<i>ldhL-ldb0094</i>基因敲除对保加利亚乳杆菌产L-乳酸的影响 <sup>*</sup>

doi:10.13523/j.cb.20190809

中国生物工程杂志

2019, Vol. 39

Issue (8): 66-73 DOI: 10.13523/j.cb.20190809

技术与方法

ldhL-ldb0094基因敲除对保加利亚乳杆菌产L-乳酸的影响 ^*

王刚^1,²,肖雨¹,李义²,刘志刚²,裴成利²,武丽达²,李艳丽¹,王希庆¹,张明磊¹,陈光^1,^3,^**(

),佟毅^2,^**(

)

1 吉林农业大学生命科学学院长春 130118
2 中粮集团吉林中粮生化有限公司长春 130118
3 吉林农业大学秸秆生物学与利用教育部重点实验室长春 130118

Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp. blgaricus Producing L-lactic Acid

WANG Gang^1,²,XIAO Yu¹,LI Yi²,LIU Zhi-gang²,PEI Cheng-li²,WU Li-da²,LI Yan-li¹,WANG Xi-qing¹,ZHANG Ming-lei¹,CHEN Guang^1,^3,^**(

),TONG Yi^2,^**(

)

1 College of Life Science, Jilin Agricultural University,Changchun 130118,China
2 Cofco Biochemical Co, LTD of Jilin, Changchun 130118,China
3 Key Laboratory for Straw Biology and Utilization of the Ministry of Education, Jilin Agricultural University,Changchun 130118,China

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摘要：

以保加利亚乳杆菌Lactobacillus delbrueckii subsp. bulgaricus CICC21101为出发菌株,利用PCR扩增L-乳酸脱氢酶(ldhL)基因上下游序列ldhL1、ldhL2,获得ldhL基因缺失且包含上下游序列的片段,连接到乳酸菌专用温敏性基因敲除质粒pGhost4,将构建好的敲除载体电转入保加利亚乳杆菌CICC21101,低温筛选。结果表明,成功获得敲除ldhL基因的敲除突变株,敲除后的工程菌D-乳酸产量由30.5g/L降为4.8g/L,L-乳酸的产量由25.4g/L增至58.3g/L,光学纯度由54.56%增至90%。同时发现ldhL-ldb0094基因的敲除致使ldhL-ldb1020表达的上调,D-乳酸脱氢酶(ldbD)基因表达量没有变化,ldhL基因敲除株的成功构建将为进一步研究该基因在保加利亚乳杆菌中的功能及后续高光学活性D-乳酸工程菌构建奠定基础。

关键词： 保加利亚乳杆菌; L-乳酸脱氢酶; 同源重组; 敲除; pGhost4

Abstract:

To construct ldhL gene deletion strain in Lactobacillus delbrueckii subsp. bulgaricus.Bulgaria lactobacillus CICC21101 was employed as the original strain, amplified the upstream sequence ldhL1 and downstream sequence ldhL2 by PCR, obtained successfully ldhL gene deletion fragment which includes both upstream and downstream sequence, then connected to knockout plasmid pGhost4, electricity to Bulgaria lactobacillus CICC21101, screening at low temperature. The results indicated that the ldhL gene knocked out deficient mutant was obtained.By gene knockout, the yield of D-lactic acid decreased from 30.5g/L to 4.8g/L, the yield of L-lactic acid increased from 25.4g/L to 58.3g/L, and the optical purity increased from 54.56% to 90%. It was a foundation to study the detail functions of ldh in Lactobacillus delbrueckii subsp. bulgaricus,it will lay the foundation of constuction on D-lactic acid engineering bacteria.

Key words: Lactobacillus delbrueckii subsp.bulgaricus L-lactate dehydrogenase Homologous recombination Knock out pGhost4

收稿日期: 2019-01-22 出版日期: 2019-09-18

ZTFLH:

Q93

基金资助: *吉林省科技厅科技支撑项目(17MY057Z);长春市科技局地院合作项目(17DY015);吉林省秸秆科技创新平台项目[(2014)C-1];吉林省教育厅项目(JJKH20180683KJ)

通讯作者: 陈光,佟毅 E-mail: chg61@163.com;tongyi@cofco.com

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	王刚
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	李艳丽
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	张明磊
	陈光
	佟毅

引用本文:

王刚,肖雨,李义,刘志刚,裴成利,武丽达,李艳丽,王希庆,张明磊,陈光,佟毅. ldhL-ldb0094基因敲除对保加利亚乳杆菌产L-乳酸的影响 ^*[J]. 中国生物工程杂志, 2019, 39(8): 66-73.

WANG Gang,XIAO Yu,LI Yi,LIU Zhi-gang,PEI Cheng-li,WU Li-da,LI Yan-li,WANG Xi-qing,ZHANG Ming-lei,CHEN Guang,TONG Yi. Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp. blgaricus Producing L-lactic Acid. China Biotechnology, 2019, 39(8): 66-73.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190809 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I8/66

图1 保加利亚乳杆菌丙酮酸代谢途径

图2 基于red-重组技术的ldh基因敲除策略

图3 重组克隆质粒PCR验证结果

图4 重组克隆质粒双酶切结果

图5 重组敲除质粒的验证电泳图

图6 工程菌pGhost-Δ ldh-L-F、pGhost-Δ ldh-R-R验证结果

图7 野生型与工程菌的生长曲线

表1 敲除ldb0094对产酸性能的影响

图8 工程菌各基因表达量的测定结果

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