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Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber

Ursula Pieper , Alexander Steinbüchel
DOI: http://dx.doi.org/10.1111/j.1574-6968.1992.tb05396.x 73-79 First published online: 1 September 1992

Summary

The first polyhydroxy acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthesis of A. eutrophus and Pseudomas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61 000 protein was identified as the PHA syntase of R. ruber by N-terminal amino acid sequence determination.

Key words
  • PHA synthase
  • Poly(3-hydroxyalkanoic acid)
  • Biodegradable polyester
  • PHA granule
  • Rhodococcus ruber
  • Alcaligenes eutrophus

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