Effects of Spore Formation Related Gene Deletion on Biomass and Extracellular Enzyme Expression of Bacillus amyloliquefaciens

doi:10.13560/j.cnki.biotech.bull.1985.2020-0852

Abstract

Abstract:

As a recognized safe（GRAS）bacterial hosts,Bacillus amyloliquefaciens has great development potential and application prospects in the efficient expression of heterologous enzyme. In this study,a series of B. amyloliquefaciens TCCC111018 mutant strains（BAΔspo0A,BAΔsigF,and BAΔsigE）were constructed by knocking out genes（spo0A,sigF and sigE）related to spore formation,and their biomasses and expressions of extracellular enzymes were analyzed and compared. The results showed that the biomass and extracellular enzyme expression of the strain BAΔspo0A were significantly reduced when compared with the starting strain BAΔupp,no obvious improvement for BAΔsigE,while the production performance of strain BAΔsigF was significantly improved. The stable phase of bacterial growth was prolonged by about 6 h,the time of producing enzyme was also advanced and the activity of acid-resistant α-amylase and alkaline protease were 25.23% and 21.3% higher than that of the control bacteria,respectively. The successful construction of BAΔsigF provides a new choice for industrial enzyme production hosts,also proposes a new strategy for the efficient production of heterologous enzymes.

Key words: Bacillus amyloliquefaciens, spore formation, biomass, acid-resistant α-amylase, alkaline protease

LI Xin-yue, ZHANG Jin-fang, XU Xiao-jian, LU Fu-ping, LI Yu. Effects of Spore Formation Related Gene Deletion on Biomass and Extracellular Enzyme Expression of Bacillus amyloliquefaciens[J]. Biotechnology Bulletin, 2021, 37(3): 35-43.

Figures/Tables 7

References 42

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	菌株/质粒	特征/目的	来源
菌株	E.coli JM109	敲除载体构建	中国科学院
	E.coli EC135 pM.Bam	对质粒DNA进行甲基化修饰	中国科学院
	B.amyloliquefaciens TCCC111018Δupp（BA Δupp）	出发菌株	本研究
	B.amyloliquefaciens TCCC111018Δspo0A（BA Δspo0A）	spo0A 基因敲除	本研究
	B.amyloliquefaciens TCCC111018ΔsigE（BA ΔsigE）	sig E基因敲除	本研究
	B.amyloliquefaciens TCCC111018ΔsigF（BA ΔsigF）	sigF 基因敲除	本研究
质粒	pWH-T2	穿梭表达载体	湖北大学
	pWH-T2-Δspo0A	敲除载体,spo0A基因敲除	本研究
	pWH-T2-ΔsigE	敲除载体,sigE 基因敲除	本研究
	pWH-T2-ΔsigF	敲除载体,sigF 基因敲除	本研究
	pLY-1	耐酸性α-淀粉酶表达载体	本实验室
	pLY-2	碱性蛋白酶表达载体	本实验室

	菌株/质粒	特征/目的	来源
菌株	E.coli JM109	敲除载体构建	中国科学院
	E.coli EC135 pM.Bam	对质粒DNA进行甲基化修饰	中国科学院
	B.amyloliquefaciens TCCC111018Δupp（BA Δupp）	出发菌株	本研究
	B.amyloliquefaciens TCCC111018Δspo0A（BA Δspo0A）	spo0A 基因敲除	本研究
	B.amyloliquefaciens TCCC111018ΔsigE（BA ΔsigE）	sig E基因敲除	本研究
	B.amyloliquefaciens TCCC111018ΔsigF（BA ΔsigF）	sigF 基因敲除	本研究
质粒	pWH-T2	穿梭表达载体	湖北大学
	pWH-T2-Δspo0A	敲除载体,spo0A基因敲除	本研究
	pWH-T2-ΔsigE	敲除载体,sigE 基因敲除	本研究
	pWH-T2-ΔsigF	敲除载体,sigF 基因敲除	本研究
	pLY-1	耐酸性α-淀粉酶表达载体	本实验室
	pLY-2	碱性蛋白酶表达载体	本实验室

引物名称	寡核苷酸序列
上游同源臂	F：CCACCGCGGTGGCGGCCGCTCTAGACGATCATCCAGAACGGGAAAG
上游同源臂	R：CCAATCTCAGATCAGCAACACAAACTTTAATTTTCTCCACG
下游同源臂	F：TGTTGCTGATCTGAGATTGGAGCATAAAGCTTCATAACGC
下游同源臂	R：TTAACGAATTCCTGCAGCCCGGGAGAGTGACGATGGATATGATTATG
同源臂重叠PCR	F：CCACCGCGGTGGCGGCCGCTCTAGACGATCATCCAGAACGGGAAAG
同源臂重叠PCR	R：TTAACGAATTCCTGCAGCCCGGGAGAGTGACGATGGATATGATTATG
单交换验证（上游交换）	F：CCGCAAAAATTCCCGGCGAC
单交换验证（上游交换）	R：AGAGTGACGATGGATATGATTATGGTCAGTTTG
单交换验证（下游交换）	F：CGATCATCCAGAACGGGAAAGTCG
单交换验证（下游交换）	R：CACTCAGTTTAAAGGCGCGTATCGCTTATG
双交换验证	F：CCGCAAAAATTCCCGGCGAC
双交换验证	R：CACTCAGTTTAAAGGCGCGTATCGCTTATG

引物名称	寡核苷酸序列
上游同源臂	F：CCACCGCGGTGGCGGCCGCTCTAGACGATCATCCAGAACGGGAAAG
上游同源臂	R：CCAATCTCAGATCAGCAACACAAACTTTAATTTTCTCCACG
下游同源臂	F：TGTTGCTGATCTGAGATTGGAGCATAAAGCTTCATAACGC
下游同源臂	R：TTAACGAATTCCTGCAGCCCGGGAGAGTGACGATGGATATGATTATG
同源臂重叠PCR	F：CCACCGCGGTGGCGGCCGCTCTAGACGATCATCCAGAACGGGAAAG
同源臂重叠PCR	R：TTAACGAATTCCTGCAGCCCGGGAGAGTGACGATGGATATGATTATG
单交换验证（上游交换）	F：CCGCAAAAATTCCCGGCGAC
单交换验证（上游交换）	R：AGAGTGACGATGGATATGATTATGGTCAGTTTG
单交换验证（下游交换）	F：CGATCATCCAGAACGGGAAAGTCG
单交换验证（下游交换）	R：CACTCAGTTTAAAGGCGCGTATCGCTTATG
双交换验证	F：CCGCAAAAATTCCCGGCGAC
双交换验证	R：CACTCAGTTTAAAGGCGCGTATCGCTTATG

基因名称	6 h-fpkm	10 h-fpkm	12 h-fpkm	30 h-fpkm
spo0A	582.56	511.54	658.06	1517.13
sigE	4.97	30.06	9.21	0.93
sigF	2380.91	4785.86	3287.31	1270.81