SYNECHOCYSTIS SP. PCC6803 POSSESSES A 2-COMPONENT POLYHYDROXYALKANOICACID SYNTHASE SIMILAR TO THAT OF ANOXYGENIC PURPLE SULFUR BACTERIA

During cultivation under storage conditions with BG11 medium containin g acetate as a carbon source, Synechocystis so. PCC6803 accumulated po ly(3-hydroxybutyrate) up to 10% (w/w) Of the cell dry weight. Our anal ysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open readin g frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to th e corresponding PHA synthase subunits PhaE and PhaC of Thiocystis viol acea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis s p. PCC6803 genes were cloned using PCR and were heterologously express ed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3- hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB-4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp . PCC6803 PHA synthase. PHA granules were detected by electron microsc opy in these cells, and the PHA-granule-associated proteins were studi ed. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raise d against the PHA synthases of A. eutrophus (PhaC) and of C, vinosum ( PhaE and PhaC) revealed no immunoreaction.