Abstract
Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid.
Key points
• An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis.
• Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Funding
This research was financially supported by the National Natural Science Foundation of China (Grant No. 32070036), Sichuan Science and Technology Program (Grant No. 2023YFN0025), and Central Public-interest Scientific Institution Basal Research Fund (1610012021001_03202) to Mingxiong He; by Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2016-BIOMA) to Guoquan Hu; and by Central Public-Interest Scientific Institution Basal Research Fund (610012022009-03102) to Bo Wu.
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YHH: investigation, formal analysis, methodology, and writing-original draft; MC: writing—review and editing; GQH: funding acquisition; BW: conceptualization, funding acquisition, methodology, project administration, and writing—review and editing; MXH: conceptualization, funding acquisition, and writing—review and editing. All authors were informed and agreed with the submission.
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Huang, Y., Chen, M., Hu, G. et al. Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis. Appl Microbiol Biotechnol 107, 7151–7163 (2023). https://doi.org/10.1007/s00253-023-12783-y
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DOI: https://doi.org/10.1007/s00253-023-12783-y