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Microbial synthesis method of eugenol

A microbial synthesis, eugenol technology, applied in the direction of microorganisms, biochemical equipment and methods, botany equipment and methods, etc., can solve the problem of low eugenol content

Pending Publication Date: 2020-09-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In order to solve the problems existing in the prior art, the present invention provides a microbia

Method used

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  • Microbial synthesis method of eugenol
  • Microbial synthesis method of eugenol
  • Microbial synthesis method of eugenol

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Experimental program
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Embodiment 1

[0031] The acquisition of embodiment 1 (class) coniferyl alcohol acyltransferase

[0032]Coniferyl alcohol acylase can catalyze the acylation reaction of coniferyl alcohol to generate coniferyl acetate. Through bioinformatics analysis and sequence alignment, three coniferyl alcohol acylases were screened out. They are CFAT (GenBank: ABG75942.1 ), CAAT1 (GenBank: KF543260.1) and AT (NCBI: NP_178020.1). The following takes coniferyl alcohol acylase PhCFAT as an example to illustrate how to obtain (like) coniferyl alcohol acylase.

[0033] Combined with the amino acid sequence of coniferyl alcohol acyltransferase CFAT and the codon preference of Escherichia coli, common restriction sites were removed, and a full-length CFAT gene was designed. The nucleic acid sequence is shown in SEQ ID No.2. The CFAT gene was designed to be connected to the downstream of the first T7 promoter of the plasmid pCDFDuet-1, and the P T7 - CFAT gene fragment, the obtained gene fragment is named as s...

Embodiment 2

[0037] The acquisition of embodiment 2 eugenol synthase

[0038] Eugenol synthase catalyzes the reduction reaction of coniferyl acetate to produce eugenol. Four eugenol synthases were screened out through bioinformatics analysis and sequence alignment mining. They are EGS1 (GenBank: ABR24113.1), EGS2 (GenBank: AKB11750.1), APS1 (GenBank: KF543262.1) and AIS1 (GenBank: ACL13526.1), respectively. The following takes eugenol synthase EGS2 as an example to illustrate how to obtain eugenol synthase.

[0039] Combined with the amino acid sequence of eugenol synthase EGS2 and the codon preference of Escherichia coli, the commonly used enzyme cutting sites were removed, and the full-length EGS2 gene was designed. The nucleic acid sequence is shown in SEQ ID No.6. The EGS2 gene was designed to be connected downstream of the second T7 promoter of the plasmid pCDFDuet-1, and the P T7 -EGS2 gene fragment, the obtained gene fragment is named synEGS2.

[0040] The synEGS2 fragment obtai...

Embodiment 3

[0043] Embodiment 3 Construction of recombinant expression vector

[0044] The optimized two types of genes were combined and connected to the plasmid pCDFDuet-1, in which coniferyl alcohol acyltransferase was inserted between the restriction sites Nco I and BamH I, and eugenol synthase was inserted into the restriction site Nde I and Kpn I between. The following takes the recombinant expression vector pCPG1 as an example to illustrate how to construct the recombinant expression vector.

[0045] The synPhCFAT fragment was digested with FastDigest endonuclease Nco I and BamH I, and the reaction system was: 5 μL 10*FD buffer, 2 μL Nco I endonuclease, 2 μL BamH I endonuclease, and 41 μL synCFAT fragment. The reaction conditions are: 37°C, 2h. And use the kit to purify and recover to obtain the synCFAT fragment cut with Nco I and BamH I. The synCFAT fragment obtained after purification and recovery was ligated with the linearized vector backbone pCDFDuet-1 (including the replic...

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Abstract

The invention discloses a microbial synthesis method of eugenol. The amino acid sequence and the nucleotide sequence of coniferyl alcohol acyltransferase CFAT are shown as SEQ ID No.1 and SEQ ID No.2respectively; and the nucleotide sequence of eugenol synthetases EGS2, APS1 and AIS1 are shown as SEQ ID No. 6, SEQ ID No. 9 and SEQ ID No. 11 respectively. 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 a foundation is laid for industrial production of microbial fermentation eugenol.

Description

technical field [0001] The invention belongs to the technical field of biopesticides, and relates to the application of a coniferyl alcohol acyltransferase and three eugenol synthetases in transforming coniferyl alcohol into eugenol in microorganisms. Background technique [0002] Eugenol is a highly efficient botanical biopesticide, which is mainly used to control important agricultural diseases such as tomato gray mold, grape downy mildew and potato late blight. At present, eugenol is prepared by extracting from cloves and other plants in China, but there are problems of raw material sources such as low content and slow growth. [0003] According to literature reports, coniferyl alcohol acyltransferase (CFAT) can catalyze the acylation reaction of coniferyl alcohol to generate coniferyl acetate, and eugenol synthase (EGS) catalyzes the reduction reaction of coniferyl acetate to generate eugenol. figure 1 shown. [0004] Coniferyl alcohol acyltransferase (CFAT) belongs to...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12N15/70C12N15/53C12N15/54
CPCC12P7/22C12N15/70C12N9/0006C12N9/1029C12Y101/01318C12N2800/22
Inventor 赵广荣曹嘉誉
Owner TIANJIN UNIV
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