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Microzyme for producing taxadiene and construction method thereof

A taxadiene and yeast technology is applied in the field of taxadiene-producing yeast and construction to achieve the effect of low cost

Inactive Publication Date: 2015-05-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the production of taxadiene in Escherichia coli has reached a high level, when the research group carried out the subsequent synthesis of taxadiene-5α-ol, only a small amount of the product was detected, which may be due to the P450 Enzymes struggle to perform their catalytic functions effectively in the context of E. coli

Method used

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  • Microzyme for producing taxadiene and construction method thereof
  • Microzyme for producing taxadiene and construction method thereof
  • Microzyme for producing taxadiene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Obtaining genes related to taxadiene synthesis pathway in yeast

[0037] A. Acquisition of tHMGR gene (yeast truncated HMG-CoA reductase gene)

[0038] Primers were designed according to the yeast HMG-CoA reductase gene sequence, SEQ ID NO: 1tHM-F: 5'-ATGGTTTTAACCAATAAAACAGTCATTTCT-3' and SEQ ID NO: 2tHM-R: 5'-TTAGGATTTAATGCAGGTGACG-3', using the W303-1A strain genome As a template, PCR was performed using pfu enzyme (95°C, 3min; 95°C, 30s, 57°C, 35s, 72°C, 2min, 32cycles; 72°C, 5min; 4°C, +∞) to amplify to obtain a 1509bp fragment. It was cloned into pMD18-T vector and sequenced to confirm that no mutation occurred. The nucleotide sequence is shown in SEQ ID NO:3, and the amino acid sequence is SEQ ID NO:4.

[0039] B. Acquisition of ERG20 gene (yeast FPP and GPP synthase gene)

[0040] Primers were designed according to the yeast ERG20 gene sequence, SEQ ID NO: 5E20-F: 5'-ATGGCTTCAGAAAAGAAAT-3' and SEQ ID NO: 6E20-R: 5'-CTATTTGCTTCTCTTGTAAACTT-3', using ...

Embodiment 2

[0045] Example 2: Construction of the vector

[0046] A. Construction of the vector SyBE_001187 (the expression vector of truncated HMG-CoA synthase gene and ERG20 gene)

[0047] ①The yeast constitutive promoter TDH3p, tHMGR gene and terminator were spliced ​​together by OE-PCR method to obtain fragments containing XhoI and ApaI sites at both ends, which were connected to the integrated vector pRS403;

[0048] ②The yeast constitutive promoter TDH3p, ERG20 gene, and terminator were spliced ​​together by OE-PCR method to obtain fragments containing SacI and XhoI at both ends, which were connected to the vector obtained in step ① to obtain the vector SyBE_001187 (see figure 2 );

[0049] B. Construction of the vector SyBE_001173 (containing the optimized taxus-derived GGPP synthase gene and the optimized taxadiene synthase gene expression vector):

[0050] ①The promoter TDH3p (GAL1p, etc. can also be selected), GGPP synthase gene and terminator are spliced ​​together by OE-PCR...

Embodiment 3

[0054] Example 3: Acquisition of Taxadiene-Producing Yeasts

[0055] Yeast transformation of the vector was performed using the lithium acetate method. The integrative plasmid was linearized in advance to integrate into the corresponding site of Saccharomyces cerevisiae BY4742 genome, and the episomal plasmid was directly transformed into Saccharomyces cerevisiae BY4742. After transformation, the yeast were screened using SD-drop solid medium (deaminated yeast nitrogen source, 6.7g / l; glucose, 20g / l; Dropout mix, 0.2%; solid supplemented with 2% agar powder), and the obtained transformants Transfer to liquid medium and cultivate for 36h, extract yeast plasmid or genome as template, and carry out PCR verification to eliminate the interference of false positives. To confirm the correct positive strain, plate streak or glycerol bacteria preservation.

[0056] Using the MVA pathway existing in yeast, the key genes HMG-CoA reductase gene (HMGR) and FPP synthase gene (ERG20) invol...

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Abstract

The invention discloses a microzyme for producing taxadiene and a construction method thereof. The method comprises the following steps: (1) constructing a carrier SyBE_001173; (2) constructing a carrier SyBE_001187; and (3) introducing the carriers SyBE_001173 and SyBE_001187 into Saccharomyces cerevisiae W303-1A or Saccharomyces cerevisiae BY4742 to obtain the microzyme for producing taxadiene. Compared with the prior art, the microzyme for producing taxadiene disclosed in the invention has the advantages of environmental protection and lower cost, and provides a feasible method for producing taxadiene.

Description

technical field [0001] The invention relates to a yeast for producing taxadiene and a construction method. Background technique [0002] Taxol is an effective anticancer drug. The traditional method of obtaining it includes plant extraction of Taxus chinensis, but the content of Taxus chinensis is not only extremely low, but also the deforestation of Taxus chinensis trees is an environmentally unfriendly behavior; although the method of plant cell culture The problem of large-scale deforestation of yew is solved, but the efficiency of obtaining paclitaxel is not high due to the long growth cycle of plant cells; the chemical synthesis method not only has complicated synthesis steps, but also has a low yield in each step. Large-scale production is bound to generate a large amount of polluting waste and is expensive. [0003] Major demands in the fields of human health, energy, and the environment are also driving the rapid development of synthetic biology. Functional gene mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/81C12N1/19C12R1/865
Inventor 元英进闫慧芳丁明珠贾云婧
Owner TIANJIN UNIV
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