Genetic organization of pha gene locus affects phaC expression, poly(hydroxyalkanoate) composition and granule morphology in Pseudomonas corrugata

The complete sequence of the pha locus responsible for the biosynthesis of poly(hydroxyalkanoates) (PHAs) in Pseudomonas corrugata 388 was determined. As with the other known pseudomonad pha gene loci, the one in P. corrugata 388 also consists of phaC1 (1,680 bps; PHA synthase 1), phaZ (858 bp; PHA depolymerase) and phaC2 (1,683 bp; PHA synthase 2) genes. A BLAST search showed that the nucleotide sequences of these genes and the amino-acid sequences of their respective gene products are homologous to those of P. corrugata CFBP5454 and P. mediterranea CFBP5447. A putative intrinsic transcription terminator consisting of a dyad symmetry (24 bp; ΔG = -41.8 kcals) that precedes a stretch of dA residues was located in the phaC1-phaZ intergenic region. P. corrugata mutant-clones XI 32-1 and XI 32-4 were constructed in which this intergenic region was replaced with a selectable kanamycin-resistance gene. These mutant clones when grown on oleic acid for 48 h showed 4.7-to 7.0-fold increases of phaC1 and phaC2 relative expression in comparison to the initial inoculants, whereas the parental strain showed only 1.2- to 1.4-fold increases. Furthermore, in comparison to parental P. corrugata with only a few large PHA inclusion bodies, the mutants grown on oleic acid produce numerous smaller PHA granules that line the periphery of the cells. With glucose as a substrate, XI 32-1 and XI 32-4 clones produce mcl-PHA with a high content (26-31 mol%) of the mono-unsaturated 3-hydroxydodecenoate as a repeat-unit monomer. Our results show for the first time the effects of the phaC1-phaZ intergenic region on the substrate-dependent temporal expression of phaC1 and phaC2 genes, the repeat-unit composition of mcl-PHA, and the morphology of the PHA granules.