Metabolic engineering of Halomonas for the utilisation of organic acids and CO2 as carbon sources

Halomonas is a robust microbial chassis currently in use in the industrial scale production of bioplastics (poly-3-hydroxybutyrate). New biotechnological applications for Halomonas currently under development include its use in the production of bio-LPG (propane and butane), monoterpenoid-based biofuel additives and mandelate. Effective sustainable and scalable microbial production strategies require considerable reductions in fermentation costs to enable scaled production to be competitive with existing biological and/or chemical production routes. Towards this end, Halomonas is a promising industrial chassis as it can be cultivated in seawater/waste water on renewable waste biomass under non-sterile conditions with minimal downstream processing, and (limited) use of fresh water. To improve the sustainability and low carbon strategy of Halomonas cultivation, a more efficient utilisation of mixed carbon sources is needed to maximise the biomass (and secondary product) yield per unit waste. A major waste product of of interest to C3 BIOTECH is acetic acid, which accumulates in some of its production processes. In this project, a synthetic biology approach will be used to engineer a genome integrated recombinant acetate utilisation route within Halomonas. Production and utilisation in E. coli are known to be carefully regulated via reversible acetylation by accessory proteins and other mechanisms. The comparable systems in Halomonas are unknown, so the E. coli system will be incorporated within Halomonas, including a variants of a key enzyme acetyl-CoA synthetase to allow the rerouting of acetate as a fermentation waste product into the central metabolite acetyl-CoA. This will increase overall carbon utilisation, both by recycling acetate generated during fermentation and the consumption of acetate naturally present in the waste biomass feed. The reduction in cytotoxic acetate accumulation within the culture should significantly improve Halomonas growth, which is likely to impact favourably on secondary product titres. A second target waste carbon source for Halomonas is atmospheric carbon dioxide. Prior studies with E. coli showed that chemolithoautotrophic CO2 fixation pathways could be constructed using native enzymes, which would function with the addition of an external energy source (e.g. hydrogen, formate and sulphur compounds). Multiple chemolithoautotrophic CO2 fixation pathways have been described, such as those based on the Calvin-Benson-Bassham cycle, reductive acetyl-CoA pathway, dicarboxylate/4-hydroxybutyrate cycle and the 3-hydroxypropionate/4-hydroxybutyrate cycle. This project will aim to incorporate one of the simpler designed CO2 fixation pathways, utilising thiosulphate as the energy source. This pathway will be initially tested for effectiveness on a plasmid-based system, before integration into the genome of an industrial strain of Halomonas. Successful implementation of these low carbon strategies will ultimately provide economic, sustainable, secure and clean alternatives to extant petrochemical LPG supplies and other industrially useful secondary products. This fits within the DTP stream of Industrial Biotechnology and Bioenergy by tackling the need for sustainable biotechnological solutions towards chemicals and biofuels production. At the same time, it addresses key climate control targets by envisioning a carbon neutral solution of generating an industrial carbon fixating strain of Halomonas, capable of delivering cost-competitive, non-fossil fuel derived biological routes towards useful compounds.