自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性

doi:10.11924/j.issn.1000-6850.casb2023-0610

中国农学通报 ›› 2024, Vol. 40 ›› Issue (18): 96-104.doi: 10.11924/j.issn.1000-6850.casb2023-0610

自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性

谢乐乐(), 何平, 唐小越, 葛菁萍, 凌宏志()

黑龙江大学，生命科学学院，农业微生物技术教育部工程研究中心，黑龙江省寒区植物基因与生物发酵重点实验室，黑龙江省普通高校微生物重点实验室，哈尔滨 150080

收稿日期:2023-08-22 修回日期:2023-11-16 出版日期:2024-06-25 发布日期:2024-06-18
通讯作者:
凌宏志，男，1979年出生，黑龙江哈尔滨人，教授，博士，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号黑龙江大学生命科学学院，Tel：0451-86609016，E-mail：linghongzhi@163.com。
作者简介:
谢乐乐，女，1999年出生，四川蓬安人，硕士研究生，研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江哈尔滨南岗区学府路74号黑龙江大学生命科学学院，E-mail：2221601@s.hlju.edu.cn。
基金资助:
国家自然科学基金(31570492)

Alcohol Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae by Suicide Plasmids Homologous Recombination: Construction and Biological Characteristics

XIE Lele(), HE Ping, TANG Xiaoyue, GE Jingping, LING Hongzhi()

Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education/ Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/ Key Laboratory of Microbiology, College of Heilongjiang Province/ School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2023-08-22 Revised:2023-11-16 Published:2024-06-25 Online:2024-06-18

摘要/Abstract

摘要：

本研究旨在探究敲除阴沟肠杆菌(Enterobacter cloacae)代谢通路上乙醇脱氢酶基因对乙偶姻代谢合成的影响，进一步提高乙偶姻产量。实验基于E. cloacae SDM中的乙醇脱氢酶adh基因序列设计特异性引物，并构建adh基因自杀质粒pKR6K-∆adh。利用热激法将自杀质粒转入E. cloacae ∆budC-ldh，成功得到多基因缺失菌株E. cloaca ∆budC-ldh-adh，并进行发酵性能测试。研究结果显示，与出发菌株相比，新构建的重组菌株在敲除了负责乙醇合成的关键酶乙醇脱氢酶adh基因后，其乙偶姻的生产强度提高了20.6%，产量提高了22.6%，同时乙醇产量提高了92.8%。此外，由于通过基因改造阻断了多条分支代谢路径，流向丁二酸代谢路径的碳通量明显变多，丁二酸产量提高101.3%。本研究成果不仅为构建高效乙偶姻生产菌株提供了有价值的参考，也为乙偶姻的大规模工业生产奠定了基础。

关键词: 乙偶姻, 乙醇脱氢酶, 同源重组, 阴沟肠杆菌, 基因缺失, 基因敲除, 代谢工程

Abstract:

The aim of this study was to investigate the effects of knockout of ethanol dehydrogenase gene in Enterobacter cloacae metabolic pathway on the metabolism and synthesis of acetoin, and further improve the production of acetoin. Based on the ethanol dehydrogenase (adh) gene sequence from E. cloacae SDM, a primer was designed to construct the adh gene suicide plasmid pKR6K-∆adh. Subsequently, this suicide plasmid was introduced into E. cloacae ∆budC-ldh through heat shock method. The resulting polygene deletion strain E. cloaca ∆budC-ldh-adh was successfully constructed and subjected to fermentation analysis. The findings revealed that the recombinant strain exhibited a 20.6% increase in acetoin production intensity, a 22.6% increase in yield, and a remarkable 92.8% increase in ethanol yield compared to control strains due to successful knockout of ethanol dehydrogenase (adh) gene which in charge of microbial ethanol synthesis pathways. Furthermore, as a consequence of blocking multiple branch metabolic pathways through genetic modification, carbon flux towards succinic acid metabolic pathway increased significantly leading to an impressive 101.3% enhancement in succinic acid yield. This experiment has provided valuable insights for constructing high-yielding strains for acetoin production and establishing large-scale industrial manufacturing processes.

