Abstract

Polyhydroxyalkanoates (PHAs) are linear polyesters, which are naturally produced by several species of bacteria, including Pseudomonas, Burkholderia and Cupriavidus. PHAs have been proposed as substitutes for petroleum-derived polymers due to their biodegradable and biocompatible properties. A major drawback of PHA production is their high costs, and therefore a commercial use of PHA is dependent at low production costs and the feasibility of mass production, which are highly related to the productivity of bacterial strains. For such reasons, several genetic approaches have been developed to enhance the synthesis and accumulation of PHAs in bacterial strains. The aims of this work were (i) to generate PHA overproducing mutants from Burkholderia cepacia 2G57 (BC_2G57), previously isolated from Colombian soils; and (ii) to scale up the fermentation parameters to a 100 L stirrer tank bioreactor. Initially, the genome sequence for BC_2G57 was obtained, assembled and mapped to the reference genome of Burkholderia cenocepacia J2315, and PHA coding genes were identified and located. Two mutagenesis approaches were then used to generate overproducing strains: First, random mini-Tn5 transposon mutagenesis was employed to generate a mutant library in BC_2G57. 10000 mutants were screened for high PHA accumulation in MMS media, using Nile Blue and Red Blue. Results suggested that 10 mutant strains evidenced an increase in PHA accumulation when compared with the wild type strain, ranging between 83 % to 53%. In all strains, the miniTn5 insertion site was determined by arbitrary PCR. Simultaneously, a null mutant in the negative regulator phaF was obtained by a suicide pKnock-Cm vector. As expected, this latter strain accumulated 62% more PHA than the wild type strain. Fermentation conditions were subsequently evaluated for the three strains with highest accumulation in a 100 L stirrer tank bioreactor, and the best conditions were identified. Currently, PHA physical properties are being evaluated, as well as the stability of the overproducing strains. Further studies are required to determine if such strains are suitable for an industrial process, and if the polymer maintains its properties along the fermentation process.However, our results suggest that random mutagenesis is an effective strategy to obtain PHA overproducing strains.