Comprehensive assessment of fungal peroxygenases for the selective synthesis of 25-hydroxyvitamins D₂ and D₃

The metabolite 25-hydroxyvitamin D is significantly better for clinical use in several diseases and food supplementations than vitamin D (calciferol) itself. The present study reports an efficient biotechnological method for the synthesis of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃, in one step, starting from the readily available and comparatively inexpensive vitamin D₂ (from fungi and yeast) and vitamin D₃ (from animals).

A widespread investigation of a series of up to 71 unspecific peroxygenases (UPOs), including UPO samples from 41 fungal species (together with several isozymes and variants) expressed in Komagataella phaffii (formerly Pichia pastoris), has been performed for the regioselective C25-hydroxylation of vitamin D (D₂ and D₃). First experiments revealed that 42 UPOs were able to transform vitamin D₂ into its 25-hydroxy derivative within a wide range of conversion rates (from 5% to > 99%), with strict regioselectivity in most cases, determined using GC-MS. The 16 best UPOs in terms of conversion and regioselectivity were tested for vitamin D₃ hydroxylation. With vitamin D₃, other hydroxylated derivatives were produced by UPOs in addition to the 25-hydroxy derivative, with the only exception of the UPO from Hypoxylon sp. (HspUPO). Finally, the synthesis of both 25-hydroxyvitamin D₂ and D₃ was scaled up (up to 20 mM of substrate loading) with HspUPO, using different cosubstrates, such as H₂O₂ or O₂/reductants, with ascorbic acid, gallic acid, or pyrogallol as reductants. High conversions (68-97% for vitamin D₂ and 63-75% for vitamin D₃) and product yields (14-19 mM for vitamin D₂ and 13-15 mM for D₃) were attained with all cosubstrates, with pyrogallol/O₂ and H₂O₂ showing the best results with both vitamins.

This approach represents a green and sustainable alternative to the chemical synthesis of these hydroxylated compounds that is tedious, occurs with little regioselectivity, and includes several steps and strong oxidizing agents. Moreover, K. phaffii a widely used yeast platform for recombinant protein production ‒ including high-yield secretory expression of industrial enzymes ‒ has been firmly established as a favorable host for recombinant UPO production in recent years.