Solaiman_2000_Biotechnol.Lett_22_789

Pseudomonas resinovorans phaC1(Pre) and phaC2(Pre) genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by PCR and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that beta-hydroxydecanoate (67-75 mol%) and beta-hydroxyoctanoate (25-33 mol%) are the major monomers of the PHA produced in cells grown on decanoate. Sequence analysis showed that the gene products of phaC1(Pre) and phaC2(Pre) had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved alpha/beta-hydrolase fold in the carboxy-terminal portion of the proteins. Switching the alpha/beta-hydrolase folds of phaC1(Pre) and phaC2(Pre) yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1(Pre) and phaC2(Pre) that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the alpha/beta-hydrolase folds of phaC1(Pre) and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells.