Study of the Biosynthesis of Poly(3-hydroxybutyrate- <i>co</i> -3-hydroxyvalerate) with High Content in 3-Hydroxyvalerate Units by <i>Cupriavidus necator</i> H1 G ⁺ 3 (DSM 545)

Poly(3-hydroxybutyrate) (PHB) has long been recognized as a promising biobased and potentially biodegradable polymer; however, its brittleness and narrow processing window limit broader implementation. Copolymerization with 3-hydroxyvalerate (3HV) improves flexibility and thermal behavior, yet controlling elevated 3HV incorporation in wild-type strains remains metabolically constrained. This work systematically investigates poly(3-hydroxybutyrate-<i>co</i>-3-hydroxyvalerate) (PHBHV) biosynthesis in <i>Cupriavidus necator</i> H1 G⁺3 DSM 545 as a benchmark production system, focusing on process limitations rather than record composition. The influence of cultivation mode (batch vs fed-batch), precursor chemistry (valeric acid vs sodium valerate), and pulse concentration was evaluated in relation to copolymer composition, molar mass distribution, and thermal properties. Under optimized conditions, 3HV contents up to 64 mol % were obtained. Increasing valeric acid concentrations led to reduced cell density and decreased 3HV incorporation, revealing a narrow precursor tolerance window. Using sodium valerate tends to promote biomass formation and 3HB biosynthesis at the expense of 3HV incorporation. A rapid, nondestructive FTIR method was developed to estimate 3HV molar fraction. These results clarify intrinsic metabolic boundaries governing 3HV incorporation and establish a framework for controlled structure-property investigation of PHBHV copolymers.