阅读说明：本技术 一种丁香酚的微生物合成方法 (Microbial synthesis method of eugenol ) 是由 赵广荣 曹嘉誉 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种丁香酚的微生物合成方法。本发明提供的松柏醇酰基转移酶CFAT,其氨基酸序列和其核苷酸序列分别用SEQ ID No.1和SEQ ID No.2所示,丁香酚合成酶EGS2、APS1和AIS1,其核苷酸序列分别用SEQ ID No.6、SEQ ID No.9和SEQ ID No.11所示。松柏醇酰基转移酶和丁香酚合成酶EGS2组合,或松柏醇酰基转移酶和丁香酚合成酶APS1组合,或松柏醇酰基转移酶和丁香酚合成酶AIS1组合,能高效催化松柏醇合成丁香酚,减少中间体积累,为微生物发酵丁香酚的工业化生产奠定了基础。(The invention discloses a microbial synthesis method of eugenol. The amino acid sequence and the nucleotide sequence of coniferyl alcohol acyltransferase CFAT provided by the invention are respectively shown by SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequences of eugenol synthetase EGS2, APS1 and AIS1 are respectively shown by SEQ ID No.6, SEQ ID No.9 and SEQ ID No. 11. The combination of coniferyl alcohol acyltransferase and eugenol synthetase EGS2, or the combination of coniferyl alcohol acyltransferase and eugenol synthetase APS1, or the combination of coniferyl alcohol acyltransferase and eugenol synthetase AIS1 can efficiently catalyze coniferyl alcohol to synthesize eugenol, reduce the accumulation of intermediates, and lay the foundation for the industrial production of eugenol by microbial fermentation.)

1. A microbial synthesis method of eugenol is characterized in that coniferyl alcohol acyltransferase CFAT and eugenol synthetase EGS2 are expressed in a combined mode, and the coniferyl alcohol is used as a substrate to directly synthesize the eugenol.

2. The microbial synthesis method of eugenol according to claim 1, wherein the amino acid sequence and gene sequence of coniferyl alcohol acyltransferase CFAT are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and the amino acid sequence and gene sequence of eugenol synthase EGS2 are shown as SEQ ID No.5 and SEQ ID No.6, respectively.

3. A microbial synthesis method of eugenol is characterized in that coniferyl alcohol acyltransferase CFAT and eugenol synthetase APS1 are expressed in a combined mode, and the coniferyl alcohol is used as a substrate to directly synthesize the eugenol.

4. The microbial synthesis method of eugenol according to claim 3, wherein the amino acid sequence and gene sequence of coniferyl alcohol acyltransferase CFAT are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and the gene sequence of eugenol synthase APS1 is shown as SEQ ID No. 9.

5. A microbial synthesis method of eugenol is characterized in that coniferyl alcohol acyltransferase CFAT and eugenol synthetase AIS1 are expressed in a combined mode, and the coniferyl alcohol is used as a substrate to directly synthesize the eugenol.

6. The microbial synthesis method of eugenol according to claim 5, wherein the amino acid sequence and the gene sequence of coniferyl alcohol acyltransferase CFAT are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and the amino acid sequence and the gene sequence of eugenol synthase AIS1 are shown as SEQ ID No.10 and SEQ ID No.11, respectively.

7. The coniferyl alcohol acyltransferase CFAT is combined with eugenol synthetase EGS2 or APS1 or AIS1, coniferyl alcohol is used as a substrate, and the application in the synthesis of eugenol is realized.

Technical Field

The invention belongs to the technical field of biological pesticides, and relates to application of coniferyl alcohol acyltransferase and three eugenol synthetases in synthesis of eugenol by converting coniferyl alcohol in microorganisms.

Background

Eugenol is a high-efficiency botanical biopesticide and is mainly used for preventing and treating important agricultural diseases such as tomato gray mold, grape downy mildew, potato late blight and the like. At present, eugenol is extracted and prepared from plants such as clove at home, and the problems of low content, slow growth and other raw material sources exist.

It is reported in the literature that coniferyl alcohol acyltransferase (CFAT) can catalyze the acylation reaction of coniferyl alcohol to generate coniferyl acetate, and eugenol synthetase (EGS) can catalyze the reduction reaction of coniferyl acetate to generate eugenol, and the pathway is shown in FIG. 1.

Coniferyl alcohol acyltransferase (CFAT) belongs to BAHD acyltransferase family, and has been identified from petunia, Prunus cerasifera, and Malus pumila and enzymatically characterized^[1-4]. Eugenol synthase is an NADPH-dependent reductase that reduces ester linkages on coniferyl acetate side chains to allyl groups. At present, eugenol synthetase derived from various plants such as basil, hop, morning glory, Chinese rose, strawberry, gymnadenia conopsea has been identified [5-9]. The yield of allogenic synthesis of eugenol in Escherichia coli is low and is below 20 mg/L. For example, the combination of LtCAAT1/LtAPS1 synthesizes about 27mg/L eugenol [ 10%]The yield is the highest reported in the literature at present.

[1]Dexter R,Qualley A,Kish C M,et al.Characterization of a petuniaacetyltransferase involved in the biosynthesis of the floral volatileisoeugenol[J].Plant J.,2007,49(2):265-275.

