Characterization of polyhydroxyalkanoate synthases from Halomonas sp. O-1 and Halomonas elongata DSM2581: Site-directed mutagenesis and recombinant expression

Abstract

Polyhydroxyalkanoates (PHAs) are a group of microbial intracellular biopolyesters that have wide potential applications for the plastics industry. Here, we report the results of molecular analyses of PHA synthases from Halomonas species, which are halophilic eubacteria known as promising industrial PHA producers, but the PHA synthase gene of Halomonas has not been cloned previously. In this study, the putative PHA synthase genes (phaC1 and phaC2) from two Halomonas strains (a new isolate, Halomonas sp. O-1, and the genome-sequenced strain, Halomonas elongata DSM2581) were cloned and characterized. We identified a gene related to the H. elongata DSM2581 PHA synthase gene (phaC1He) in the Halomonas sp. O-1 genome (phaC1HO1). PHA synthases usually contain a lipase box-like sequence Gly-X-Cys-X-Gly in their active sites. However, the equivalent sequence found in PhaC1HO1 and PhaC1He was determined to be Ser-X-Cys-X-Gly, with serine replacing the first glycine. The cloned phaC1HO1 and phaC1He genes were found to be functional when expressed in Escherichia coli JM109 and Ralstonia eutropha PHB-4. Site-directed mutagenesis studies showed that the cysteine in the Ser-X-Cys-X-Gly sequence was the catalytic center of Halomonas PhaC1 proteins and that replacement of serine with glycine slightly affected PHA biosynthesis and copolymer composition. In addition, a second potential PHA synthase gene (phaC2) was cloned and its polymerization activity was evaluated. The results herein provide an important molecular basis for PHA production by Halomonas species.