Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

Synthetic fungicides have effectively controlled plant pathogenic fungi but their continued use has been detrimental to natural biological systems, and sometimes resulted into 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. 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.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][1] II s restriction enzymes and [T4][2] DNA ligase was used to clone the complete 9-LOX gene into the expression plasmid vector pPICZαB. Protein expression in Pichia pastoris yeast cells was induced by addition of absolute methanol every after 24h for up to four days. Analysis of protein expression from cell lysates was achieved by SDS-PAGE and Western blotting probed with anti-histidine antibody which showed putative protein bands of 97kDa representing recombinant 9-LOX protein. It is recommended that optimization studies on the yeast kex2 convertase and the α- secretion factor can be done to enable secretion of recombinant Solanum tuberosum 9-LOX protein since the protein in this study was recovered from cell lysates. ### Competing Interest Statement The authors have declared no competing interest. [1]: https://en.wikipedia.org/wiki/Restriction_enzyme#Type_II [2]: https://en.wikipedia.org/wiki/DNA_ligase#T4_DNA_ligase