Production and characterization of copolymers consisting of 3-hydroxybutyrate and adjustable lactate by engineered Halomonas bluephagenesis from glucose

Polyhydroxyalkanoates (PHAs) constitute a diverse family of biodegradable and biocompatible polymers with potential as sustainable alternatives to petroleum-based plastics. Microbial poly(3-hydroxybutyrate-co-lactate), abbreviated as P(3HB-co-LA), as member of the PHA family exhibiting a wide range of lactate (LA) molar ratios, was biosynthesized by engineered Halomonas bluephagenesis (H. bluephagenesis). With genome integration of four copies of mutated PHA synthase PhaC1_Ps (E130D, S325T, S477G and Q481K) and propionyl-CoA transferase (Pct₅₄₀), the resulting H. bluephagenesis produced 6.1 g L^-1 cell dry weight (CDW), with 48.4 wt% P(3HB-co-27.6 mol% LA). H. bluephagenesis CJN29 with deletion in ppc, pta and dld genes, encoding phosphoenolpyruvate carboxylase, phosphate acetyltransferase, and lactate dehydrogenase, respectively, reached 6.8 g L^-1 CDW containing 54.8 wt% P(3HB-co-35.9 mol% LA). After medium optimization, H. bluephagenesis CJN29 produced 10.8 g L^-1 CDW containing 54.1 wt% P(3HB-co-36.2 mol% LA). Deletion of mreB encoding a cytoskeletal protein significantly enlarged the cells for convenient downstream purification. Using glucose as the sole carbon source in a 7-L fermenter, H. bluephagenesis CJN29 achieved 93.8 g L^-1 CDW, with 57.3 wt% P(3HB-co-31.6 mol% LA). The record-high P(3HB-co-LA) production achieved in this study, coupled with its adjustable material properties, establishes engineered H. bluephagenesis as a customized P(3HB-co-LA) producer under non-sterile conditions, enabling cost-effective bioproduction.