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Biofuels Bioprod."],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:sub>2<\/jats:sub>\n                      and 25-hydroxyvitamin D\n                      <jats:sub>3<\/jats:sub>\n                      , in one step, starting from the readily available and comparatively inexpensive vitamin D\n                      <jats:sub>2<\/jats:sub>\n                      (from fungi and yeast) and vitamin D\n                      <jats:sub>3<\/jats:sub>\n                      (from animals).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>Komagataella phaffii<\/jats:italic>\n                      (formerly\n                      <jats:italic>Pichia pastoris<\/jats:italic>\n                      ), has been performed for the regioselective C25-hydroxylation of vitamin D (D\n                      <jats:sub>2<\/jats:sub>\n                      and D\n                      <jats:sub>3<\/jats:sub>\n                      ). First experiments revealed that 42 UPOs were able to transform vitamin D\n                      <jats:sub>2<\/jats:sub>\n                      into its 25-hydroxy derivative within a wide range of conversion rates (from 5% to\u2009&gt;\u200999%), with strict regioselectivity in most cases, determined using GC\u2013MS. The 16 best UPOs in terms of conversion and regioselectivity were tested for vitamin D\n                      <jats:sub>3<\/jats:sub>\n                      hydroxylation. With vitamin D\n                      <jats:sub>3<\/jats:sub>\n                      , other hydroxylated derivatives were produced by UPOs in addition to the 25-hydroxy derivative, with the only exception of the UPO from\n                      <jats:italic>Hypoxylon<\/jats:italic>\n                      sp. (\n                      <jats:italic>Hsp<\/jats:italic>\n                      UPO). Finally, the synthesis of both 25-hydroxyvitamin D\n                      <jats:sub>2<\/jats:sub>\n                      and D\n                      <jats:sub>3<\/jats:sub>\n                      was scaled up (up to 20\u00a0mM of substrate loading) with\n                      <jats:italic>Hsp<\/jats:italic>\n                      UPO, using different cosubstrates, such as H\n                      <jats:sub>2<\/jats:sub>\n                      O\n                      <jats:sub>2<\/jats:sub>\n                      or O\n                      <jats:sub>2<\/jats:sub>\n                      \/reductants, with ascorbic acid, gallic acid, or pyrogallol as reductants. High conversions (68\u201397% for vitamin D\n                      <jats:sub>2<\/jats:sub>\n                      and 63\u201375% for vitamin D\n                      <jats:sub>3<\/jats:sub>\n                      ) and product yields (14\u201319\u00a0mM for vitamin D\n                      <jats:sub>2<\/jats:sub>\n                      and 13\u201315\u00a0mM for D\n                      <jats:sub>3<\/jats:sub>\n                      ) were attained with all cosubstrates, with pyrogallol\/O\n                      <jats:sub>2<\/jats:sub>\n                      and H\n                      <jats:sub>2<\/jats:sub>\n                      O\n                      <jats:sub>2<\/jats:sub>\n                      showing the best results with both vitamins.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      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,\n                      <jats:italic>K. phaffii<\/jats:italic>\n                      a widely used yeast platform for recombinant protein production \u2012 including high-yield secretory expression of industrial enzymes \u2012 has been firmly established as a favorable host for recombinant UPO production in recent years.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13068-026-02792-0","type":"journal-article","created":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T13:35:42Z","timestamp":1783690542000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comprehensive assessment of fungal peroxygenases for the selective synthesis of 25-hydroxyvitamins D2 and D3"],"prefix":"10.1186","volume":"19","author":[{"given":"Alejandro","family":"Gonz\u00e1lez-Benjumea","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andr\u00e9s","family":"Olmedo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharina","family":"Ebner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anton","family":"Glieder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Guti\u00e9rrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,10]]},"reference":[{"key":"2792_CR1","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1007\/s11154-017-9424-1","volume":"18","author":"MF Holick","year":"2017","unstructured":"Holick MF. 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