, Volume 54, Issue 5, pp 665-670

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

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Abstract

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 acetate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG. Southern hybridization experiments with digoxigenin-labeled phaG Pp from P. putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads. Although P. oleovorans does not accumulate PHAMCL from non-related carbon sources, its genomic DNA reveals a strong hybridization signal. We employed PCR to amplify this phaG homologue. The respective PCR product comprising the coding region of phaG Po was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaGPo from P. oleovorans exhibited about 95% amino acid sequence identity to PhaGPp from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG Po was not transcribed even under inducing conditions, i.e. in the presence of gluconate as carbon source, whereas induction of phaG Pp transcription was obtained in P. putida. When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR84 complemented the phaG-negative mutant PhaGN-21 from P. putida. Interestingly, reintroduction of phaG Po under lac promoter control into the natural host P. oleovorans established PHAMCL synthesis from non-related carbon sources in this bacterium. These data indicated that phaG Po in P. oleovorans is not functionally expressed and does not exert its original function.

Received: 19 February 2000 / Received revision: 29 May 2000 / Accepted: 3 June 2000