| Abstract [eng] |
Bacterial lipoxygenase (LOX) is an enzyme that has the potential to play a significant role in various industrial applications. It exhibits unique catalytic properties and substrate specificity, making it an attractive tool for the production of high-value products such as oleochemicals, biofuels, flavor and aroma compounds, food additives, and many others. However, there are only a few successful attempts to achieve a high recombinant LOX yield and it is not commercially available yet in proper amounts. The aim of this research was to investigate the heterologous expression of bacterial LOX from Pseudomonas aeruginosa in different yeast genera and to construct a system that would allow secretion of the protein into the extracellular space. This study was carried out with non- methylotrophic Saccharomyces cerevisiae and methylotrophic Pichia pastoris yeast strains. An episomal shuttle pFX7αF-LOX vector was used for LOX expression in S. cerevisiae. The impact of using different mutant strains for LOX production was explored. The integrating pPIC9K-LOX vector for testing the LOX expression in P. pastoris was constructed by homologous recombination method in E.coli. Using AOX1 promotor and methanol as an inducer a successful active LOX expression was achieved with the majority of the protein located in the culture supernatant. Adjacent to this, for LOX expression in P. pastoris without the need of external inducers, a new expression plasmid was constructed using Golden Gate based cloning strategy. |
