Abstract

Polyhydroxyalkanoates (PHA) are biopolymers produced and accumulated naturally by several bacterial strains. These biopolymers exhibit great potential to substitute petroleum-based plastics, being thermoplastic, biodegradable, biocompatible and produced from renewable sources. The monomer composition in the polymer chain determines the characteristics and various applications of the biopolymer. PHAs can be formed by short (HA_SCL) or medium chain (HA_MCL) monomers. Copolymers combining HA_SCL and HA_MCL result on a polymer whose properties are different from the pure monomer ones and are suitable for a wide range of applications. They are comparable to oil derived polymers and easily processed, attracting great interest from industry due to its similarity to low density polyethylene. Pseudomonas spp. are aerobic Gram-negative bacteria capable of producing PHA_MCL by beta-oxidation pathway, which supplies medium-chain monomers polymerized by the PHA synthase enzyme class II (phaC2) from various carbon sources. In this study we used the mini-Tn7 system to insert PHA synthase of Ralstonia eutropha (phaC1_Re) into the genome of an UV mutant Pseudomonas strain, unable to produce PHAs, in order to produce P(HB-co-HA_MCL). A genome insertion exclude drawbacks by the plasmid presence in the bacterial cell, as the gene multicopy state and the necessity of continuous selection using antibiotics. In mini-Tn7 system the elements are readily transferred to the formation of mutants, to promote a single site and orientation with specific insertion in a downstream position of the glmS operon. The strains obtained were phenotype and genotypically tested to confirm the presence of phaC1_Re in the genomic DNA and the lack of resistance marker resulting from genetic manipulations. To evaluate the ability of obtained recombinant strains regarding the accumulation of PHA, shaken flasks experiments were conducted. The composition of produced PHA was evaluated and compared with previously obtained results.