Efficient production of extracellular pullulanase in Bacillus subtilis ATCC6051 using the host strain construction and promoter optimization expression system

枯草芽孢杆菌 质粒 细胞外 蛋白酵素 生物 莎梵婷 表达式向量 分子生物学 普鲁兰酶 重组DNA 微生物学 生物化学 细菌 基因 遗传学
作者
Xin Liu,Hai Wang,Bin Wang,Li Pan
出处
期刊:Microbial Cell Factories [Springer Nature]
卷期号:17 (1) 被引量:41
标识
DOI:10.1186/s12934-018-1011-y
摘要

Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Extracellular expression of pullulanase from recombinant B. subtilis is still limited due to the issues on promoters of B. subtilis expression system. This study was undertaken to develop a new, high-level expression system in B. subtilis ATCC6051.To further optimize B. subtilis ATCC6051 as a expression host, eight extracellular proteases (aprE, nprE, nprB, epr, mpr, bpr, vpr and wprA), the sigma factor F (spoIIAC) and a surfactin (srfAC) were deleted, yielding the mutant B. subtilis ATCC6051∆10. ATCC6051∆10 showed rapid growth and produced much more extracellular protein compared to the widetype strain ATCC6051, due to the inactivation of multiple proteases. Using this mutant as the host, eleven plasmids equipped with single promoters were constructed for recombinant expression of pullulanase (PUL) from Bacillus naganoensis. The plasmid containing the PspovG promoter produced the highest extracellular PUL activity, which achieved 412.9 U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter PamyL-PspovG produced the maximum extracellular PUL activity (625.5 U/mL) and showed the highest expression level (the dry cell weight of 18.7 g/L).Taken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter PamyL-PspovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051.
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