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.
Highlights
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
Respective thermophilic chassis offer a number of attractions over current model organisms, such as Escherichia coli and Saccharomyces cerevisiae.[1−4]
To identify the most effective promoter systems to accomplish this, a novel reporter system was first established in P. thermoglucosidasius based on a thermostable fluorescent protein, eCGP123.36,37 Purified eCGP123 protein has previously been shown to fluoresce at 80 °C36 under in vitro conditions and to function in vivo in the thermoacidophilic archaeon Sulfolobus acidocaldarius[38] but has yet to be evaluated in a bacterium
Summary
Authors Matthew S. H. Lau − BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Lili Sheng − BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Ying Zhang − BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Complete contact information is available at: https://pubs.acs.org/10.1021/acssynbio.1c00138
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