Metabolic engineering of Salinivibrio sp. TGB10 for PHBV biosynthesis with a high 3-hydroxyvalerate fraction from starch and propionate.

Polyhydroxyalkanoates (PHA) are environmentally friendly biopolymers that have the potential to replace non-degradable plastics, yet large-scale industrial PHA production remains unattainable due to their high costs. Halophilic bacteria capable of growing under high-salt conditions are regarded as novel hosts for the economical production of PHA. Salinivibrio sp. TGB10, a moderately halophilic bacterium, efficiently accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using glucose and propionate as substrates. The genetic engineering of Salinivibrio species has not been reported due to the lack of molecular biology tools. Here, Salinivibrio sp. TGB10 was metabolically engineered using bacterial conjugation and gene knockout strategies based on markerless genomic DNA replacement. Through this approach, deletion of the native 2-methylcitrate synthase gene (prpC) increased the 3-hydroxyvalerate (3 HV) monomer content in the PHBV copolymer to nearly 90 mol%. Furthermore, the heterologous expression of a NaCl-tolerant amylase from Vibrio alginolyticus enabled efficient starch utilization, resulting in a PHBV titer of 3.35 g/L with 77.89 mol% 3 HV when starch and propionate were used as carbon sources. To the best of our knowledge, this is the first study to report the metabolic engineering of the Salinivibrio genus. The resulted Salinivibrio strains demonstrate significant potential for industrial production of PHBV with a high 3 HV composition.