Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

Abstract

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 PCRamplified 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.

Development and optimization of Lysis gene E as a counter‐selection marker with high stringency

“…MG‐3, MG‐10, and CWE‐4 were previously constructed in our lab. [ 24 ] E. coli strain MG1655 was a generous gift from Professor Sheng Yang. E. coli strains harboring a λ kil counter‐selection system and the S. marcescens strain GY1 were isolated previously in our lab.…”

“…Single‐stranded DNA, sspigA‐F, used to delete P BAD ‐E‐Gm R or P BAD ‐kil‐sd‐E‐Gm R selection/counter‐selection cassettes inserted into the pigA locus of S. marcescens was reported in our previous study. [ 24 ]…”

“…E. coli strains harboring a λ kil counter-selection system and the S. marcescens strain GY1 were isolated previously in our lab. [24,26,27] Plasmids pKD46 and pSim6 are generous gifts from Doctor Donald L. Court. Plasmid pKDsgack was purchased from Addgene (www.addgene.org, 62654).…”

Development and optimization of Lysis gene E as a counter-selection marker with high selection stringency

“…After purification following PCR amplification, linear dsDNA was electroporated into cells for Red recombination. Single-stranded DNA, sspigA-F, used to deleteP BAD -E-Gm R or P BAD -kil-sd-E-Gm R selection/counter-selection cassettes inserted into the pigAlocus of S. marcescens was reported in our previous study (W. Chen, Chen, & Cao, 2021).…”

“…Unfortunately, high-temperature condition like 42°C is not suitable for the growth ofS. marcescens (W. Chen et al, 2021), We resorted to the AraC/P BAD regulatory system which function well in this species. At first, E-Gm R fragment with short homologous arms was amplified and transformed into competent MG1655[pKD46] to substitute the CDS of Red recombinases in plasmid pKD46 to construct the goal plasmid pKD-EG.…”