Identification and Characterization of a type II Polyhydroxyalkanoate Synthase in Pseudomonas extremaustralis.

CATONE, MV; LÓPEZ, NI

Villa Carlos Paz, Córdoba, Argentina

Congreso; VI Congreso Argentino de Microbiología General (SAMIGE).; 2009

SAMIGE

Polyhydroxyalkanoates (PHAs) are polyesters produced by many bacterial species as intracellular storage compounds under unbalanced growth conditions. According to the number of carbon atoms that form the monomer units, PHA canb be classified into two groups: short-chain-length (scl) with C3 to C5 monomer units and medium-chain length (mcl) PHA with C6 to C14 monomer units.Both kinds of polymers have different characteristics: scl-PHAs have properties close to conventional plastics while the mcl-PHAs are regarded as elastomers and rubbers.Type I and type II synthases are the key enzymes that catalyze the biosynthesis of scl and mcl PHA, respectively.Pseudomonas species usually have a mcl-PHA cluster that involves two type II synthase genes (phaC1and phaC2) separated by the gene encoding the depolymerase (phaZ). However,Pseudomonas extremaustralis has a complete cluster for polyhydroxybutyrate (PHB) production, the most common PHA, including a type I synthase. While the wild type strain produces only PHB, the phaCscl mutant showed the ability to produce PHAmcl. We used a PCR cloning strategy, based on the design of degenerated primers of the neighbour gene sequences of phaC2 synthase,phaZandphaD, of complete genome sequenced Pseudomonas, to screen for a type II synthase inP. extremaustralis. The resulting amplicon was sequenced and two open reading frames (ORF) were detected, one of 1683 nt and other of 170 nt belonging to a putative completephaC2and a putative incompletephaZ, respectively. Sequence showed a 90% of similarity with phaC2 of P. fluorescens SBW25 and 92% withphaZofComamonas testosteroni. In order toprove its functionality, the putative phaC2 gene was subcloned into the plasmid pSJ33 and introduced by transformation in P. putidaGPp104, a mutant strain unable to produce PHA.phaC2 was able to complement the PHA negative phenotype. PHA granules were observed by microscopy after staining with Nile blue when the strain was grown on sodium octanoate. PHA was not accumulated when glucose or gluconate were used as carbon source. In a previous work we observed that the complementation of P. putida KT2440, a mcl-PHA producer, with type I synthase (phaC) of P. extremaustralis leads to the production of PHB instead of PHAmcl,suggesting mainly a cynetic control, more than regulatory, between type I and II synthases. The presence of a type II PHA synthase in the genome of P.extremaustralis that is expressed when type I synthase is inactive, constitute the first report of this feature in Pseudomonas spp. One interesting result of this work is that genetic manipulation allowed the production of polymers with different properties in the same strain, which could be useful for biotechnological applications.