MicroFuelPro: Microbial fuel development framework using synthetic biology for next generation drop-in renewable fuel production

Abstract

The use of renewable biofuels has emerged as a valid alternative to the exploitation of depletable fossil-fuel carbons. Amongst biofuels, first-generation (1G) bioethanol is one of the most widely used renewable transport fuels. However, since its production depends on the exploitation of simple sugars from crops, a transition to using waste biopolymers is essential. In the present research, we aimed at engineering the ethanologenic Gram-negative bacterium Zymomonas mobilis towards the efficient production of ethanol from waste biopolymers. As robust molecular tools for the engineering of Z. mobilis were absent, we developed a broad library of constitutive synthetic promoters and terminators for fine transcriptional regulation, and tested bicistronic designs (BCDs), used to uncouple the translational strength of a ribosome binding site (RBS) from its context. Additionally, we demonstrated that the promoters regulation in Z. mobilis does not vary between aerobic and anaerobic conditions. The characterised molecular tools were made compatible with a high-throughput combinatorial assembly method and their functionality confirmed with the heterologous expression of the 2,3-butanediol pathway. As polymers cannot enter bacterial cells, the secretion of hydrolytic enzymes is imperative. An investigation to identify the Type One Secretion tags was performed through bioinformatics analysis and construction of a mutant. A putative tag was identified and used for the secretion of a heterologous β-galactosidase. The findings presented in this study are a benchmark for efficient engineering in Z. mobilis, and pave the way for its application in converting waste biopolymers into fermentable sugars for sustainable bioethanol production.

Date of Award	22 May 2025
Original language	English
Awarding Institution	Northumbria University
Supervisor	Ulugbek Azimov (Supervisor), Jose Munoz (Supervisor) & Ciaran Kelly (Supervisor)