Metabolic engineering of Aeromonas hydrophila for the enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Wild-type Aeromonas hydrophila 4AK4 produced 35–45 wt.% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) consisting of 10–15 mol% 3-hydroxyhexanoate (3HHx). To enhance PHBHHx production, vgb gene encoding Vitreoscilla haemoglobin or fadD gene encoding Escherichia coli acyl-CoA synthase was co-expressed with polyhydroxyalkanoates (PHA) synthesis-related genes including phbAB from Wautersia eutropha and phaPCJ from A. hydrophila. Expression of vgb increased PHBHHx content from 46 to 53 wt.% without affecting the polymer monomers composition, whereas fadD increased both PHBHHx content from 46 to 64 wt.% and its 3HHx fraction from 15 to 24 mol%. Co-expression of vgb or fadD gene with PHA-synthesis-related genes generally increased PHBHHx content over 60 wt.%. Co-expression of phbAB with vgb increased PHBHHx content and concentration up to about 70 wt.% and 4.0 g l⁻¹, respectively. Fermentor study also showed that in the recombinants harboring vgb, CDW, PHBHHx concentration and productivity were significantly elevated up to 54 g l⁻¹, 28.5 g l⁻¹ and 0.791 g l⁻¹ h⁻¹, respectively, suggesting that vgb could promote PHA synthesis. In this strain, lac promoter could be used to constitutively express foreign genes such as phbA and phbB encoding β-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase of W. eutropha, respectively, without use of IPTG. The results showed that combined expression of different genes was a successful strategy to enhance PHA production, which could be useful for strain development to construct other recombinant PHA-producing strains.