Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.

The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.

Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.

The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.

pMTL60000: A modular plasmid vector series for Parageobacillus thermoglucosidasius strain engineering.

Parageobacillus thermoglucosidasius is a promising chassis for producing chemicals and fuels. Here we designed, built and tested the pMTL60000 modular plasmids containing standardised Gram-positive and Gram-negative replicons, selectable markers and application-specific modules. The pMTL60000 modular plasmids were characterised with regard to transformation efficiency, segregational stability, copy number and compatibility.

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.

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.