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Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains

Lau, Matthew S. H.; Sheng, Lili; Zhang, Ying; Minton, Nigel P.

Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains Thumbnail


Authors

Matthew S. H. Lau

Lili Sheng



Abstract

The relentless rise in the levels of atmospheric greenhouse gases caused by the exploitation of fossil fuel necessitates the development of more environmentally friendly routes to the manufacture of chemicals and fuels. The exploitation of a fermentative process that uses a thermophilic chassis represents an attractive option. Its use, however, is hindered by a dearth of genetic tools. Here we expand on those available for the engineering of the industrial chassis Parageobacillus thermoglucosidasius through the assembly and testing of a range of promoters, ribosome binding sites, reporter genes, and the implementation of CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases. The latter were used to demonstrate that the deletion of the two native plasmids carried by P. thermoglucosidasius, pNCI001 and pNCI002, either singly or in combination, had no discernible effects on the overall phenotypic characteristics of the organism. Through the CRISPR/Cas9-mediated insertion of the gene encoding a novel fluorescent reporter, eCGP123, we showed that pNCI001 exhibited a high degree of segregational stability. As the relatively higher copy number of pNCI001 led to higher levels of eCGP123 expression than when the same gene was integrated into the chromosome, we propose that pNCI001 represents the preferred option for the integration of metabolic operons when stable commercial strains are required.

Citation

Lau, M. S. H., Sheng, L., Zhang, Y., & Minton, N. P. (2021). Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains. ACS Synthetic Biology, 10(7), 1739-1749. https://doi.org/10.1021/acssynbio.1c00138

Journal Article Type Article
Acceptance Date May 14, 2021
Online Publication Date Jul 1, 2021
Publication Date Jul 16, 2021
Deposit Date May 25, 2021
Publicly Available Date Jul 2, 2022
Journal ACS Synthetic Biology
Electronic ISSN 2161-5063
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Volume 10
Issue 7
Pages 1739-1749
DOI https://doi.org/10.1021/acssynbio.1c00138
Keywords CRISPR/Cas9; genetic tools; thermophile; stable plasmid expression; synthetic biology; curing plasmids
Public URL https://nottingham-repository.worktribe.com/output/5570614
Publisher URL https://pubs.acs.org/doi/abs/10.1021/acssynbio.1c00138

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