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Journal of Bacteriology, June 2008, p. 4173-4180, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00134-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei ^,

Qiuhe Lu,^1,2, Jing Han,^1,2, Ligang Zhou,^1,2 Jian Zhou,¹ and Hua Xiang^1*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences,¹ Graduate University of Chinese Academy of Sciences, Beijing, Peoples Republic of China²

Received 25 January 2008/ Accepted 31 March 2008

The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC_Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE_Hme and phaC_Hme genes were constitutively expressed, and both the PhaE_Hme and PhaC_Hme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC_Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC_Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC_Hme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE_Hme/PhaC_Hme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC_Hme and PhaE_Hme, accounted for the PHBV synthesis in H. mediterranei.

Published ahead of print on 11 April 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

Q.L. and J.H. contributed equally to this paper.