Molecular cloning and recombinant protein expression of 9-lipoxygenase gene from solanum tuberosum in pichia pastoris yeast cells

Abstract

Lipoxygenases (LOX) are ubiquitous non-heme Iron-containing dioxygenases that catalyze the addition of molecular oxygen to Polyunsaturated Fatty Acids (PUFAs) such as Linoleic acid to form Oxylipins that possess anti-microbial activity. Synthetic fungicides have effectively controlled plant pathogenic fungi but their continued use has been detrimental to natural biological systems, and sometimes resulted in the development of fungal resistance. They also have undesirable effects on non-target organisms and foster environmental and human health concerns thus new biodegradable alternatives have to be investigated. The aim of this study was to generate a recombinant 9-Lipoxygenase protein for chemo enzymatic synthesis of Oxylipin based biodegradable fungicides. Golden gate assembly, a molecular cloning method that allows assembly of many DNA fragments into a complete piece using Type II s restriction enzymes and T4 DNA ligase was used to clone the large size of 9-LOX gene insert (2629bp). Three 9-LOX fragments were amplified from PET-28a-9-LOX recombinant plasmid vector and sub cloned individually into 3 intermediary level 0 vector pAGM35763. The recombinant intermediary plasmid vectors containing the 9-LOX fragments were used in a restriction-ligation reaction to assemble the 9- LOX fragments in the right sequence order into the destination plasmid vector pPICZaB. Chemically competent DH10B Escherichia coli cells were transformed with the ligation product, transformants selected and recombinant plasmid extracted and quantified. To achieve protein expression, the extracted recombinant plasmid vector pPICZaB was linearized and transformed into electrocompetent Pichia pastoris X-33 cells and grown on YPDS agar. Five Zeocin resistant transformant colonies were grown in BMMY media at 30°C and 250rpm for 4 days. Protein expression was induced by the addition of absolute methanol (0.5%) and methanol added every after 24h for up to 4 days to maintain protein expression. Analysis of protein expression from cell lysates was achieved by running an SDS-PAGE and Western blotting which showed a putative protein band of 97kDa representing 9-LOX protein. Results confirmed successful expression of recombinant 9-LOX protein in Pichia pastoris strain X-33. It is recommended that optimization studies on the yeast kex2 convertase and the a- secretion factor can be done to reinforce the secretion of recombinant Solanum tuberosum 9-LOX protein since the protein in this study was recovered from cell lysates.