Molecular Characterization of the phaEC Hm Genes, Required for Biosynthesis of Poly(3-Hydroxybutyrate) in the Extremely Halophilic Archaeon Haloarcula marismortui

Abstract

ABSTRACT Although many haloarchaea produce biodegradable polyhydroxyalkanoates (PHAs), the genes involved in PHA 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 PhaE Hm 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 PHA 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 PHA synthase in the Haloarcula species.