALDI-MS) to analyze, HPLC methods are shown in Table 3.

Table 3 is used for the HPLC method [solvent orange 2 As of Mass Spectrometer Method：10mM ammonium acetates (NH₄OAc) the aqueous solution, solvent B：Acetonitrile (CH₃CN), flow velocity=1ml/min, in 260nm detection reaction products (adenine, CoA groups)]

Table 3

Use Prominence Modular HPLC (Japanese Shimadzu Corporation) and Dikma reverse-phase chromatographic columns (Agilent 1200, ZORBAX Eclipse XDB-C18,250 × 4.6mm, 5 μm), couple LTQ-Obitrap Velos Pro mass spectrographs (Thermo Scientific companies), sample is analyzed by HPLC-HR-ESIMS, and mass-to-charge ratio (m/z) is swept in the positive-ion mode Scope 110-1200 is retouched, testing result is as shown in Figure 3；

As a result show, test group 2) occur newly-increased product peak A (2- enoyl CoAs in heptan) in retention time 16.75min, Its m/z observation is 878.1981 (theoretical values 878.19565, error only has 2.8ppm).Meanwhile, test group 2) and control 1) group observes substrate peak B (CoA, m/z observation 766.4, theoretical value 766.1).

Amplification prepares the reaction system of 2- enoyl CoAs in heptan as it was previously stated, difference is：Reacted system is passed through Enzyme is inactivated, ultrafiltration removing protein, then separates 2- enoyl CoA in heptan products with preparation HPLC, dense using MAXI dry lyo vacuum Contracting/freeze-drying system (Heto-Holten, Denmark) vacuum freezedrying, the encapsulation of gained chromatographically pure 2- enoyl CoAs in heptan freeze-dried powder - 80 DEG C of preservations are put in ampoule bottle.

The in vitro test of the hexamethylene methylacyl coenzyme A biosynthesis of embodiment 3

Reaction system is in addition to substrate need to replace with final concentration 16mM naphthenic acids (NaOH adjusts pH to 7.2), remaining component It is identical with table 3 with reaction condition.Follow-up preparation process is with reference to embodiment 2, isolated 10mg hexamethylenes methylacyl coenzyme A sterlings.Inspection Survey result as follows：

¹H-NMR (nuclear magnetic resonance) data (300MHz, D₂O)：δ:0.56(s,H-10'),0.69(s,H-11'),1.55(m, ), H-5 2.26 (t, J=6.9Hz, H-4'), 2.40 (t, J=6.9, H-6), 2.55 (t, J=8.1Hz, H-4), 2.75 (t, J= 6.0Hz, H-1'), 3.11 (t, J=6.0, H-3and H-7), 3.27 (m, H-2'and H-5'), 3.35 (m, H-5 "), 3.64 (m,H-5”),3.82(s,H-7'),4.04(br s,CH₂- 9'), 4.39 (br s, H-4 "), 5.98 (d, J=7.5Hz, H- 1”),8.08(s,adenine-CH),8.35(s,adenine-CH).ESI-MS mass spectrometric datas [negative ion mode (negative Ion mode), m/z]：876.0[M-H]^-, 437.5 [M-2H]^2-。

Comparative example

According to coming from Rhodopseudomonas palustris (Rhodopseudomonas disclosed in U.S. Crosby in 2012 et al. PalustrisCGA009 the preparation method of malonyl coenzyme A synzyme) prepares malonyl coenzyme A synzyme, using embodiment 2 and embodiment 3 in method prepare 2- enoyl CoAs in heptan and hexamethylene methylacyl coenzyme A.

After testing, 2- enoyl CoAs in heptan and hexamethylene methylacyl coenzyme A generation are not found in reaction system.Marsh is red false single The malonyl coenzyme A synzyme of born of the same parents bacterium can not be catalyzed 2- heptenoic acids, naphthenic acid it is such in short unsaturated straight chain or with ring The monocarboxylic acid of shape structure is changed into the reaction of corresponding acyl-CoA.

Described above is only one embodiment of the present of invention, it is noted that for those skilled in the art For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications should also be regarded For protection scope of the present invention.

