芽胞形成相关基因缺失对解淀粉芽胞杆菌生物量及胞外酶表达的影响

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

摘要/Abstract

摘要：

解淀粉芽胞杆菌作为公认的安全生产宿主菌（GRAS）,在高效表达异源蛋白方面具有巨大的发展潜力和应用前景。本研究以解淀粉芽胞杆菌TCCC111018为出发菌株,通过敲除芽胞形成相关基因（spo0A,sigF和sigE）,构建了一系列突变菌株（BAΔspo0A,BAΔsigF,BAΔsigE）,并对突变株生物量和胞外酶表达量进行分析比较。结果表明,与出发菌株BAΔupp相比,菌株BAΔspo0A的生物量及胞外酶表达量显著降低,BAΔsigE无明显变化,而菌株BAΔsigF的生产性能有显著提升,菌体生长的稳定期延长约6 h,产酶时间提前,耐酸性α-淀粉酶与碱性蛋白酶酶活分别提高了25.2%和21.3%,该菌株的成功构建为工业酶生产宿主提供了新的选择,也为异源酶的高效生产提出了一种新的策略。

关键词: 解淀粉芽胞杆菌, 芽胞形成, 生物量, 耐酸性α-淀粉酶, 碱性蛋白酶

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

李昕悦, 张金方, 徐小健, 路福平, 李玉. 芽胞形成相关基因缺失对解淀粉芽胞杆菌生物量及胞外酶表达的影响[J]. 生物技术通报, 2021, 37(3): 35-43.

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.

图/表 7

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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