Synthesis of Secreted Heterologous Actinobacterial Cholesterol Oxidase from Nocardioides simplex in Cells of Methylotrophic Yeast Pichia pastoris (Komagataella phaffii) GS115

Cholesterol oxidase (ChO) belongs to the family of flavin-containing enzymes that catalyze the oxidation of 3β-hydroxy-5-ene steroids with the formation of hydrogen peroxide and the products with a 3‑keto-4-ene structure. This study dealt with obtaining a secreted mature form of heterologous ChO from the actinobacterium Nocardioides simplex (ChONs) in the cells of a recombinant methylotrophic yeast Pichia pastoris GS115. Based on the plasmid vector pPICZαA, genetic constructs were designed for heterologous expression of the target gene choN (KR76_09550) from N. simplex in recombinant yeast. We compared the activity of the cell-free culture supernatant of recombinant P. pastoris strains containing secreted heterologous ChONs with respect to the conversion of cholesterol to cholestenone, depending on the presence and position of a 6 × His tag in the ChONs molecule. The conditions for synthesis of the most active heterologous secreted ChONs in the selected recombinant yeast strain were optimized.