SEQUENCE LISTING

<110>Liu, surpasses

<120>A kind of application of middle short chain acyl CoA synthase

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1458

<212> DNA

<213> Streptomyces coelicolor

<400> 1

atgtcctctc tcttcccggc cctctccccg gccccgaccg gcgccccggc cgaccggccc 60

gcgctgcggt tcggcgagcg ctccctgacc tacgcggaac tcgccgcggc ggcgggcgcc 120

acggccgggc ggatcggcgg cgccggccgg gtcgcggtct gggccacccc ggcgatggag 180

accggcgtcg ccgtggtggc ggcgctgctg gccggggtcg ccgccgtacc gctcaacccg 240

aagtccggcg acaaggaact cgcgcacatc ctctccgaca gcgcgccctc gctcgtcctg 300

gcgcccccgg acgcggaact cccgcccgcc ctcggggccc tggagcgcgt cgacgtcgac 360

gtgcgggccc gcggggcggt ccccgaggac ggtgccgacg acggcgaccc cgcgctcgtc 420

gtctacacct cgggcaccac gggaccgccg aagggcgccg tcatcccccg gcgggcgctc 480

gccacgaccc tggacgcgct cgccgacgcg tggcagtgga ccggcgagga cgtgctggtg 540

caggggctgc cgctgttcca cgtgcacggg ctggtcctcg gcatcctcgg cccgctgcgc 600

cggggcgggt ccgtgcggca cctgggccgg ttctccaccg agggtgcggc gcgggagctg 660

aacgacggcg cgaccatgct gttcggggtg ccgacgatgt accaccggat cgccgagacg 720

ctccccgccg acccggagct ggcgaaggcg ctcgccgggg cccggctgct ggtgtcgggg 780

tcggccgcgc tgccggtgca cgaccacgag cgcatcgccg ccgccaccgg gcgccgggtg 840

atcgagcggt acggcatgac cgagacgctg atgaacacca gcgtgcgcgc cgacggcgag 900

ccgcgcgccg ggacggtggg cgtgccgctg cccggtgtgg agctgcggct ggtggaggag 960

gacggcacgc cgatcgcggc gctcgacggg gagagcgtcg gcgagatcca ggttcgcggc 1020

ccgaacctgt tcaccgagta cctgaaccgc cccgacgcca ccgccgccgc cttcaccgag 1080

gacggcttct tccgcaccgg cgacatggcg gtgcgcgacc ccgacggcta tgtccgcatc 1140

gtcggccgca aggccaccga cctgatcaag agcggcggtt acaagatcgg ggccggggag 1200

atcgagaacg ccctgctcga acacccggag gtccgggagg ccgccgtcac cggcgaaccc 1260

gaccccgacc tcggggaacg gatcgtggcc tggatcgtcc cggccgaccc cgccgccccg 1320

cccgccctcg gcacgctggc cgaccacgtc gccgcccggc tcgccccgca caagcggccg 1380

cgcgtcgtcc ggtacctcga cgcggtgccc cgcaacgaca tggggaagat catgaagcgg 1440

gcgctgaacc gtgactga 1458

<210> 2

<211> 485

<212> PRT

<213> Streptomyces coelicolor

<400> 2

Met Ser Ser Leu Phe Pro Ala Leu Ser Pro Ala Pro Thr Gly Ala Pro

1 5 10 15

Ala Asp Arg Pro Ala Leu Arg Phe Gly Glu Arg Ser Leu Thr Tyr Ala

20 25 30

Glu Leu Ala Ala Ala Ala Gly Ala Thr Ala Gly Arg Ile Gly Gly Ala

35 40 45

Gly Arg Val Ala Val Trp Ala Thr Pro Ala Met Glu Thr Gly Val Ala

50 55 60

Val Val Ala Ala Leu Leu Ala Gly Val Ala Ala Val Pro Leu Asn Pro

65 70 75 80

Lys Ser Gly Asp Lys Glu Leu Ala His Ile Leu Ser Asp Ser Ala Pro

85 90 95

Ser Leu Val Leu Ala Pro Pro Asp Ala Glu Leu Pro Pro Ala Leu Gly

100 105 110

Ala Leu Glu Arg Val Asp Val Asp Val Arg Ala Arg Gly Ala Val Pro

115 120 125

Glu Asp Gly Ala Asp Asp Gly Asp Pro Ala Leu Val Val Tyr Thr Ser

130 135 140

Gly Thr Thr Gly Pro Pro Lys Gly Ala Val Ile Pro Arg Arg Ala Leu

145 150 155 160

Ala Thr Thr Leu Asp Ala Leu Ala Asp Ala Trp Gln Trp Thr Gly Glu

165 170 175

Asp Val Leu Val Gln Gly Leu Pro Leu Phe His Val His Gly Leu Val

180 185 190

Leu Gly Ile Leu Gly Pro Leu Arg Arg Gly Gly Ser Val Arg His Leu

195 200 205

Gly Arg Phe Ser Thr Glu Gly Ala Ala Arg Glu Leu Asn Asp Gly Ala

210 215 220

Thr Met Leu Phe Gly Val Pro Thr Met Tyr His Arg Ile Ala Glu Thr

225 230 235 240

Leu Pro Ala Asp Pro Glu Leu Ala Lys Ala Leu Ala Gly Ala Arg Leu

245 250 255

Leu Val Ser Gly Ser Ala Ala Leu Pro Val His Asp His Glu Arg Ile

