Compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by replacement of granule-associated protein in Ralstonia eutropha.

BACKGROUND: Phasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1(Re) locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil.

RESULTS: Considering the high expression level of phaP1(Re), phaJ(Ac) [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1(Re) on chromosome 1 of R. eutropha strain NSDG harboring phaC(NSDG) (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJ(Ac) within pha operon. Insertion of the second phaC(NSDG) along with phaJ(Ac) at the phaP1(Re) locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1(Re) with phaP(Ac) (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester.

CONCLUSIONS: The downstream of phaP1(Re) was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters.