用于在枯草芽孢杆菌和短小芽孢杆菌中进行基因表达的新型穿梭表达载体的构建。

Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

作者信息

Shao Huanhuan, Cao Qinghua, Zhao Hongyan, Tan Xuemei, Feng Hong

机构信息

Key Laboratory of Bio-Resource & Eco-Environment, Ministry of Education, Sichuan Key Laboratory of Molecular Biology & Biotechnology, College of Life Sciences, Sichuan University.

出版信息

J Gen Appl Microbiol. 2015;61(4):124-31. doi: 10.2323/jgam.61.124.

DOI:10.2323/jgam.61.124

PMID:26377132

Abstract

A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

摘要

通过枯草芽孢杆菌S1-4菌株的基因组测序检测到一种天然质粒（pSU01）。构建了两种基于pSU01的穿梭表达载体pSU02-AP和pSU03-AP，使其能够在枯草芽孢杆菌WB600中稳定复制。这些载体含有报告基因aprE，其编码来自短小芽孢杆菌BA06的碱性蛋白酶。表达载体pSU03-AP仅具有最小复制元件（rep、SSO、DSO），并且在结构上表现出更高的稳定性，这表明pSU01中的其余基因（ORF1、ORF2、mob、hsp）对于枯草芽孢杆菌中质粒的结构稳定性并非必需。此外，使用pSU03-AP能更高效地实现碱性蛋白酶的重组生产，其拷贝数估计每条染色体超过100个。此外，在选择压力下，pSU03-AP也可用于短小芽孢杆菌BA06的转化和复制。总之，pSU03-AP有望成为枯草芽孢杆菌和短小芽孢杆菌中基因表达的有用工具。