260 265 270

Ala Ala Ala Thr Gly Arg Arg Val Ile Glu Arg Tyr Gly Met Thr Glu

275 280 285

Thr Leu Met Asn Thr Ser Val Arg Ala Asp Gly Glu Pro Arg Ala Gly

290 295 300

Thr Val Gly Val Pro Leu Pro Gly Val Glu Leu Arg Leu Val Glu Glu

305 310 315 320

Asp Gly Thr Pro Ile Ala Ala Leu Asp Gly Glu Ser Val Gly Glu Ile

325 330 335

Gln Val Arg Gly Pro Asn Leu Phe Thr Glu Tyr Leu Asn Arg Pro Asp

340 345 350

Ala Thr Ala Ala Ala Phe Thr Glu Asp Gly Phe Phe Arg Thr Gly Asp

355 360 365

Met Ala Val Arg Asp Pro Asp Gly Tyr Val Arg Ile Val Gly Arg Lys

370 375 380

Ala Thr Asp Leu Ile Lys Ser Gly Gly Tyr Lys Ile Gly Ala Gly Glu

385 390 395 400

Ile Glu Asn Ala Leu Leu Glu His Pro Glu Val Arg Glu Ala Ala Val

405 410 415

Thr Gly Glu Pro Asp Pro Asp Leu Gly Glu Arg Ile Val Ala Trp Ile

420 425 430

Val Pro Ala Asp Pro Ala Ala Pro Pro Ala Leu Gly Thr Leu Ala Asp

435 440 445

His Val Ala Ala Arg Leu Ala Pro His Lys Arg Pro Arg Val Val Arg

450 455 460

Tyr Leu Asp Ala Val Pro Arg Asn Asp Met Gly Lys Ile Met Lys Arg

465 470 475 480

Ala Leu Asn Arg Asp

485

<210> 3

<211> 37

<212> DNA

<213>It is artificial synthesized

<400> 3

tcgattgcac atatgtcctc tctcttcccg gccctct 37

<210> 4

<211> 39

<212> DNA

<213>It is artificial synthesized

<400> 4

atcggatagc tcgagtcagt cacggttcag cgcccgctt 39

Claims

1. a kind of application of middle short chain acyl CoA synthase in 2- enoyl CoAs in heptan, hexamethylene methylacyl coenzyme A is prepared.

2. application as claimed in claim 1, it is characterised in that step is as follows：

100mM 4- hydroxyethyl piperazineethanesulfonic acids；The glycerine of quality percent by volume 10%, unit g/ml；100mM magnesium chlorides；15~ 50mM2- heptenoic acids, the sodium salt of 2- heptenoic acids, the sodium salt of naphthenic acid or naphthenic acid；20mM atriphos；5mM is auxiliary Enzyme A；The malonyl coenzyme A synzyme of 10 μM of purifying；pH 7.5；

(3) above-mentioned reaction system reacts 7.5~10h under the conditions of 20~24 DEG C, and terminating reaction removes zymoprotein, purified, does It is dry, produce.

3. application as claimed in claim 2, it is characterised in that in the step (1), prepare amino acid sequence such as SEQ ID Middle short chain acyl CoA synthase shown in NO.2 is adopted with the following method：

The malonyl coenzyme A synthase gene matB for expanding streptomyces coelicolor is reacted through PCR using primer matBF and matBR, Then by construction of expression vector and engineering bacteria, induced expression, then through Ni-NTA ni-sepharose purification malonyl coenzyme A synzyme, Produce；

The nucleotide sequence of the primer matBF and matBR is as follows：

matBF:5’-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3’

matBR:5’-ATCGGATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3’。

4. application as claimed in claim 2, it is characterised in that in the step (3), it is using ultrafiltration centrifugation to remove zymoprotein Filtering is removed.

5. application as claimed in claim 2, it is characterised in that in the step (3), described purifying is to use HPLC chromatogram Purified.

6. application as claimed in claim 2, it is characterised in that in the step (3), the drying is to be done using vacuum refrigeration Drying method is carried out.