Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, 510006, China.
Appl Biochem Biotechnol. 2021 Sep;193(9):2916-2931. doi: 10.1007/s12010-021-03576-y. Epub 2021 May 10.
Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCR-amplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible P operon, which demonstrates obvious lethality to S. marcescens. Subsequently, Gm-kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.
尽管粘质沙雷氏菌在工业应用中有很大的潜力，但缺乏强大的遗传修饰工具限制了对有用代谢物的调控网络的理解，因此限制了它们的大规模生产。为了满足这一迫切需求，我们在本研究中基于 Red 重组建立了粘质沙雷氏菌的基因组编辑策略。无需预先对宿主进行修饰，通过 PCR 扩增的抗性基因取代了 nucA 和 pigA。抗性基因两侧不需要长同源臂。使用该程序，很容易删除粘质沙雷氏菌中长达 20kb 的片段。然后，我们构建了一个在诱导型 P 操纵子控制下构建的反向选择基因 kil，它对粘质沙雷氏菌表现出明显的致死性。随后，Gm-kil 双选择盒被插入 pigA 基因的 CDS 中。通过单链 DNA 介导的重组，通过 kil 反向选择有效地修复了这种插入突变。为粘质沙雷氏菌成功建立了基于 Red 重组系统的强大遗传修饰平台。在该物种中，可以轻松进行多种类型的修饰和多种重组策略。我们希望这项研究将有助于粘质沙雷氏菌的理论研究和代谢工程研究。
