Method for improving isoprene synthesis capability of saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and isoprene, which is applied in the field of bioengineering, can solve problems such as improving the synthesis ability of weak isoprene, and achieve the effects of improving the synthesis ability of isoprene, enhancing the strength, and enhancing the transcription level.

Active Publication Date: 2017-02-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The weak strength of the downstream isoprene synthesis pathway has become a bottleneck problem limiting the improvement of isoprene synthesis capacity

Method used

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  • Method for improving isoprene synthesis capability of saccharomyces cerevisiae
  • Method for improving isoprene synthesis capability of saccharomyces cerevisiae
  • Method for improving isoprene synthesis capability of saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of isoprene synthesis pathway in Saccharomyces cerevisiae

[0048] The isoprene synthase gene from Populus alba was optimized with codon optimization software, and the optimized sequence was artificially synthesized. The optimized IspS was cloned into p416xwp01 plasmid with primers IspS-F(26) and IspS-R(27), and transformed into Saccharomyces cerevisiae to synthesize isoprene.

Embodiment 2

[0049] Example 2: A method for constructing a GAL4 overexpression strain.

[0050] The plasmid PLGL-△GAL1 / 7 / 10-PGAL4-GAL4 used to construct the GAL4 overexpression strain is as follows:

[0051] Using the plasmid PUG6 as a template, the loxp-KanMX-loxp fragment was amplified with primers loxp-F1(30) and loxp-R1(31). Using the plasmid pESC-URA as a template, use primers loxp-F2 (32) and loxp-R2 (33) to amplify the ori fragment, and use primers TADH1-F2 (34) and TCYC1-R2 (35) to amplify the T ADH1 -MCS1-P GAL10 -P GAL1 -MCS2-T CYC1 segment. The three fragments were fused to construct plasmid PMRI-28.

[0052] Using the Saccharomyces cerevisiae BY4741 genome as a template, the upstream 534bp of the GAL1 / 7 / 10 segment was amplified with primers △GAL1 / 7 / 10-UP-F1(22) and △GAL1 / 7 / 10-UP-R1(23) , using primers ΔGAL1 / 7 / 10-DN-F1 (24) and ΔGAL1 / 7 / 10-DN-R1 (25) to amplify the downstream 671bp. The two fragments have homologous sequences, and the two fragments are fused by overlapping...

Embodiment 3

[0062] Example 3: Determination of IspS transcript levels.

[0063] The starting strain (C) and the GAL4 overexpression strain (E1) were cultured in liquid SS-URA medium. During the shake flask fermentation process, 2 mL of fermentation broth was taken respectively at 18 hours, 24 hours, 48 ​​hours and 72 hours.

[0064] Total RNA of each sample was extracted with an RNA extraction kit (Takara). Genomic DNA was removed using a reverse transcription kit (Takara) and reverse transcribed to obtain cDNA. The experimental operation was carried out according to the instructions of the kit.

[0065] Using the ACT1 gene as an internal reference, real-time fluorescent quantitative PCR was performed on IspS to obtain an amplification curve and record the CT value. use 2 -△△CT Relative transcript levels of IspS were calculated.

[0066] Table 1 Relative transcript levels of IspS

[0067]

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Abstract

The invention provides a method for improving an isoprene synthesis capability of saccharomyces cerevisiae. An IspS (isoprene synthase) gene sequence SEQ ID NO: 1 from a white poplar is cloned to a plasmid through enzyme digestion and connection and is converted into a saccharomyces cerevisiae parent body, so that isoprene is synthesized in the saccharomyces cerevisiae; a transcriptional level of IspS gene is enhanced through GAL4 over-expression; the catalytic activity of IspS is improved through orthogenesis. The saccharomyces cerevisiae and the isoprene synthase are combined together, so that the yield of isoprene is improved to be 8.2 times as much as the original yield.

Description

technical field [0001] The invention relates to a method for improving the synthesis ability of isoprene in Saccharomyces cerevisiae, belonging to the field of bioengineering. Background technique [0002] Isoprene is an important monomer for synthetic rubber, and its annual consumption exceeds 1 million tons. There are many plants in nature that release isoprene. Isoprene is released into the atmosphere as a gas and cannot be easily collected, making it difficult to use in industrial production. The main source of isoprene at present is the C5 fraction. Due to the non-renewable petroleum resources and the increasingly serious environmental pollution caused by petrochemical industry, the synthesis of isoprene using microorganisms as cell factories has become an important research topic. Most microorganisms have 2-methylerythritol phosphate (MEP) or mevalonate (MVA) pathways, which can synthesize dimethylallyl pyrophosphate (DMAPP), a precursor of isoprene. Transferring t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P5/00C12R1/865
CPCC07K14/395C12N9/88C12P5/007C12Y402/03027
Inventor 于洪巍叶丽丹王凡
Owner ZHEJIANG UNIV
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