Characterising and engineering the extremophilic chassis Halomonas TD1.0 for biomanufacturing

Abstract

Replacing petrochemicals with sustainable alternatives is essential to limit the effects of climate change but the widespread employment of microbial manufacturing is currently prevented by its high cost. The industrially relevant chassis Halomonas bluephagenesis TD1.0 could greatly reduce operating costs by not requiring sterile conditions or fresh water. It remains poorly understood, however, and its genetic toolkit, limited. This work mapped TD1.0’s proteome during growth on key constituents of waste-derived anaerobic digestate (acetate, propionate and butyrate), including their assimilation pathways. Through ALE, a strain was evolved with up to 4-fold higher maximum specific growth rate on propionate and tolerance of 100 g L-1 propionate. An operon upregulated in propionate was adapted for novel expression systems for TD1.0. These showed low detection limits (0.1 g L-1) and up to 218-fold induction, 7-fold higher than the best alternatives derived from existing E. coli systems. Furthermore, the genes, mRNAs and enzymes for the majority of TD1.0 vitamin B12 biosynthetic reactions were identified. Most genes specific to cobalamin (Cbl) synthesis were organised as a single cluster with two predicted cobalamin riboswitches. An assay was developed that confirmed AdoCbl’s presence in TD1.0 lysate and has potential medical applications given its fast output (45 min) and high sensitivity (

Date of Award	28 Feb 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Main Supervisor) & Sam Hay (Co Supervisor)