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

Poly(3-hydroxybutyrate-<i>co</i>-3-hydroxyvalerate) (PHBV) with a 0-30 mol% controllable range of 3HV ratios was produced by <i>Halomonas bluephagenesis</i> (<i>H. bluephagenesis</i>) and characterized. An endogenous plasmid containing <i>scpA</i> and <i>scpB</i> encoding methylmalonyl-CoA epimerase and methylmalonyl-CoA decarboxylase, respectively, redirects succinyl-CoA toward propionyl-CoA, enabling <i>de novo</i> PHBV synthesis with a 1.7 mol% 3HV. Deletion of <i>sdhE</i> and <i>prpC</i> encoding succinate dehydrogenase and 2-methylcitrate synthase, respectively, further enhanced the 3HV to 4 mol%. <i>H. bluephagenesis</i> 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. <i>H. bluephagenesis</i> 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.