Unsterile production of a polyhydroxyalkanoate copolymer by Halomonas cupida J9

Microbial production of bioplastics polyhydroxyalkanoates (PHA) has opened new avenues to resolve "white pollution" caused by petroleum-based plastics. PHAs consisting of short- and medium-chain-length monomers, designated as SCL-co-MCL PHAs, exhibit much better thermal and mechanical properties than PHA homopolymers. In this study, a halophilic bacterium Halomonas cupida J9 was isolated from highly saline wastewater and proven to produce SCL-co-MCL PHA consisting of 3-hydroxybutyrate (3HB) and 3-hydroxydodecanoate (3HDD) from glucose and glycerol. Whole-genome sequencing and functional annotation suggest that H. cupida J9 may possess three putative PHA biosynthesis pathways and a class I PHA synthase (PhaC_J9). Interestingly, the purified His₆-tagged PhaC_J9 from E. coli BL21 (DE3) showed polymerizing activity towards 3HDD-CoA and a phaC_J9-deficient mutant was unable to produce PHA, which indicated that a low-substrate-specificity PhaC_J9 was exclusively responsible for PHA polymerization in H. cupida J9. Docking simulation demonstrated higher binding affinity between 3HB-CoA and PhaC_J9 and identified the key residues involved in hydrogen bonds formation between 3-hydroxyacyl-CoA and PhaC_J9. Furthermore, His489 was identified by site-specific mutagenesis as the key residue for the interaction of 3HDD-CoA with PhaC_J9. Finally, PHA was produced by H. cupida J9 from glucose and glycerol in shake flasks and a 5-L fermentor under unsterile conditions. The open fermentation mode makes this strain a promising candidate for low-cost production of SCL-co-MCL PHAs. Especially, the low-specificity PhaC_J9 has great potential to be engineered for an enlarged substrate range to synthesize tailor-made novel SCL-co-MCL PHAs.