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

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

Abstract

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

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.

Figures/Tables 12

References 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