Synthesis of tunable copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate by engineered Halomonas bluephagenesis and...

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a 0-30 mol% controllable range of 3HV ratios was produced by Halomonas bluephagenesis (H. bluephagenesis) and characterized. An endogenous plasmid containing scpA and scpB encoding methylmalonyl-CoA epimerase and methylmalonyl-CoA decarboxylase, respectively, redirects succinyl-CoA toward propionyl-CoA, enabling de novo PHBV synthesis with a 1.7 mol% 3HV. Deletion of sdhE and prpC encoding succinate dehydrogenase and 2-methylcitrate synthase, respectively, further enhanced the 3HV to 4 mol%. H. bluephagenesis GZ05 was engineered for late-phase-specific MreB (a cytoskeletal protein) degradation, enlarged intracellular PHBV granules for enhanced PHBV synthesis, and convenient downstream. A series of growth experiments was conducted in a 7 L bioreactor fed with valerate to produce PHBV with various 3HV molar ratios (2-27 mol%). A quantitative relationship between valerate concentration and the final 3HV molar ratio was established with an R² = 0.9833, enabling precise control of the 3HV ratio in PHBV. H. bluephagenesis GZ05 was grown to 100 g L^-1 cell dry weight (CDW) containing 73 wt% PHBV consisting of 1.6 mol% 3HV in a 5000 L bioreactor. Thermal analysis demonstrated enhanced flexibility with higher 3HV content in PHBV.