Polyhydroxyalkanoate synthase from Bacillus sp. INT005 is composed of PhaC and PhaR

Abstract

A polyhydroxyalkanoate (PHA) biosynthesis gene locus from Bacillus sp. INT005 strain, which had been isolated from a gas field, was cloned and analyzed at the molecular level. We found that a 3.8-kbp DraI-digested fragment of genomic DNA of Bacillus sp. INT005 conferred PHA-producing ability to Escherichia coli, which was PHA-negative. The DNA fragment contained three genes, phaR, -B and -C. The activity of 3-ketoacyl-CoA reductase with NADPH was detected in the lysate from recombinant E. coli carrying the phaB gene. Although PHA synthase activity could be detected in the extract from E. coli carrying phaR, -B and -C genes, no such activity could be detected in that from E. coli carrying only the phaC gene. However, the mixture of the crude extracts of E. coli expressing phaR or phaC revealed very high PHA synthase activity. Furthermore, when His-tagged PhaC was purified by Ni-affinity chromatography from the mixture of crude extracts containing His-tagged PhaC or native PhaR, the eluate contained His-tagged PhaC and native PhaR. On the other hand, PhaR did not bind to the column directly. This purified PhaC with PhaR had 160-fold higher specific activity of PHA synthase than that without PhaR. In addition, the kinetics of the purified PhaC with PhaR revealed a lag phase that preceded the linear phase. It has been known that class III PHA synthase is composed of two different subunits, PhaC and PhaE, and phaC and phaE genes are directly linked in the genomes. Furthermore, the PHA synthase has no lag phase. We hence concluded that the PHA synthase of Bacillus sp. INT005 consists of PhaC and PhaR, and has characteristics different from class III PHA synthase.