Production of PHA copolymers consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) by recombinant Halomonas bluephagenesis

Abstract

Microbial synthesized poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), namely PHBHHx, with different 3HHx monomer ratios have shown biocompatibility, biodegradability and flexibility for commercial uses. This study aims at the low-cost production of PHBHHx with customized and/or rich 3HHx by engineered Halomonas bluephagenesis (H. bluephagenesis), which is a rising-star chassis for Next Generation Industrial Biotechnology (NGIB). Recombinant H. bluephagenesis harboring heterologous PHA synthase (PhaCac) and enoyl coenzyme-A hydratase (PhaJac) could produce PHBHHx from glucose and sodium hexanoate. After rationally fine-tuning the expression levels of phaCac and phaJac employing two inducible promoters, Plac and Plux induced by IPTG and AHL, respectively, the resulting H. bluephagenesis TDC-CJ produced 55 wt% P(3HB-co-14.21 mol% 3HHx). Furthermore, two inducible promoters were replaced by constitutive ones, Pporin68 and Pporin58, respectively, with similar expression strength to remove the additions of inducers. Notably, PHBHHx of high 3HHx ratios, reaching up to 49 mol%, was further obtained by weakening the native β-oxidation pathway. Finally, chromosomally engineered H. bluephagenesis G34 with two expression cassettes of phaCJ on loci G3 and G4 was constructed to achieve the synthesis of P(3HB-co-36 mol% 3HHx) without supplementing antibiotic. For lab-scale fermentation in a 7-L bioreactor, H. bluephagenesis G34 produced PHBHHx with controllable molar fraction of 3HHx from 0-to-37 mol% by designing glucose/hexanoate ratio. These efforts demonstrate that engineering H. bluephagenesis is of great potential for PHBHHx production of low cost for industrial-scale biomanufacturing purpose.