单倍体酿酒酵母的丙酮酸脱羧酶基因(pdc1)的敲除与鉴定

doi:10.11924/j.issn.1000-6850.casb20190600332

中国农学通报 ›› 2020, Vol. 36 ›› Issue (24): 91-98.doi: 10.11924/j.issn.1000-6850.casb20190600332

所属专题：生物技术

单倍体酿酒酵母的丙酮酸脱羧酶基因(pdc1)的敲除与鉴定

康杰¹^,²(), 王长丽¹^,², 葛菁萍¹^,²()

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

收稿日期:2019-06-25 修回日期:2019-07-10 出版日期:2020-08-25 发布日期:2020-08-20
通讯作者: 葛菁萍
作者简介:康杰,男,1995年出生,研究生,研究方向：微生物学。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学224信箱,Tel：0451-86609016,E-mail：kangjie182@126.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);黑龙江省教育厅重点项目“利用肺炎克雷伯氏菌发酵生产2,3-丁二醇及机理探讨”(HDJCCX-2016Z05);黑龙江省自然科学基金重点项目“乙酸代谢与副干酪乳杆菌群体感应互作探究及对细菌素产生的影响”

Pyruvate Decarboxylase Gene (pdc1) of Haploid Saccharomyces Cerevisiae: Knockout and Identification

Kang Jie¹^,²(), Wang Changli¹^,², Ge Jingping¹^,²()

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

Received:2019-06-25 Revised:2019-07-10 Online:2020-08-25 Published:2020-08-20
Contact: Ge Jingping

摘要/Abstract

摘要：

为了抑制单倍体酿酒酵母H14的副产物乙醇的合成,使得2,3-丁二醇产量的提升。利用基因工程手段构建载体pWCL-pdc1,获得两端含40 bp pdc1的同源重组片段-loxP-kanMX-loxP。利用Cre/loxP技术获得pdc1缺失菌株S. cerevisiae H14-01 (△pdc1)。并以野生型菌株S. cerevisiae H14为对照,进行摇瓶发酵试验。S. cerevisiae H14-01长势明显略低于原始菌株。在整个发酵期间,2,3-丁二醇的最高产量和转化率分别为0.373±0.016 g/L和0.005 g/g,分别较原始菌株提高了37.30%和4.66%,但原始菌株没有检测到乙偶姻和2,3-BD生成。另外S. cerevisiae H14-01的乙醇转化率降低了33.24%,但甘油产量提高了15.76%。说明了碳流流向乙醇被阻断之后,会增加2,3-丁二醇的产量,同时会使此部分碳流流向甘油。因此,并为进一步获得高产2,3-丁二醇的工程微生物群体奠定了基础。

关键词: 酿酒酵母, 2,3-丁二醇, Cre/loxP技术, 丙酮酸脱羧酶, 基因敲除

Abstract:

The aim is to inhibit the synthesis of ethanol, a by-product of haploid Saccharomyces cerevisiae H14, and increase the yield of 2,3-butanediol. The vector pWCL-pdc1 was constructed by genetic engineering to obtain the homologous recombinant fragment loxP-kanMX-loxP containing 40 bp pdc1 at both ends. The pdc1 deleted strain S. cerevisiae H14-01 (Δpdc1) was obtained by Cre/loxP technique. The shaking flask fermentation experiment was carried out with the wild type strain S. cerevisiae H14 as the control. The growth of S. cerevisiae H14-01 was significantly lower than that of the original strain. During the whole fermentation period, the maximum yield and conversion rate of 2,3-butanediol were 0.373 ±0.016 g/L and 0.005 g/g, respectively, which were 37.30% and 4.66% higher than that of the original strain, respectively. However, acetoin and 2,3-BD production was not detect in the original strain. In addition, the ethanol conversion of S. cerevisiae H14-01 decreased by 33.24%, but the glycerol production increased by 15.76%. It showed that after the carbon flow to ethanol was blocked, the production of 2, 3-butanediol could be increased, and part of the carbon flow will flow to glycerol. Therefore, the study lays a good foundation for further obtaining an engineering microbial population with high yield of 2,3-butanediol.

Key words: Saccharomyces cerevisiae, 2,3-butanediol, Cre/loxP technology, pyruvate decarboxylase, gene knockout

中图分类号:

Q933

康杰, 王长丽, 葛菁萍. 单倍体酿酒酵母的丙酮酸脱羧酶基因(pdc1)的敲除与鉴定[J]. 中国农学通报, 2020, 36(24): 91-98.

Kang Jie, Wang Changli, Ge Jingping. Pyruvate Decarboxylase Gene (pdc1) of Haploid Saccharomyces Cerevisiae: Knockout and Identification[J]. Chinese Agricultural Science Bulletin, 2020, 36(24): 91-98.

图/表 12

图1. 转化子的PCR验证示意图

图2. 阳性质粒单酶切验证 M1、M2：DNA Marker DL 15000和DL 2000;1、2：pMDTM18-T和pTWCL-pdc1酶切产物

图3. pdc1-loxP-kanMX转化S. cerevisiae H14 A对照组,B实验组

图4. 阳性克隆子的菌液PCR验证 M：DNA Marker DL 2000;1、2、3、4、8：阳性克隆菌液PCR扩增kanMX

图5. pdc1-B1和pdc1-C1引物对PCR扩增验证 M：DNA Marker DL 2000;2、5、6、9、10、11：PCR扩增基因组kanMX片段

图6. 基因组PCR扩增验证 M：DNA Marker DL 2000;1~4：基因组PCR扩增产物;A：引物对pdc1-A1和pdc1-B1;B：引物对pdc1-A1和pdc1-D1;C：引物对pdc1-C1和pdc1-D1

图7. 含pSH69质粒的阳性转化子的筛选△pdc1/H14

图8. 消除G418抗性菌株的筛选△pdc1/H14

图9. 琼脂糖凝胶电泳验证pdc1基因 M：DNA Marker DL2000;1~3：基因组PCR扩增产物

图10. H14/H14-△pdc1的乙醇转化率比较

图11. H14/H14-△pdc1-1的OD600、葡萄糖剩余量、乙偶姻和2,3-丁二醇浓度比较

图12. H14/H14-△pdc1-1的甘油和乙醇含量比较

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单倍体酿酒酵母的丙酮酸脱羧酶基因(pdc1)的敲除与鉴定

Pyruvate Decarboxylase Gene (pdc1) of Haploid Saccharomyces Cerevisiae: Knockout and Identification

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