Biosynthesis of polyhydroxyalkanoate copolyesters by Aeromonas hydrophila mutant expressing a low-substrate-specificity PHA synthase PhaC2Ps

A polyhydroxyalkanoate (PHA) synthase negative mutant termed Aeromonas hydrophila CQ4 was constructed from its wild type strain A. hydrophila 4AK4. Heterologous expression of a low-substrate-specificity PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 in A. hydrophila CQ4 could copolymerize short-chain-length (SCL) 3-hydroxybutyrate (3HB) and medium-chain-length (MCL) 3-hydroxyalkanoates (3HA). Co-expressing (R)-specific enoyl-CoA hydratase and PHA synthase PhaC2Ps in A. hydrophila CQ4 (phaJAh, phaC2Ps) led to accumulation of 20.86 wt% PHA copolyesters containing 59 mol% 3HB, 37 mol% 3-hydroxyhexanoate (3HHx) and 4 mol% of other MCL 3HA in shake flask culture. When grown in fermentor, cellular dry weight (CDW) and PHA content were 22.75 g L−1 and 20.08 wt%, respectively, with 72.38 mol% 3HB, 25.18 mol% 3HHx and 2.45 mol% other MCL 3HA in the copolymer. If beta-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha were co-expressed with PhaC2Ps, A. hydrophila CQ4 (phaC2Ps, phbARe, phbBRe) accumulated PHA copolyesters containing enhanced 3HB content up to 79.95 mol% and reduced 3HHx fraction of 8.55 mol%. When grown in fermentor it produced 16.28 g L−1 CDW containing 11.64 wt% PHA consisting of 87.38 mol% 3HB, 9.75 mol% 3HHx and 2.87 mol% other MCL 3HA. These results further demonstrate that A. hydrophila is a good candidate for metabolic engineering for diverse PHA production.