Abstract
Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C. glutamicum. Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C. glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C. glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five genes (up to ~ 13.0 kb) and knock-in of one DNA fragment. Furthermore, through kanamycin resistance repair, the ssDNA recombineering parameters were optimized. We hope the highly efficient method will be helpful for the studies of C. glutamicum, and potentially, to other bacteria.
Key Points
• Counterselection marker I-SceI-mediated C. glutamicum genome engineering
• A suicide vector contains I-SceI expression elements and its recognition sites
• Gene deletions and knock-in were conducted; efficiency was as high as 90%
• Through antibiotic resistance repair, ssDNA recombineering parameters were optimized
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Acknowledgments
We gratefully acknowledge Dr. John Cronan, Dr. Lothar Eggling, Dr. Jan Nešvera, and Dr. Pablo Ravasi for providing the strain and plasmids.
Funding
Funding was provided by the National Natural Science Foundation of China (NSFC 81273412).
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GS conceived and designed research. MW, YX and JY conducted experiments. GS contributed reagents. All authors participated in the data analysis. GS wrote the manuscript with help from other authors. All authors read and approved the final manuscript.
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Wu, M., Xu, Y., Yang, J. et al. Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering. Appl Microbiol Biotechnol 104, 3597–3609 (2020). https://doi.org/10.1007/s00253-020-10517-y
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DOI: https://doi.org/10.1007/s00253-020-10517-y