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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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-015-0380-8) contains supplementary material, which is available to authorized users.

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