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Saccharomyces cerevisiae engineering bacteria for producing farnesene and application thereof

A technology of Saccharomyces cerevisiae and engineering bacteria, which is applied in the field of synthetic biology, can solve the problem that the influence of farnesene synthesis is not explained, and achieve the effect of simple gene manipulation

Pending Publication Date: 2020-12-15
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, there are also studies that use overexpression of all genes of the mevalonate pathway to increase the metabolic flux of the mevalonate pathway, but so far, there has been no study on the effects of different mevalonate pathway enhancement methods and the expression of farnesene synthase. Related reports on the influence of farnesene synthesis

Method used

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  • Saccharomyces cerevisiae engineering bacteria for producing farnesene and application thereof
  • Saccharomyces cerevisiae engineering bacteria for producing farnesene and application thereof
  • Saccharomyces cerevisiae engineering bacteria for producing farnesene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Construction of Saccharomyces cerevisiae farnesene synthetic strain

[0046] 1.1 Codon optimization of plant-derived farnesene synthase encoding gene and construction of integrated expression plasmid

[0047] Apple (Fsap, GenBank: AY787633.1), Soybean (Fsso, Glyma.13G321100), Artemisia annua (Fsaa, GenBank: AF374462.1) and Orange (Fscj, GenBank: AY835398.1) sources were obtained through NCBI and SoyBase. The gene sequence encoding ene synthase was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and gene synthesis. The codon-optimized Fsap (SEQ ID NO.1), Fsso (SEQ ID NO.2), Fsaa (SEQ ID NO.3) and Fscj (SEQ ID NO.4) were respectively connected to the pUC57 vector to obtain the plasmid pUC57 -Fsap, pUC57-Fsso, pUC57-Fsaa, pUC57-Fscj, the preservation host is Escherichia coli JM109. Plasmids pUC57-Fsso, pUC57-Fsaa, and pUC57-Fscj were digested with SalI and NheI, and fragments Fsso, Fsaa, and Fscj were obtained by gel recovery. pUC57-Fsa...

Embodiment 2

[0053] Example 2: Enhancing the Mevalonate Metabolic Pathway and Improving the Synthesis Ability of Engineering Bacteria Farnesene

[0054] 2.1 Mevalonate pathway gene enhanced integration expression plasmid construction

[0055] Using the genome of Saccharomyces cerevisiae YPH499 as a template, primers P5 and P6, P7 and P8, and P9 and P10 were used to amplify HMG1, P GAL10 -P GAL1 and ERG20; use primers P5 and P10 to obtain HP20 by fusion PCR amplification, and connect it to the pMD-19Tsimple vector; after the sequence is correct, digest it with SmaI, and recover from the gel to obtain HP20. Using the genome of Saccharomyces cerevisiae CICC 31906 as a template, primers P1 and P2, P3 and P4 were respectively amplified and then fused, connected to pMD-19T simple vector to obtain plasmid Ts-HIS3(R), digested with SmaI and connected with HP20 to obtain Integrate the expression plasmid Ts-HIS3-HPE ( Figure 7 ).

[0056] Using the genome of Saccharomyces cerevisiae YPH499 as a...

Embodiment 3

[0063] Example 3: Effects of Chromosomal Integration of Different Copy Numbers of HMG1 on Farnesene Synthesis

[0064] 3.1 Construction of Chromosomal Integration HMG1 Expression Plasmid

[0065] Using the genome of Saccharomyces cerevisiae YPH499 as a template, primers P5 and P6, P7 and P48 amplified HMG1 and P GAL10 -P GAL1 , HP was amplified by fusion PCR, connected to the pMD-19T simple vector, digested with SmaI and SalI after correct sequencing, recovered from gel to obtain HP, and filled with sticky ends as blunt ends. Using Ts-HIS3(R) as a template, reverse PCR amplification was performed with primers P2 and P3, and the vector was obtained by digestion with SmaI and SalI. The fragment was connected with the vector to obtain the integrated expression plasmid Ts-HIS3-HP ( Figure 13 ).

[0066] Using the genome of Saccharomyces cerevisiae YPH499 as a template, primers P47 and P6, P7 and P49 were used to amplify HMG1, P GAL10 -P GAL1 , HP was amplified by fusion PCR...

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Abstract

The invention discloses saccharomyces cerevisiae engineering bacteria for producing farnesene and application thereof, and belongs to the technical field of synthetic biology. According to the saccharomyces cerevisiae engineering bacteria, farnesene synthetase Fsso with high conversion efficiency in saccharomyces cerevisiae is obtained through screening; all gene-enhanced farnesene synthetic strains with different copy numbers of HMG1 and mevalonic acid paths are constructed, and that single-copy and double-copy HMG1 are most beneficial to continuous synthesis and accumulation of farnesene isconfirmed; and the accumulation amount of farnesene can be further increased by simultaneously controlling expression of HMG1 and Fsso through a GAL promoter. The constructed farnesene synthetic strains are easy in gene operation, and 1.11g / L farnesene can be accumulated at most through shake flask fermentation.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae engineering bacterium for producing farnesene and an application thereof, belonging to the field of synthetic biology. Background technique [0002] In nature, farnesene exists in two forms: α-farnesene and β-farnesene. In agriculture, farnesene can resist the attack of pests on crops; in industry, farnesene can be used as a substitute for petroleum because of its high hydrophobicity and high-density energy storage; in the pharmaceutical industry, farnesene can be used as a synthetic vitamin Precursor of E. The content of farnesene in plants is very low, and it takes a lot of manpower and material resources to extract it from plants; it is difficult to obtain monoploid products through chemical synthesis; microorganisms have become the most popular choice because of their rapid growth, strong synthesis specificity, and low substrate cost. Research focus on farnesene synthesis. Saccharomyces cerevis...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/60C12N15/53C12N15/81C12P5/02C12R1/865
CPCC12N9/88C12N9/0006C12N15/81C12P5/026C12Y101/01034Y02E50/10
Inventor 石贵阳王均华李由然朱惠霖张梁丁重阳徐沙顾正华
Owner JIANGNAN UNIV
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