Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

• Published in: Applied biochemistry and biotechnology 2021-09, Vol.193 (9), p.2916-2931
• Main Authors: Chen, Wei, Chen, Ruyi, Cao, Jianyun
• Format: Article
• Language: English
• Subjects: Bacteria; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Deoxyribonucleic acid; DNA; Genes; Genetic modification; Genome editing; Genomes; Homologous recombination; Homology; Industrial applications; Insertion; Lethality; Mass production; Metabolic engineering; Metabolites; Mutation; Original Article; Phosphatidylinositol N-acetylglucosaminyltransferase; Serratia marcescens; Single-stranded DNA
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-021-03576-y

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 BAD operon, which demonstrates obvious lethality to S. marcescens . Subsequently, Gm R -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 .