Ji Minghua, Liu Yunhui, Wu Haiying, Li Sijie, Duan Haiyan, Shi Jiping, Sun Junsong
Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
J Ind Microbiol Biotechnol. 2021 Jul 1;48(5-6). doi: 10.1093/jimb/kuab024.
Catalases are a large group of enzymes that decompose hydrogen peroxide to oxygen and hydrogen, and have been applied widely in numerous areas. Bacillus subtilis ATCC 6051a is a well-known host strain for high level secretion of heterologous peptides. However, the application of 6051a was seriously hampered by insufficient transformation efficiency. In this study, D-xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, generating 164S, a mutant owns a transformation efficiency of 1 000-fold higher than its parent strain, thus allowing gene replacement by double crossover recombination using linear dsDNAs. The efficiency of the flanking arms for homologous recombination was then analyzed. We found that 400 bp was the minimal length of homologous fragments required to initiate efficient recombination in the 164S strain. In addition, DNA cassettes encoding two mesophilic catalases (Orf 2-62 and Orf 2-63) from B. licheniformis were integrated onto 164S. The catalytic properties of recombinant Orf 2-62 and Orf 2-63 were analyzed, and were found to be predominantly secreted into the fermentation broth, although they obviously lack any known secretory signal peptide. This work demonstrated that B. subtilis 164S is an excellent cell tool, not only for its superior secretion capacity, but also for its convenience in genetic modification.
过氧化氢酶是一类可将过氧化氢分解为氧气和氢气的酶，已在众多领域得到广泛应用。枯草芽孢杆菌ATCC 6051a是一种用于高效分泌异源肽的知名宿主菌株。然而，其转化效率不足严重阻碍了6051a的应用。在本研究中，将D-木糖诱导的comK整合到枯草芽孢杆菌ATCC 6051a的基因组中，产生了164S，这是一种突变体，其转化效率比亲本菌株高1000倍，因此可以使用线性双链DNA通过双交换重组进行基因替换。然后分析了同源重组侧翼臂的效率。我们发现400 bp是在164S菌株中启动高效重组所需的同源片段的最小长度。此外，将编码来自地衣芽孢杆菌的两种嗜温过氧化氢酶（Orf 2-62和Orf 2-63）的DNA盒整合到164S上。分析了重组Orf 2-62和Orf 2-63的催化特性，发现它们主要分泌到发酵液中，尽管它们明显缺乏任何已知的分泌信号肽。这项工作表明，枯草芽孢杆菌164S是一种优秀的细胞工具，不仅因其卓越的分泌能力，还因其在基因改造方面的便利性。
