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Saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and construction method thereof

A technology of Saccharomyces cerevisiae and construction method, applied in the field of recombinant Saccharomyces cerevisiae and construction, and can solve the problems of low purity, unreported zingerone, high cost and the like

Pending Publication Date: 2020-04-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Terpenoids accumulate less in plants, and the extraction and purification of plant raw materials often have low yields, low purity and require a large amount of plant raw materials; the synthesis of terpenoids by chemical synthesis has been widely used, but for terpenoids with complex structures compounds, which remain difficult and costly
Zingerone is only found in Zingiberaceae plants. Although the biosynthetic pathway has been explained, its synthesis regulation mechanism in plant hosts has not been reported. It is still difficult to improve the production of Zingiberone by transforming the original host by means of metabolic engineering.
Therefore, it is imperative to develop a method that can efficiently synthesize natural products, but up to now, there has been no report on the synthesis of zingerone by using synthetic biology technology

Method used

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  • Saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and construction method thereof
  • Saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and construction method thereof
  • Saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Preparation method of each fragment source

[0046](1) The sequence information of the gene element ZSS1 used in the present invention comes from Zingiber zerumbet (Zingiber zerumbet, GenBank: AB247331.1) synthesized by the company through codon optimization of Saccharomyces cerevisiae, and the optimized α-humulene synthase encoding gene ZSS1 The nucleotide sequence is shown in SEQ ID NO.1;

[0047] The sequence information of CYP71BA1 comes from Zingiber zerumbet (GeneBank: AB331234.1), synthesized by the company through Saccharomyces cerevisiae codon optimization, and the optimized nucleotide sequence of the cytochrome P450 enzyme coding gene CYP71BA1 is shown in SEQ ID NO.2 shown.

[0048] The sequence information of AtCPR1 comes from Arabidopsis thaliana (GeneBank: BT008426), which was synthesized by the company through codon optimization of Saccharomyces cerevisiae, and the nucleotide sequence of the optimized cytochrome P450 reductase encoding gene A...

Embodiment 2

[0075] Example 2, Construction of Saccharomyces cerevisiae recombinant strain 1 producing α-humulene, 8-hydroxy-α-humulene and zingerone

[0076] (1) δ-up, P ADH2 -ZSS1-T CYC1 , construction of HIS3-δ-down

[0077] Perform PCR with the PCR templates and primers described in Table 1, respectively, to obtain DNA fragments: M1 (δ-up), M2 (P ADH2 ), M3 (ZSS1), M4 (T CYC1 ), M5 (HIS3), M6 (δ-down).

[0078] Table 1 Primer Sequence

[0079]

[0080] The fragments M2, M3, and M4 are fused into an expression cassette P by fusion PCR ADH2 -ZSS1-T CYC1 ;

[0081] Fragments M5 and M6 were fused into a HIS3-δ-down fragment by fusion PCR.

[0082] The fragments undergoing fusion PCR have 20-30bp homologous sequences to each other, overlap and complement each other, and the fragments can serve as primers and templates for each other. The fused fragments were mixed in an equimolar ratio, and the total amount was greater than 800ng. Add dNTPs, 2×Phanta Max Buffer, DNA polymerase, ...

Embodiment 3

[0113] Example 3, Construction of Saccharomyces cerevisiae recombinant strain 2 producing α-humulene, 8-hydroxy-α-humulene and zingerone

[0114] (1) Construction of tHMG1 expression cassette and ERG20 expression cassette

[0115] Carry out PCR with the PCR template described in table 3 and primer respectively, obtain DNA fragment: M19(P TDH3 ), M20(tHMG1), M21(T CYC1 ), M22 (P PGK1 ), M23 (ERG20), M24 (T CYC1 ).

[0116] Table 3 Primer Sequence

[0117]

[0118] The fragments M19, M20, and M21 were fused into tHMG1 expression cassette P by fusion PCR TDH3 -tHMG1-T CYC1 ; Fragments M22, M23, M24 are passed through fusion PCR, ERG20 expression cassette P PGK1 -ERG20-T CYC1 ;

[0119] For the fragment fusion method, refer to Example 2(1).

[0120] (2) Construction of expression vector P1 containing tHMG1 expression cassette

[0121] Digest P with restriction endonucleases ApaI and PstI TDH3 -tHMG1-T CYC1 and PRS304 plasmid (ATCC, USA), and recover and purify enzy...

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Abstract

The invention discloses a saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and a construction method thereof. The method comprises the following steps: introducing an optimized alpha-humulene synthase encoding gene ZSS1, an optimized cytochrome P450 enzyme encoding gene CYP71BA1, an optimized cytochrome P450 reductase encoding gene AtCPR1 and an optimized dehydrogenase encoding gene ZSD1S114A into saccharomyces cerevisiae by utilizing homologous recombination; and obtaining a saccharomyces cerevisiae recombinant bacterium 1 capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone. Experiments prove that the saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone, constructed by the invention, can produce alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone, and lays a foundation for synthesizing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone by artificial cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Saccharomyces cerevisiae producing α-humulene, 8-hydroxy-α-humulene and zingerone and a construction method. Background technique [0002] Zingerone is the main component of sesquiterpenes in the rhizome of Ginger Ginger. Recent studies have found that Zingerone has anti-inflammatory and anti-cancer effects, so it has attracted much attention. The biosynthesis pathway of zingerone has been clarified, similar to the biosynthesis of other sesquiterpene compounds, its precursor farnesyl pyrophosphate (FPP) comes from the mevalonate pathway (MVA). FPP is catalyzed by α-humulene synthase ZSS1 to synthesize α-humulene. α-humulene, also known as α-caryophyllene or humulene, is a natural monocyclic sesquiterpene compound. The substance is found in the essential oil of hops of the Humulaceae plant, so it is also called humulene. α-humulene in beer has anti-cold effects. α-humu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P5/00C12P7/26C12R1/865
CPCC12N9/88C12N9/0081C12N9/0042C12N9/0004C12Y402/03104C12Y114/15006C12Y106/02004C12P5/002C12P7/26
Inventor 卢文玉张传波
Owner TIANJIN UNIV
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