A Toolkit for Engineering Geobacillus and Parageobacillus as a Chassis for Bio-Valorisation of Waste Produced in the Built Environment and Production of Industrially Relevant Chemicals

Abstract

The petrochemical industry is dependent on fossil fuels to produce commodity and high-value chemicals. Production of these chemicals from these non-renewable sources releases huge amounts of greenhouse gases. Unsustainable production, coupled with unsustainable methods of waste management is accelerating climate change and causing ecological damage. Microbial cell factories can be used as sustainable routes for bio-valorisation of waste, with Parageobacillus and Geobacillus as potential candidates due to their ability to utilise biomass and alkane feedstocks for growth. This work aimed to expand the genetic tools for engineering these species and use these tools for improved waste degradation and production of an industrially relevant compound.
Improving predictability of gene expression for a particular promoter was explored using synthetic RBS sequences, coupled with the self-cleaving ribozyme RiboJ to act as a buffer. We observed more similarity in expression of Dasher-GFP and mOrange for each promoter compared to native RBS and synthetic RBS sequences lacking RiboJ. Twelve constitutive promoters conserved across the genus were characterised in G. subterraneus, P. toebii, G. uzenensis and P. thermoglucosidasius, using Dasher-GFP, with the library displaying a diverse range of expression levels in each species. Additionally, a library of signal peptides and Rho-independent terminators were identified and characterised in P. thermoglucosidasius DSM2542. Finally, level 1 and level 2 modular cloning vectors were developed for producing vectors with single and multiple transcriptional units using Golden gate modular cloning.
Using these tools, the alkane utilisation ability of G. uzenensis was successfully expanded through the expression of LadA, and the bacteria was used for degradation of waste produced by cold-plasma assisted pyrolysis of HDPE plastic. Finally, P. thermoglucosidasius was used as a chassis for production of D-glucaric acid. The work undertaken will allow the engineering of Geobacillus as a microbial cell factory for waste valorisation.

Date of Award	22 May 2025
Original language	English
Awarding Institution	Northumbria University
Supervisor	Paul James (Supervisor)