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

Background 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 D 2 and 25-hydroxyvitamin D 3 , in one step, starting from the readily available and comparatively inexpensive vitamin D 2 (from fungi and yeast) and vitamin D 3 (from animals). Results 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 (D 2 and D 3 ). First experiments revealed that 42 UPOs were able to transform vitamin D 2 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 D 3 hydroxylation. With vitamin D 3 , other hydroxylated derivatives were produced by UPOs in addition to the 25-hydroxy derivative, with the only exception of the UPO from Hypoxylon sp. ( Hsp UPO). Finally, the synthesis of both 25-hydroxyvitamin D 2 and D 3 was scaled up (up to 20 mM of substrate loading) with Hsp UPO, using different cosubstrates, such as H 2 O 2 or O 2 /reductants, with ascorbic acid, gallic acid, or pyrogallol as reductants. High conversions (68–97% for vitamin D 2 and 63–75% for vitamin D 3 ) and product yields (14–19 mM for vitamin D 2 and 13–15 mM for D 3 ) were attained with all cosubstrates, with pyrogallol/O 2 and H 2 O 2 showing the best results with both vitamins. Conclusions 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.