CLONING AND CHARACTERIZATION OF THE METHYLOBACTERIUM-EXTORQUENS POLYHYDROXYALKANOIC-ACID-SYNTHASE STRUCTURAL GENE

A cosmid gene bank of partially EcoRI-digested genomic DNA from Methyl obacterium extorquens IBT no. 6 was screened for DNA fragments restori ng polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mut ant Alkaligenes eutrophus H16 PHB-4. The M. extorquens PHA-synthase st ructural gene phaC(Mex) was mapped on a 23-kbp EcoRI fragment by compl ementation studies, by hybridization experiments with heterologous DNA probes from A. eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis. Evidence for the presence of genes for a beta-ketothiolase or an acetoacetyl-coenzyme A reductase on this fr agment was not obtained. The nucleotide sequence of a 3.7-kbp region w as obtained. It contained the entire 1.815-kbp phaC(Mex) plus approxim ately each 900-bp upstream and downstream of phaC(Mex). PhaC(Mex) enco ded a protein of 605 amino acids with a relative molecular mass (M(r)) of 66742, which exhibited 38.1% amino acid identity with the A. eutro phus PHA synthase. Determination of the N-terminal amino acid sequence of an M(r) 65000 protein, which was enriched concomitantly with the p urification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviou sly removed post-translationally. Enzyme analysis, which was done with the native gene and a phaC(Mex)'-'IacZ fusion gene, gave no evidence for expression of phaC(Mex) in Escherichia coli.