Molecular characterization of the phaECHm, genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic Archaeon Haloarcula marismortui

Although many haloarchaea produce biodegradable polyhydroxyalkanoates (PHAs), the genes involved in PRA synthesis in the domain of Archaea have not yet been experimentally investigated yet. In this study, we revealed that Haloarcula marismortui was able to accumulate poly (3-hydroxybutyrate) (PHB) up to 21% of cellular dry weight when cultured in a minimal medium with excessive glucose and identified the phaE(Hm) and phaC(Hm) genes, probably encoding two subunits of a class III PHA synthase. These two genes were adjacent and directed by a single promoter located 26 bp upstream of the transcriptional start site and were constitutively expressed under both nutrient-rich and -limited conditions. Interestingly, PhaC(Hm) was revealed to be strongly bound with the PHB granules, but PhaEH,m seemed not to be. Introduction of either the phaE(Hm) or phaC(Hm) gene into Haloarcula hispanica, which harbors highly homologous phaEC(Hh) genes, could enhance the PHB synthesis in the recombinant strains, while coexpression of the both genes always generated the highest PHB yield. Significantly, knockout of the phaEC(Hh) genes in H. hispanica led to a complete loss of the PRA synthase activity. Complementation with phaEC(Hm) genes, but not a single one, restored the capability of PHB accumulation as well as the PHA synthase activity in this phaEC-deleted haloarchaeon. These results indicated that the phaEC genes are required for biosynthesis of PHB and might encode an active PRA synthase in the Haloarculd species.