CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响

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

中国农学通报 ›› 2020, Vol. 36 ›› Issue (29): 69-77.doi: 10.11924/j.issn.1000-6850.casb2020-0127

所属专题：生物技术

CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响

刘磊¹^,²(), 李娜¹^,², 姜雪雍¹^,², 孙健¹^,², 吕雨泽¹^,², 葛菁萍¹^,²()

¹黑龙江大学农业微生物技术教育工程研究中心,哈尔滨 150500
²黑龙江大学生命科学学院微生物省高校重点实验室,哈尔滨 150080

收稿日期:2020-05-25 修回日期:2020-07-27 出版日期:2020-10-15 发布日期:2020-10-16
通讯作者: 葛菁萍
作者简介:刘磊,男,1996年出生,山东菏泽人,硕士,研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号224信箱黑龙江大学生命科学学院,Tel：0451-86609016,E-mail：liuleiheida@163.com
基金资助:
国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492);黑龙江省麻类（工业）产业技术协同创新体系;黑龙江大学研究生创新科研项目(YJSCX2020-204HLJU)

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

Liu Lei¹^,²(), Li Na¹^,², Jiang Xueyong¹^,², Sun Jian¹^,², Lv Yuze¹^,², Ge Jingping¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
² Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2020-05-25 Revised:2020-07-27 Online:2020-10-15 Published:2020-10-16
Contact: Ge Jingping

摘要/Abstract

摘要：

旨在利用CRISPR/Cas9基因编辑技术敲除酿酒酵母甘油-3-磷酸脱氢酶基因(gpd2),探究其对2,3-丁二醇产量的影响。根据酿酒酵母(Saccharomyces cerevisiae)W5甘油-3-磷酸脱氢酶基因(gpd2)设计供体片段及gRNA片段,将gRNA片段与可表达Cas9蛋白的敲除载体相连,之后将重组质粒及供体DNA片段转化到S. cerevisiae W5细胞中,根据表型筛选及PCR验证获得gpd2基因缺失菌株。结果表明目的基因gpd2敲除成功,基因缺失菌株与原始菌株经发酵实验相比,甘油产量下降22.01%,乙醇产量提高24.65%,2,3-丁二醇产量下降10.60%。gpd2基因的敲除并没有提高2,3-丁二醇的产量,原因可能是逐渐积累的NADH会优先被细胞内大量的乙醇脱氢酶所氧化,作用于乙醇的产生,而不是优先作用于2,3-丁二醇的合成。本实验构建了适用于酿酒酵母的基因敲除系统,该系统对进一步探究酿酒酵母其他代谢产物与2,3-丁二醇合成之间的关系具有实际的借鉴意义。

关键词: 酿酒酵母, CRISPR/Cas9, 2,3-丁二醇, 载体构建, 代谢工程

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

中图分类号:

S182

刘磊, 李娜, 姜雪雍, 孙健, 吕雨泽, 葛菁萍. CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响[J]. 中国农学通报, 2020, 36(29): 69-77.

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.

图/表 10

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

表1. 本实验所用的质粒

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

表2. 本实验所用的引物

图1. 6-HH-20-HDV片段的结构

图2. 重组表达质粒pUDP004-gpd2的PCR验证 M：DL 2000 Marker;1~3：阳性重组表达质粒pUDP004-gpd2的PCR产物

图3. 基因敲除质粒的结构

图4. GPD2-repair-T质粒的PCR验证 M：DL 2000 Marker;1：阳性质粒GPD2-repair-T的PCR产物

图5. 转化株的生长情况 A：转化子在SM-Ac固体培养基上的菌落分布;B：转化子的菌落形态观察;红色箭头标记的是可能的阳性转化子

图6. 验证引物在gpd2基因上的位置

图7. gpd2基因缺失菌株PCR验证 M：DL 2000 Marker;1-5：引物GPD2-300F、GPD2-300R;6-10:引物GPD2-ORF-F、GPD2-ORF-R;1、6：同一单菌落;2、7：同一单菌落;3、8：同一单菌落;4、9：同一单菌落;5、10：对照菌株

图8. S. cerevisiae SJ-Δgpd2与S. cerevisiae W5的摇瓶发酵实验 A为S. cerevisiae SJ-Δgpd2与S. cerevisiae W5的OD600与pH变化曲线;B为二者的葡萄糖剩余量与乙醇产量变化曲线;C为二者的甘油与2,3-丁二醇产量变化曲线

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CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响

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

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