Improving poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis with the construction of a genome-scale metabolic model for Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are biodegradable plastics emerging as sustainable alternatives to petroleum-based plastics. Haloferax mediterranei has shown strong potential for large-scale production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using cheap carbon sources as substrates. To facilitate rational strain engineering, we constructed the first genome-scale metabolic model for H. mediterranei, named iHM951, comprising 1862 reactions and 1827 metabolites. The simulated carbon source utilization capacity and cell growth rate on glucose using iHM951 were consistent with experimental observations. Furthermore, iHM951-predicted flux distributions showed good agreement with experimental data from ¹³C-metabolic flux analysis. Model predictions highlighted the critical role of triosephosphate isomerase (TpiA) in supporting cell growth and PHBV production. In line with these predictions, the ΔtpiA mutant exhibited markedly reduced growth and diminished PHBV synthesis. Conversely, overexpression of tpiA, achieved by replacing its native weak promoter with one of moderate strength, led to a 26 % increase in biomass and a 47 % enhancement in PHBV production. Supporting these findings, RNA-seq analysis revealed upregulation of both the semi-phosphorylated Entner-Doudoroff pathway and PHBV biosynthetic genes in the tpiA-overexpressing strain. Taken together, the iHM951 model of H. mediterranei provides a valuable framework for optimizing PHBV biosynthesis and offers new insights into archaeal strain engineering.