Construction and application of a Saccharomyces cerevisiae engineered strain producing farnesene
A technology for Saccharomyces cerevisiae and engineering bacteria, applied in the field of synthetic biology, can solve the problems of increased burden of bacterial growth, complicated steps, and inability to provide enough microbial cell farnesene.
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Embodiment 1
[0035] Example 1: Overexpression of tHMG1 enhances farnesene synthesis in Saccharomyces cerevisiae
[0036] 1.1 Codon optimization and integrated expression plasmid construction of plant-derived farnesene synthase encoding gene
[0037] The sequence of soybean-derived farnesene synthase encoding gene (Glyma.13G321100) was obtained through SoyBase, and the sequence was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and gene synthesis. The optimized gene was named Fsso, and Fsso was connected to the pUC57 vector by Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the plasmid pUC57-Fsso, and the storage host was Escherichia coli JM109.
[0038] The plasmid pUC57-Fsso was extracted, and the fragment Fsso was recovered by digesting the gel with SalI and NheI.
[0039] Using the genome of Saccharomyces cerevisiae YPH499 as a template, primers F13 and F14, F11 and F12 were amplified to obtain P TDH3 and T TPI1 ; Using the PMGKR plasmid as a template, lox...
Embodiment 2
[0047] Example 2: Effect of GAL promoter on farnesene synthesis ability
[0048] 2.1 P GAL1 -Fsso-T TPI1 Construction of integrated expression plasmid
[0049] Using the genome of Saccharomyces cerevisiae YPH499 as a template, primers F23 and F24, F11 and F22 were amplified to obtain P GAL10 -P GAL1 and T TPI1 ; Using the PMGKR plasmid as a template, loxp-KANMX-loxp was obtained by amplification with primers F9 and F10; loxp-KANMX-loxp, P GAL10 -P GAL1 and T TPI1 Fusion PCR with primers to obtain loxp-KANMX-loxp-T TPI1 -P GAL10 -P GAL1 , ligated to pMD-19T simple vector to obtain Ts-XTP101. Inverse PCR amplification was performed with primers F25 and F26, and digested with EcoRI and BamHI to obtain the digested Ts-XTP101. Using pUC57-Fsso as a template, Fsso was obtained by amplification with primers F27 and F28, and then connected to pMD-19T simple vector to obtain Ts-Fsso. Fsso was linked with Ts-XTP101 to obtain Ts-XTP101so; Ts-XTP101so was digested with XbaI an...
Embodiment 3
[0053] Example 3: ERG20 fusion expression with Fsso reduces FPP loss
[0054] 3.1 P TDH3 -ERG20-Fsso-T TPI1 Construction of integrated expression plasmid
[0055] Using the plasmid Ts-XTP prepared in Example 1 as a template, reverse PCR was performed with primers F25 and F29, and the PCR product was digested with BamHI and EcoRI to obtain the digested Ts-XTP. Using Saccharomyces cerevisiae YPH499 genomic DNA as template, ERG20 was obtained by amplification with primers F30 and F31. Using plasmid pUC57-Fsso as template, Fsso was obtained by amplification with primers F32 and F33. Using ERG20 and Fsso as templates, primers F30 and F33 were fused and amplified to obtain ERG20-Fsso. ERG20-Fsso was digested with BamHI and EcoRI and ligated with the digested Ts-XTP vector to obtain Ts-XTPEso. Digest with XbaI and AflII, and recover the fragment XTPEso by gel recovery. Using Ts-GAL80 as a template, reverse PCR was performed with primers F19 and F21, and the PCR product was dige...
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