Lau, Matthew Simon Harold
(2019)
The expansion of available bioparts and the development of CRISPR/Cas9 for genetic engineering of Parageobacillus thermoglucosidasius.
PhD thesis, University of Nottingham.
Abstract
Growing public concern over the environmental and health impacts caused by the use of fossil fuels, coupled with diminishing reserves, is now compelling the pursuit for an alternative source. The search for alternative methods to manufacture products conventionally derived from fossil fuels, has led to research into microbial conversion of lignocellulosic biomass into analogous derivatives. Though many well established renewable energy methods currently exist, research into the production of petroleum-based products, such as transportation fuel and platform chemicals, is currently insufficient. Second generation biofuels have successfully overcome numerous problems associated with original biofuel production; however, many challenges are prevalent for established mesophilic organisms such as Escherichia coli or Saccharomyces cerevisiae. Thermophilic species have been presented as a potential platform strain to circumvent these problems. Typically non-fastidious and catabolically flexible organisms, thermophiles can ferment both hexose and pentose sugars, facilitate lower overall fermentation costs and are generally more robust organisms, a trait valuable for industrial processes. Unfortunately, current genetic tools are not well established to achieve the necessary yields to replace both the fossil fuel industry and the currently used organisms.
This study details the identification and subsequent testing of additional genetic tools, to expand the ability for genetic engineering in the thermophile Parageobacillus thermoglucosidasius. The capacity to express exogenous DNA is often predicated on the availability of necessary gene expression elements for complete optimisation of synthetic systems. The identification and expression of the first two thermostable fluorescent proteins in P. thermoglucosidasius allowed the characterisation of several important gene expression elements, which included a collection of promoters, ribosomal binding sites and transcriptional terminators. Subsequently, these tools can now be utilised for precise expression of both endogenous and exogenous DNA in P. thermoglucosidasius.
The initial subset of libraries were further successfully utilised to enable the first expression of a CRISPR/Cas9 complex, isolated from Streptococcus thermophilus, in P. thermoglucosidasius. This bacterial adaptive immunity system was then repurposed to facilitate successful genome editing in P. thermoglucosidasius, verified by the removal of the endogenous acetate kinase gene. The efficacy of CRISPR/Cas9 for genome engineering was further demonstrated by its ability to integrate exogenous DNA into both the chromosome and the extrachromosomal megaplasmid present in P. thermoglucosidasius. Cas9 was also validated as an effective method of curing plasmids, through the removal of the megaplasmids, pNCI001 and pNCI002, present in P. thermoglucosidasius. The curing displayed no essential function of either megaplasmid under the conditions tested, but implied the presence of a carbon utilisation pathway, for degradation of aromatic hydrocarbons. The curing of the megaplasmids indicated no beneficial effects and therefore displayed their potential as a vehicle for heterologous expression, due to their stability and increased copy number.
Validation of the Cas9-based technology was further achieved through the deletion of the native R-M systems, which revealed an increase in transformation efficiency. This increase can be co-opted to offset the reduction in transformation efficiency observed through the introduction of Cas9 and furthermore, facilitate more efficient allele exchange.
The ability to increase succinate production was a further demonstration of the applicability of these developed tools for genome engineering of P. thermoglucosidasius. The production of this important platform chemical, typically produced from petroleum, was achieved through the deletion of several genes from within the central carbon utilisation pathways, using the Cas9 technology exploited in this study. Deletion of succinate dehydrogenase resulted in a 3.5-fold increase compared to WT and produced a base-line strain for further deletions, which will enable the diversion of carbon flux towards succinate production.
Both the tools and strains presented in this study can be built upon to synthetically produce an industrially useful strain of P. thermoglucosidasius. This demonstrates significant potential for the future production of biofuels and platform chemicals from lignocellulosic biomass.
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