Watzlawick Hildegard, Altenbuchner Josef
Institute of Industrial Genetics, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
AMB Express. 2019 Sep 30;9(1):158. doi: 10.1186/s13568-019-0884-4.
The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.
利用CRISPR/Cas9系统，将来自枯草芽孢杆菌的编码用于降解半乳甘露聚糖的β-半乳糖苷酶的ganA基因整合到枯草芽孢杆菌染色体的不同位点。由此，将总共五个其中ganA基因与葡糖醇启动子融合的ganA盒插入到受体染色体中，其中假定的、芽孢形成和蛋白酶基因被删除。具有五个ganA表达盒拷贝的菌株显示出的β-半乳糖苷酶活性与在源自pUB110的多拷贝质粒上具有相同基因且在葡糖醇启动子和GutR激活剂的相同调控下的菌株相似。当在诱导条件下且无抗生素选择的情况下进行生长时，具有多拷贝质粒的菌株中β-半乳糖苷酶的产量迅速下降。相比之下，染色体中具有五个ganA拷贝的菌株产生β-半乳糖苷酶至少40代。这表明CRISPR/Cas9系统是构建稳定生产菌株的一种有价值且简便的工具。与在表达载体中克隆相比，将多个靶基因整合到染色体中需要付出更大的努力，但靶基因更高的稳定性和缺乏抗生素抗性基因证明了这种努力是合理的。
