Production and Characterization of Copolymers Consisting of 3-Hydroxybutyrate and Increased 3-Hydroxyvalerate by β-Oxidation Weakened Halomonas.

Abstract

Polyhydroxyalkanoates (PHA) with high 3-hydroxyvalerate (3HV) monomer ratios lead to their accelerated biodegradation and improved thermal and mechanical properties. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a broad range of 3HV ratios were produced and characterized using the next generation industrial biotechnology (NGIB) chassis Halomonas bluephagenesis (H. bluephagenesis). Wild type H. bluephagenesis was found to produce P(3HB-co-66.31mol% 3HV) when cultured in the presence of valerate. Deletion on the functional enoyl-CoA hydratase (fadB₁) increased to 93.11 mol% 3HV in the PHBV copolymers. Through tuning the glucose and valerate co-feeding, PHBV with controllable 3HV ratios were adjusted to range from 0-to-93.6 mol% in shake-flask studies. Metabolic weakening of the β-oxidation pathway paired with flux limitation to the native 3HB synthesis pathway were used to reach the highest reported 98.3 mol% 3HV by H. bluephagenesis strain G34B grown in shake flasks. H. bluephagenesis strain G34B was grown to 71.42 g/L cell dry weight (CDW) containing 74.12 wt% P(3HB-co-17.97 mol% 3HV) in 7 L fermentors. Mechanical properties of PHBV with 0, 22.81, 42.76, 73.49 and 92.17 mol% 3HV were characterized to find not linearly related to increased 3HV ratios. Engineered H. bluephagenesis has demonstrated as a platform for producing PHBV of various properties.