Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.