Effects on 2,3-butanediol Production of Saccharomyces cerevisiae: gpd2 Gene Knockout by CRISPR/Cas9 Technology

doi:10.11924/j.issn.1000-6850.casb2020-0127

Abstract

Abstract:

This study aims at using CRISPR/Cas9 gene editing technology to knock out the glycerol-3-phosphate dehydrogenase gene (gpd2) in Saccharomyces cerevisiae, and investigating its effect on 2,3-butanediol production. Designing donor and guide RNA (gRNA) based on glycerol-3-phosphate dehydrogenase gene (gpd2) of S. cerevisiae W5, and linking the knockout vector could express Cas9 protein to the gRNA fragment, then the recombinant plasmid and donor DNA fragment were transformed into S. cerevisiae W5 cells. The gpd2 gene knocked-out strain was obtained through phenotypic screening and PCR validation, indicating that the gpd2 gene was successfully knocked out. Compared with the original strain, the gpd2 gene knocked-out strain increased ethanol production by 24.65%, and decreased glycerol production by 22.01% and 2,3-butanediol production by 10.60%. The production of 2,3-butanediol did not increase by knocking out the gpd2 gene, probably because that gradually accumulated NADH was preferentially oxidized by a large amount of alcohol dehydrogenase in the cell, and affected the production of ethanol rather than the synthesis of 2,3-butanediol. In this study, a suitable gene knockout system is constructed for S. cerevisiae, which has practical reference for further exploring the relationship between other metabolites of S. cerevisiae synthesis and 2,3-butanediol synthesis.

Key words: Saccharomyces cerevisiae, CRISPR/Cas9, 2,3-butanediol, vector construction, metabolic engineering

CLC Number:

S182

Liu Lei, Li Na, Jiang Xueyong, Sun Jian, Lv Yuze, Ge Jingping. Effects on 2,3-butanediol Production of Saccharomyces cerevisiae: gpd2 Gene Knockout by CRISPR/Cas9 Technology[J]. Chinese Agricultural Science Bulletin, 2020, 36(29): 69-77.

Figures/Tables 10

References 23

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质粒名称	质粒特征	来源
pUDP004	amdS、TEF1_prom-Cas9-PHO5_term、panARSopt、GAP_prom-CYC1_term	购自ADDGENE
pUDP004-gpd2	pUDP004、GAP_prom-sgRNA(gpd2)-CYC1_term	本实验室构建
pMD18-T	Amp^R	购自宝生物工程（大连）有限公司

质粒名称	质粒特征	来源
pUDP004	amdS、TEF1_prom-Cas9-PHO5_term、panARSopt、GAP_prom-CYC1_term	购自ADDGENE
pUDP004-gpd2	pUDP004、GAP_prom-sgRNA(gpd2)-CYC1_term	本实验室构建
pMD18-T	Amp^R	购自宝生物工程（大连）有限公司

引物名称	引物序列(5′→3′)	引物长度/bp
GPD2-6F	CAAAAAGATC	10
GPD2-6R	TCAGGATCTT	10
GPD2-20F	GATCTTTTACACTCCATCAAGT	22
GPD2-20R	TTGATGGAGTGTAAAAGATCGA	22
GPD2-ORF-F	ATGCTTGCTGTCAGAAGATTAACAAGATACACATTCC	37
GPD2-ORF-R	CTATTCGTCATCGATGTCTAGCTCTTCAATCATCTC	36
GPD2-F1	CAGACGCAGCAGCAAGTAAC	20
GPD2-R1	TTCGTACACAGCGTTGACCT	20
GPD2-F2	AAAGAGGCAAGGGGAGCGAAGGAAAAGGA	29
GPD2-R2	TCGCTCCCCTTGCCTCTTTTTCCCCCAACCA	31
GPD2-300F	GATGGGTTGCTGAGGGGAAG	20
GPD2-300R	ACTGGAGAGCCGTCAGTAGT	20
HH-BbsI-F	GCAAATCGTCTTCACCTGAAGACTG	25
M13F-47	CGCCAGGGTTTTCCCAGTCACGAC	24
M13R-48	AGCGGATAACAATTTCACACAGGA	24
GPD2-P1	GCTCGTCGATCTTTTACACTCCATCAA	27
GPDpro-F	CGGTAGGTATTGATTGTAATTCTG	24
CYC1-R	GCGTGAATGTAAGCGTGAC	19

引物名称	引物序列(5′→3′)	引物长度/bp
GPD2-6F	CAAAAAGATC	10
GPD2-6R	TCAGGATCTT	10
GPD2-20F	GATCTTTTACACTCCATCAAGT	22
GPD2-20R	TTGATGGAGTGTAAAAGATCGA	22
GPD2-ORF-F	ATGCTTGCTGTCAGAAGATTAACAAGATACACATTCC	37
GPD2-ORF-R	CTATTCGTCATCGATGTCTAGCTCTTCAATCATCTC	36
GPD2-F1	CAGACGCAGCAGCAAGTAAC	20
GPD2-R1	TTCGTACACAGCGTTGACCT	20
GPD2-F2	AAAGAGGCAAGGGGAGCGAAGGAAAAGGA	29
GPD2-R2	TCGCTCCCCTTGCCTCTTTTTCCCCCAACCA	31
GPD2-300F	GATGGGTTGCTGAGGGGAAG	20
GPD2-300R	ACTGGAGAGCCGTCAGTAGT	20
HH-BbsI-F	GCAAATCGTCTTCACCTGAAGACTG	25
M13F-47	CGCCAGGGTTTTCCCAGTCACGAC	24
M13R-48	AGCGGATAACAATTTCACACAGGA	24
GPD2-P1	GCTCGTCGATCTTTTACACTCCATCAA	27
GPDpro-F	CGGTAGGTATTGATTGTAATTCTG	24
CYC1-R	GCGTGAATGTAAGCGTGAC	19