[2]Koeduka T,Orlova I,Baiga T J,et al.The lack of floral synthesisand emission of isoeugenol in petunia axillaris subsp.Parodii is due to amutation in the isoeugenol synthase gene[J].Plant J., 2009,58(6):961-969.

[3]Zhang T,Huo T,Ding A,et al.Genome-wide identification,characterization,expression and enzyme activity analysis of coniferyl alcoholacetyltransferase genes involved in eugenol biosynthesis in Prunus mume[J].PLoS One,2019,14(10):e0223974.

[4]Yauk Y K,Souleyre E J F,Matich AJ,et al.Alcohol acyl transferase1links two distinct volatile pathways that produce esters andphenylpropenesin apple fruit[J].Plant J.,2017,91(2): 292-305.

[5]Koeduka T,Louie G V,Orlova I,et al.The multiple phenylpropenesynthases in both Clarkia breweri and Petunia hybrida represent two distinctprotein lineages[J].Plant J.,2008,54(3): 362-374.

[6] Cloning and expression analysis of the Everest eugenol synthase gene RheGS1 [ J ] Chinese agricultural science, 2012,45(3):590 Amplifier 597.

[7] Cloning and expression analysis of the eugenol synthase gene RcEGS1 from Rosa chinensis (Rosa chinensis), J.J.. Hill., J.Hill., 2012,39(7):1387.

[8]Araguez I,Osorio S,Hoffmann T,et al.Eugenol production in achenesand receptacles of strawberry fruits is catalyzed by synthases exhibitingdistinct kinetics[J].Plant Physiol.2013, 163(2):946-958.

[9]Gupta AK,Schauvinhold I,Pichersky E,et al.Eugenol synthase genesin floral scent variation in Gymnadenia species[J].Funct.Integr.Genomics,2014,14(4):779-788.

[10]Kim S J,Vassao D G,Moinuddin S G,et al.Allyl/propenyl phenolsynthases from the Creosote bush and engineering production of specialty/commodity chemicals,eugenol/isoeugenol,in Escherichia coli[J].Arch.Biochem.Biophys.,2014,541:37-46.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a microbial synthesis method of eugenol, which solves the problem of low content of eugenol prepared in the prior art.

The technical scheme of the invention is summarized as follows:

a microbial synthesis method of eugenol comprises expressing coniferyl alcohol acyltransferase CFAT and eugenol synthetase EGS2 in combination, and directly synthesizing eugenol with coniferyl alcohol as substrate. The amino acid sequence and the gene sequence of the coniferyl alcohol acyltransferase CFAT are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the amino acid sequence and the gene sequence of the eugenol synthetase EGS2 are respectively shown as SEQ ID No.5 and SEQ ID No. 6.

A microbial synthesis method of eugenol comprises expressing coniferyl alcohol acyltransferase CFAT and eugenol synthetase APS1 in combination, and directly synthesizing eugenol with coniferyl alcohol as substrate. The amino acid sequence and the gene sequence of coniferyl alcohol acyltransferase CFAT are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the gene sequence of eugenol synthetase APS1 is shown as SEQ ID No. 9.

A microbial synthesis method of eugenol comprises expressing coniferyl alcohol acyltransferase CFAT and eugenol synthetase AIS1 in combination, and directly synthesizing eugenol with coniferyl alcohol as substrate. The amino acid sequence and the gene sequence of coniferyl alcohol acyltransferase CFAT are respectively shown as SEQ ID No.1 and SEQ ID No.2, and the amino acid sequence and the gene sequence of eugenol synthetase AIS1 are respectively shown as SEQ ID No.10 and SEQ ID No. 11.

The coniferyl alcohol acyltransferase CFAT is combined with eugenol synthetase EGS2 or APS1 or AIS1, coniferyl alcohol is used as a substrate, and the application in the synthesis of eugenol is realized.

The invention has the advantages that:

the invention converts coniferyl alcohol into eugenol by a microbiological method, screens the combined expression of coniferyl alcohol acyltransferase and three eugenol synthetases, establishes a biosynthesis method, and directly synthesizes the eugenol by taking the coniferyl alcohol as a substrate. The invention screens out the optimal enzyme and the optimal combination thereof through the expression of escherichia coli, the yield of the eugenol reaches more than 200mg/L, and the invention is beneficial to the industrial production of the eugenol.

Drawings

FIG. 1 is a pathway for converting coniferyl alcohol into eugenol by a microbiological method;

FIG. 2 is a gas chromatogram of a fermentation sample of strain BEG 1;

FIG. 3 is a mass spectrum of a fermentation sample of the strain BEG 1;

FIG. 4 is a standard mass spectrum of eugenol.

Detailed Description

Coli strains e.coli BL21(DE3) and e.coli DH5 α used in the present invention were purchased from beijing hologold biotechnology limited.

The LB medium consisted of: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, the balance being water, sterilizing at 121 ℃ under 0.1MPa for 20 min.

The composition of the M9Y culture medium is as follows: 6.8g/L Na₂HPO₄、3.0g/LKH₂PO₄、1.0g/L NaCl、0.5g/L NH₄Cl, 1.0 g/L yeast powder and the balance ofSterilizing with water and culture medium under 0.1Mpa at 121 deg.C for 20 min. After sterilization, the final concentration of 2mM MgSO was added₄、0.1mM CaCl₂And 10g/L glucose.

The present invention will be further described with reference to the following examples.