ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD

doi:10.11924/j.issn.1000-6850.casb2023-0075

Abstract

Abstract:

The objective is to address the issue of Carbon Catabolite Repression (CCR) in Klebsiella pneumoniae during mixed carbon source fermentation for 2,3-butanediol (2,3-BD) production, which leads to a decrease in production efficiency. In this study, a Cm resistance gene was used as a marker, and the ptsG gene deletion strain of K. pneumoniae HD79-N was successfully constructed using the λRed homologous recombination technique. Furthermore, the fermentation results using a mixed carbon source of glucose and xylose (glucose:xylose=2:1) demonstrated that the K. pneumoniae HD79-N strain, with the ptsG gene deletion, significantly alleviated CCR, enabling simultaneous utilization of glucose and xylose for 2,3-BD production with a final yield of 9.81±0.38 g/L. Moreover, the xylose utilization rate of K. pneumoniae HD79-N strain [0.23±0.01 g/(L·h)] was also increased by 57.82% compared to that of K. pneumoniae HD79 strain [0.15±0.00 g/(L·h)]. The findings of this study provide technical insights into alleviating the CCR effect caused by the ptsG gene in K. pneumoniae strains and enhancing the production of 2,3-BD.

Key words: 2,3-butanediol, gene knockout, ptsG gene, homologous recombination, Klebsiella pneumonia

MAO Liangyang, LI Na, GE Jingping. ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD[J]. Chinese Agricultural Science Bulletin, 2024, 40(3): 95-102.

Figures/Tables 11

References 27

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序号	名称	菌株用途	菌株特点	菌株来源
1	K. pneumoniae HD79	出发菌株	野生型	黑龙江大学微生物重点实验室保存
2	E. coli DH5α	克隆和转化	ΔlacU169, Rˉ, Mˉ, Amp^r	黑龙江大学微生物重点实验室保存
3	K. pneumoniae HD79-N	用于本探究	Cm^r, ΔptsG, Cm^r	自行构建

序号	名称	菌株用途	菌株特点	菌株来源
1	K. pneumoniae HD79	出发菌株	野生型	黑龙江大学微生物重点实验室保存
2	E. coli DH5α	克隆和转化	ΔlacU169, Rˉ, Mˉ, Amp^r	黑龙江大学微生物重点实验室保存
3	K. pneumoniae HD79-N	用于本探究	Cm^r, ΔptsG, Cm^r	自行构建

序号	名称	质粒特点	质粒用途	质粒来源
1	pMD18-T	Amp^r, lacZ, 2692 bp	克隆载体	大连宝生物有限公司
2	pKD46	Amp^r, Exo, Beta, Gam, 6329 bp	同源重组	北京鼎国生物科技有限公司
3	pLysS	Cm^r, T7 lysozyme, 4886 bp	克隆cm基因	北京鼎国生物科技有限公司

序号	名称	质粒特点	质粒用途	质粒来源
1	pMD18-T	Amp^r, lacZ, 2692 bp	克隆载体	大连宝生物有限公司
2	pKD46	Amp^r, Exo, Beta, Gam, 6329 bp	同源重组	北京鼎国生物科技有限公司
3	pLysS	Cm^r, T7 lysozyme, 4886 bp	克隆cm基因	北京鼎国生物科技有限公司

引物名称	引物序列(5’-3’)	用途
ptsG₁-F	CCGTACTGCCTATCGCAGGTATC	克隆ptsG₁上游片段594 bp
ptsG₁-R	gacaccaggCGTTCCGATGTGATGCAGACC	克隆ptsG₁上游片段594 bp
ptsG₂-F	tggtgctacgcGCGATTTGGCACTCTGCTAAAC	克隆ptsG₂下游片段579 bp
ptsG₂-R	GTACGCCAGAACCTGCCACTAC	克隆ptsG₂下游片段579 bp
cm^r-F	GATCATCTGAACGCGGCACGTAAGAGGTTCC	克隆cm^r序列888 bp
cm^r-R	CACGCCATTCCCCGCTTATTATCACTTATTCA	克隆cm^r序列888 bp
pKD46-F	CCCAGCCCTGTGTATAACTCAC	验证转化结果 1387 bp
pKD46-R	CAACTCGGTCGCCGCATACA	验证转化结果 1387 bp
ptsG-F	AGCCAAAGCGAGTAAAGTTCAC	验证重组结果2507 bp
ptsG-R	GCGGACTGTTTTCAGGGTTAT	验证重组结果2507 bp
M13R	GTAAAACGACGGCCAGT	验证重组质粒
M13F	TGTAAAACGACGGCCAGT	验证重组质粒
ptsG-up	CGTGATCCTCTCCTTCATTTG	qRT-PCR检测扩增ptsG
ptsG-down	tAAGCCGCCAGACAGTTTG	qRT-PCR检测扩增ptsG
xylA-up	CGGGCGAAGTTATTGCTG	qRT-PCR检测扩增xylA
xylA-down	GGCTTTCCGTCATTACAACC	qRT-PCR检测扩增xylA
xylB-up	CCGCCGATAAGCGTGAT	qRT-PCR检测扩增xylB
xylB-down	GGTGTGGTTCCTGCCCTAT	qRT-PCR检测扩增xylB
xylE-up	TTCCGCACCGAACATCC	qRT-PCR检测扩增xylE
xylE-down	GCCCGCTTTACATTGCC	qRT-PCR检测扩增xylE
xylF-up	ACCTTCCTGCTTGGCTTCT	qRT-PCR检测扩增xylF
xylF-down	aCTGGCAGAAAGACCGTGAT	qRT-PCR检测扩增xylF
xylG-up	GCAGTTCGGATGAAATGACA	qRT-PCR检测扩增xylG
xylG-down	tCCAGCAAAAGGCGATTC	qRT-PCR检测扩增xylG
xylH-up	ACCCACCATCCGTTCCA	qRT-PCR检测扩增xylH
xylH-down	GCCATCGTCGTCATTATGC	qRT-PCR检测扩增xylH
16S rDNA-up	TGTGTAGCCCTGGTCGTAAG	qRT-PCR检测扩增内参基因
16S rDNA-down	CGGACATCCACAGAACTTAGC	qRT-PCR检测扩增内参基因