Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway

Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate ( PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6-C14), when grown on simple carbon sources such as, for example, gluconate or ace tate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synth esis are linked by the transacylase PhaG. Southern hybridization experiment s with digoxigenin-labeled phaG(Pp) from P. putida and genomic DNA from var ious pseudomonads indicate that phaG homologues are present in various othe r pseudomonads. Although P. oleovorans does not accumulate PHA(MCL) from no n-related carbon sources, its genomic DNA reveals a strong hybridization si gnal. We employed PCR to amplify this phaG homologue. The respective PCR pr oduct comprising the coding region of phaG(Po) was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaG(Po) from P. oleovorans exhibited about 95% amino acid sequence identity to Pha G(Pp) from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG(Po) was not transcribed even under inducing conditions, i.e. in the pr esence of gluconate as carbon source, whereas induction of phaG(Pp) transcr iption was obtained in P. putida. When octanoate was used as sole carbon so urce, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR 84 complemented the phaG-negative mutant PhaG(N)-21 from P. putida. Interes tingly, reintroduction of phaG(Po) under lac promoter control into the natu ral host P. oleovorans established PHAMCL synthesis from non-related carbon sources in this bacterium. These data indicated that phaG(Po) in P. oleovo rans is not functionally expressed and does not exert its original function .