Bioproduction by Halomonas bluephagenesis using Bioinformatic and Genetic Engineering Approach

Abstract

Industrial biotechnology has immense potential to drive sustainable production of valuable chemicals. Halophilic Halomonas bluephagenesis, with its ability to grow in non-sterile environments to high-density while naturally producing polyhydroxyalkanoates (PHA), offers a promising industrial chassis. However, the production of PHA is still hindered by high costs, partially attributed to complicated downstream processing and resource use. This thesis aims to address these challenges by leveraging OMICS profiling to devise cost-cutting solutions for increasing industrial viability of H. bluephagenesis for PHA production. Firstly, an interactive bioinformatics web platform specifically designed for H. bluephagenesis was established, facilitating exploration and targeted engineering efforts for non-expert researchers. Two key cost-reduction strategies emerged from the platform: (1) the genetic knockout of exopolysaccharides (EPS) to enhance cell aggregation for convenient downstream processing (DSP), and (2) the engineering of metabolic pathways to enable multi-product production in a single fermentation process. These strategies were validated in two detailed case studies. Insights from the platform indicate that genetically knocking out EPS can enhance carbon flux to PHA at the expense of energy-intensive cell wall components. Six engineered EPS-KO strains successfully redirected carbon from EPS biosynthesis to PHA production, achieving an 85% PHA content and 96 g/L cell dry weight in a 5000 L bioreactor after 40 hours growth. Additionally, downstream processing efficiency improved significantly: centrifugation time was reduced tenfold (from several hours to 15–30 minutes), water usage for PHA purification decreased by 30%, and enzyme usage fell by 35%. EPS-KO reduced expensive γ-butyrolactone supplementation, which is converted to be 4-hydroxybutyrate (4HB) monomer in PHA, by 40%, resulting in significant cost savings without sacrificing productivity. The bioinformatic platform analysis suggested co-production targets alongside PHA to lower costs for bioproduction by H. bluephagenesis. Metabolic engineering enabled the organism to co-produce propane, PHA, and mandelate in a single, semi-continuous fermentation process, with propane extracted from the headspace, mandelate from the fermentation broth, and PHA from cellular biomass. The process achieved 62 g/gDCW propane, 71 mg mandelate/L, and 69% g/gDCW PHA, demonstrating the feasibility of co-production. The two highlighted cases underscore the potential to use omics data for new strategies in industrial biotechnology. The ability to simplify downstream processing while achieving high yields of multiple products positions H. bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis capable of scalable, cost-effective bio-based production. This research provides a cohesive framework for future biotechnological innovation and offers a robust solution for the economic challenges associated with multiple product bioproduction.

Date of Award	1 Aug 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nigel Scrutton (Supervisor) & Neil Dixon (Supervisor)