Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter

Abstract

Molecular methods have been applied to analyze the expression of the Alcaligenes eutrophus poly(3-hydroxybutyrate) (PHB) synthase gene (phbC). The translational initiation codon was identified by analysis of the amino acid sequence of a PHB synthase-beta-galactosidase fusion protein. This protein was purified to almost gel electrophoretic homogeneity by chromatography on DEAE-Sephacel and on aminophenyl-beta-D-thiogalactopyranoside-Sepharose from cells of A. eutrophus which harbored a phbC'-'lacZ fusion gene. A sequence (TTGACA-18N-AACAAT), exhibiting striking homology to the Escherichia coli sigma 70 promoter consensus sequence, was identified approximately 310 bp 5' upstream from the translation initiation codon. An S1 nuclease protection assay mapped the transcription start point of phbC 6 bp downstream from this promoter. The location of the promoter was confirmed by analyzing the expression of active PHB synthase in clones of E. coli harboring 5' upstream deletions of phbC ligated to the promoter of the lacZ gene (lacZp) in a Bluescript vector. Plasmids do181 and do218, which were deleted for the first 108 or 300 bp of the phbC structural gene, respectively, conferred the ability to synthesize large amounts of different truncated PHB synthase proteins to the cells. These proteins contributed to approximately 10% of the total cellular protein as estimated from sodium dodecyl sulfate-polyacrylamide gels. The modified PHB synthase encoded by plasmid do181 was still active. Clones in which the lacZp-'phbC fusion harbored the complete phbC structural gene plus the phbC ribosome binding site did not overexpress PHB synthase.