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 (E130D, S325T, S477G and Q481K) and propionyl-CoA transferase (Pct), the resulting H. bluephagenesis produced 6.1 g L 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 CDW containing 54.8 wt% P(3HB-co-35.9 mol% LA). After medium optimization, H. bluephagenesis CJN29 produced 10.8 g L 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 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.