Adaptation of Cupriavidus necator to levulinic acid for enhanced production of P(3HB-co-3HV) copolyesters

Evolutionary engineering is an approach, which allows preparing microorganisms with desired phenotype without the need for knowledge of genetic characteristics connected with changes at phenotype level. This approach is based on cell multiplication under stress conditions and it can be performed in various designs. In connection with PHA-producing bacteria, evolutionary engineering can be used to gain microorganisms producing PHAs with required material properties. Using levulinic acid (LA) as a stress factor and Cupriavidus necator H16 as a model PHA producing bacterial strain, five evolved strains have been obtained in the present study. Compared with the parental strain, all of these showed better growth in the presence of LA, strains ALA01 and ALA04 showed higher PHA content in biomass and strains ALA03 and ALA04 showed higher 3-hydroxyvalerate (3HV) content in copolymer P(3HB-co-3HV). By DSC and FTIR analysis of polymers, connections between 3HV content, crystallinity and melting temperature have been studied. Metabolic characterization provided information about possible adaptation strategies of evolved strains, while based on methods of statistical analysis similarities and differences between individual strains were discussed. Considering obtained information, evolutionary engineering displays a useful tool for modification of microorganisms together with fine-tuning the properties of produced metabolites (PHA copolyesters).