Abstract

Polyhydoxyalkanoates (PHAs) are biodegradable biopolymers that has been expected as eco-friendly polymeric materials. Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is one of the most practical PHAs by reason of its soft and flexible properties. We have previously constructed recombinant strains of Ralstonia eutropha capable of synthesizing P(3HB-co-3HHx) efficiently from vegetable oils by replacing the native PHA synthase gene (phaC_Re) with the mutated PHA synthase gene from Aeromonas caviae (phaC_NSDG) and by modifying the (R)-3HB-CoA formation pathway and β-oxidation pathways. In this study, we examined further engineering of the gene of R. eutropha recombinant strains focusing on PHA granule-associated protein PhaP (Phasin). Recently, the activation of PhaC_Ac by PhaP in vitro was reported. We here investigated the effects of PhaPs derived from different sources on P(3HB-co-3HHx) biosynthesis from vegetable oils by R. eutropha. When phaP1_Re encoding the natively major PHA granule-associated protein was replaced by phaP from A. caviae (phaP_Ac) in R. eutropha expressing phaC_NSDG and phaJ_Ac (a gene of (R)-enoyl-CoA hydratase), the 3HHx composition in P(3HB-co-3HHx) synthesized from soybean oil was significantly enhanced with maintaining high content of cellular PHA. Interestingly, the replacement of phaP also led to increase of the molecular weight. The compositional change by the phaP replacement was enhanced when PhaC_NSDG was overexpressed from two copies of the gene. These results suggested that the 3HHx incorporation ability of PhaC_NSDG on granules covered with PhaP_Ac was higher than that of PhaC_NSDG on granules covered with PhaP1_Re. That is possibly due to some specific interactions between PhaC_NSDG and PhaP_Ac derived from the same bacterium.