Studies On Phasin PhaP, A Polyhydroxyalkanoate Sythesis Related Protein From Aeromonas Hydrophila

Aeromonas hydrophila 4AK4 is the strain that synthesizes poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), one of the member of polyhydroxyalkanoates (PHA), from lauric acid. In present study, the PHA synthesis related genes were cloned from A. hydrophila 4AK4 by PCR method and the functions of these genes were identified. The nucleotide sequence of this region showed a gene cluster consisting of phaP encoded a granule-associated protein and genes of PHA synthase and (R)-specifec enoyl-CoA hydratase. DNA sequence analysis result showed that these PHA related genes between A. hydrophila 4AK4 and Aeromonas caviae shared very high homology.PhaP, phaC and phaJ was over-expressed in A. hydrophila 4AK4, respectively. The result showed that recombinant strains harboring additional copies of phaP and phaC accumulated PHBHHx copolyesters with higher 3-hydroxyhexanoate (HHx) proportion than that of wild type strain. The molecular weight of PHBHHx produced by the above recombinants was lower than that obtained from the wild type grown under similar conditions, while phaJ_Ah had little effect. Over-expression oiphaP_Ah led to the production of more PHA granules but with reduced sizes.The proteins associated on surface of the inclusions were separated by SDS -PAGE. The result clearly indicated that PhaP_Ah and PhaC were bound on the granules. PhaP_Ah was the predominant protein present in the PHBHHx granules. The real-time PCR results suggested that phasin PhaP_Ah up-regulated phaC gene at the transcription level.In an attempt to further understand PhaP regulatory mechanism of PHA biosynthesis, the functions between PhaP_Ah from A. hydrophila 4AK4 and PhaP_We from Wautersia eutropha H16 (formerly Ralstonia eutropha) were compared. Analysis of the primary sequences of the PhaP_Ah and the PhaP_We showed that the disparity in size between the A. hydrophila and W. eutropha phasins were 13 and 20 kDa. PhaPwe with low amino acid homology to PhaP_Ah was cloned into A. hydrophila 4AK4. It exhibited similar effects on PHBHHx production and PHBHHx composition. These data suggest that the phasins could represent a protein family possessing similar functions with similar secondary structures.