Enhancing the biotransformation efficiency of human CYP17A1 in Pichia pastoris by co-expressing CPR and glucose-6-phosphate dehydrogenase simultaneously

Abstract

Steroids are a widely used class of drugs, and their hydroxylation modification has important pharmacological significance. Cytochrome P450 is the core enzyme for hydroxylation modification of steroid molecules, but its use is significantly restricted because of low activity and poor catalytic efficiency. In the present study, we optimized the codons of the human CYP17A1 (hCYP17A1) gene and performed its functional expression in Pichia pastoris GS115. The GS115-hCYP17A1 cells exhibited activities of dual function, and the following two products were detected: 17α-hydroxyprogesterone (17-HP, titer: 40.12 ± 3.16 mg/L) and androstenedione (AD, titer: 4.70 ± 0.31 mg/L). Subsequently, we compared the activity of hCYP17A1 co-expression with NADPH-cytochrome P450 oxidoreductases (CPRs) from five different strains of P. pastoris. Moreover, to strengthen the NADPH regeneration system, glucose-6-phosphate dehydrogenase (G6PDH) from three different species was introduced into the pathway from progesterone to 17-HP and AD by the GS115-hCYP17A1 cells. After optimizing CPRs and G6PDHs, three foreign proteins were co-expressed in the host cell, namely CYP17A1, CPR_YP from Yorkshire pig, and G6PDHc from Candida tropicalis. Finally, the substrate conversion efficiency was found to be increased by 2.21-fold (46.88%) of that of the starting strains. We obtained a heterologous expression system with the highest biotransformation efficiency achieved to date. This work provided an essential reference for the study of the introduction of crucial enzyme systems into heterologous model organisms to construct an efficient steroid biotransformation system.

Keywords

Cytochrome P450 catalytic system / 17α-hydroxyprogesterone / Whole-cell biotransformation / Cofactor recycling

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Funding

Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China(KLIB-KF202103); National Key R&D Program of China(2019YFA0905300); Natural Science Foundation of Jiangsu Province(BK20200618); Institute for Health Care Improvement(TSBICIP-KJGG-001-14); National Natural Science Foundation of China(31700041)