Key words: acetoin, alcohol dehydrogenase, homologous recombination, Enterobacter cloacae, gene deletion, gene knockout, metabolic engineering

谢乐乐, 何平, 唐小越, 葛菁萍, 凌宏志. 自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性[J]. 中国农学通报, 2024, 40(18): 96-104.

XIE Lele, HE Ping, TANG Xiaoyue, GE Jingping, LING Hongzhi. Alcohol Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae by Suicide Plasmids Homologous Recombination: Construction and Biological Characteristics[J]. Chinese Agricultural Science Bulletin, 2024, 40(18): 96-104.

图/表 12

参考文献 25

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序号	质粒名称	长度	用途	来源
1	pMD18-T	2692 bp	克隆载体	大连宝生物工程有限公司
2	pKR6K	5220 bp	自杀质粒	实验室提供
3	pT-∆adh1	3212 bp, Amp^r	克隆adh基因上游片段	自行构建
4	pT-∆adh2	3212 bp, Amp^r	克隆adh基因下游片段	自行构建
5	pKR6K-∆adh	6193 bp, Kan^r	敲除adh基因自杀质粒	自行构建

序号	质粒名称	长度	用途	来源
1	pMD18-T	2692 bp	克隆载体	大连宝生物工程有限公司
2	pKR6K	5220 bp	自杀质粒	实验室提供
3	pT-∆adh1	3212 bp, Amp^r	克隆adh基因上游片段	自行构建
4	pT-∆adh2	3212 bp, Amp^r	克隆adh基因下游片段	自行构建
5	pKR6K-∆adh	6193 bp, Kan^r	敲除adh基因自杀质粒	自行构建

引物	序列5’-3’	酶切位点	用途
adh1F	AATT GAATTCAATCAAAGAGCGATAAGATCACATT	EcoRI	克隆adh基因上游片段520 bp
adh1R	AATT GGATCCAATGCTCTCCTGATAATGTTTAAAC	BamHI	克隆adh基因上游片段520 bp
adh2F	AATT TCTAGATTGACAACGTGATAAAAAACCCGCC	XbaI	克隆adh基因下游片段520 bp
adh2R	AATT GCATGCAAACGGACCTGTTGCAGGATATTCG	SphI	克隆adh基因下游片段520 bp
adhF	AATCAAAGAGCGATAAGATCACATT	—	验证adh基因敲除结果
adhR	AAACGGACCTGTTGCAGGATATTCG	—	验证adh基因敲除结果

引物	序列5’-3’	酶切位点	用途
adh1F	AATT GAATTCAATCAAAGAGCGATAAGATCACATT	EcoRI	克隆adh基因上游片段520 bp
adh1R	AATT GGATCCAATGCTCTCCTGATAATGTTTAAAC	BamHI	克隆adh基因上游片段520 bp
adh2F	AATT TCTAGATTGACAACGTGATAAAAAACCCGCC	XbaI	克隆adh基因下游片段520 bp
adh2R	AATT GCATGCAAACGGACCTGTTGCAGGATATTCG	SphI	克隆adh基因下游片段520 bp
adhF	AATCAAAGAGCGATAAGATCACATT	—	验证adh基因敲除结果
adhR	AAACGGACCTGTTGCAGGATATTCG	—	验证adh基因敲除结果

产物浓度/(g/L)	菌株
产物浓度/(g/L)	E.cloacae-02	E.cloacae-03
OD₆₀₀	7.57±0.14	8.41±0.28
葡萄糖剩余量	-	3.81±1.21
乙偶姻	16.18±0.46	19.83±0.49
乳酸	-	-
2,3-BDO	3.82±0.33	4.76±0.40
丁二酸	0.75±0.06	1.51±0.07
乙酸	2.21±0.16	3.03±0.13
乙醇	5.80±0.26	0.42±0.09
乙偶姻转化率/(g/g)	0.27±0.01	0.33±0.01
乙偶姻生产强度/[g/(L•h)]	0.34±0.01	0.41±0.01

自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性

Alcohol Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae by Suicide Plasmids Homologous Recombination: Construction and Biological Characteristics

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