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

• The method for tuning gene expression levels were characterized in Halomonas sp. • The highest 3HHx ratio in PHBHHx was obtained from Halomonas sp. with fadB 1 deletion. • Recombinant Halomonas sp. G34 produced PHBHHx containing 0–37 mol% 3HHx. 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 (PhaC ac) and enoyl coenzyme-A hydratase (PhaJ ac) could produce PHBHHx from glucose and sodium hexanoate. After rationally fine-tuning the expression levels of phaC ac and phaJ ac employing two inducible promoters, P lac and P lux 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, P porin68 and P porin58 , 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. [ABSTRACT FROM AUTHOR]