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Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

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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 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 PBAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, GmR-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.

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Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

Special thanks to Professor Donald L. Court for the plasmid (pKD46 and pSim6) and Sheng Yang for the strain MG1655.

Funding

This study was supported by the Science and Technology Program of Guangdong Province (grant number 2015A010107014) and Grant-in-Aid from the Natural Scientific Foundation of China (grant number 31501895).

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Authors and Affiliations

  1. Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, China

    Wei Chen & Ruyi Chen

  2. Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, 510006, China

    Wei Chen

  3. School of Economics and Trade, South China University of Technology, Guangzhou, 510006, China

    Jianyun Cao

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  1. Wei Chen
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Contributions

W Chen and RY Chen performed the research; W Chen designed the experiments and wrote the manuscript. JY Cao supervised the research and helped to draft the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jianyun Cao.

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Key Points

• PCR-amplified products with short homology were used for recombination.

• A usable counter-selection system based on kil gene was constructed.

• Insertion mutation can be repaired efficiently with ssDNA-meditated recombination.

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Chen, W., Chen, R. & Cao, J. Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination. Appl Biochem Biotechnol 193, 2916–2931 (2021). https://doi.org/10.1007/s12010-021-03576-y

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  • DOI: https://doi.org/10.1007/s12010-021-03576-y

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