TY - GEN A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Zimmermann, Jörg A1 - Kellner, Harald A1 - Jehmlich, Nico A1 - Ullrich, René A1 - Zänder, Daniel A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - A peroxygenase from Chaetomium globosum catalyzes the selective oxygenation of testosterone T2 - ChemBioChem N2 - Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates including less activated hydrocarbons by transferring peroxide-borne oxygen. Herein, we investigated a cell-free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule of testosterone by a novel unspecific peroxygenase that was produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass of 36 kDa and specific activities of 4.4 to 12 U mg-1. Whereas well-known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with up to 7,000 total turnovers (TTN) into two oxygenated products: the 4,5-epoxide of testosterone in β-configuration and 16α-hydroxytestosterone. The reaction was performed at 100-mg scale resulting in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, which both could be isolated with purities above 96 %. Thus, CglUPO may be a promising biocatalyst for the oxyfunctionalization of bulky steroids and provide a useful tool for the synthesis of pharmaceutically relevant steroidal molecules. KW - peroxidase KW - hydroxylation Y1 - 2017 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbic.201600677/abstract U6 - https://doi.org/10.1002/cbic.201600677 SN - 1439-7633 VL - 18 IS - 6 SP - 563 EP - 569 ER - TY - CHAP A1 - Hofrichter, Martin A1 - Kellner, Harald A1 - Herzog, Robert A1 - Karich, Alexander A1 - Liers, Christiane A1 - Scheibner, Katrin A1 - Kimani, Virginia Wambui A1 - Ullrich, René ED - Nevalainen, Helena T1 - Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions. T2 - Grand Challenges in Fungal Biotechnology KW - Peroxygenase Y1 - 2020 SN - 978-3-030-29540-0 SN - 978-3-030-29541-7 U6 - https://doi.org/10.1007/978-3-030-29541-7 SN - 2367-1017 SN - 2367-1025 SP - 369 EP - 403 PB - Springer Nature CY - Cham ET - 1. Auflage ER - TY - GEN A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Kiebist, Jan A1 - Kellner, Harald A1 - Hofrichter, Martin T1 - Unspezifische Peroxygenasen - Oxyfunktionalisierung außerhalb der Pilzhyphe T2 - Biospektrum KW - Peroxygenase Y1 - 2020 U6 - https://doi.org/10.1007/s12268-020-1338-x SN - 1868-6249 SN - 0947-0867 VL - 26 IS - 1 SP - 103 EP - 106 ER - TY - GEN A1 - Gomes de Santos, Patricia A1 - Hoang, Manh Dat A1 - Kiebist, Jan A1 - Kellner, Harald A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Liers, Christiane A1 - Alcalde, Miguel T1 - Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations T2 - Applied and environmental microbiology N2 - Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. KW - Peroxygenase KW - heterologous functional expression Y1 - 2021 UR - https://journals.asm.org/doi/10.1128/AEM.00878-21 U6 - https://doi.org/10.1128/AEM.00878-21 SN - 1098-5336 SN - 0099-2240 VL - 87 IS - 19 ER - TY - GEN A1 - Schramm, Marina A1 - Friedrich, Stephanie A1 - Schmidtke, Kai-Uwe A1 - Panzer, Paul A1 - Kellner, Harald A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases T2 - Antioxidants N2 - Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts. KW - unspecific peroxygenase KW - monooxygenase KW - cell-free protein synthesis KW - in vitro translation Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/2/284 U6 - https://doi.org/10.3390/antiox11020284 SN - 2076-3921 VL - 11 IS - 2 SP - 1 EP - 15 ER - TY - GEN A1 - Hofrichter, Martin A1 - Kellner, Harald A1 - Herzog, Robert A1 - Karich, Alexander A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Ullrich, René T1 - Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases T2 - Antioxidants N2 - Unspecific peroxygenases (UPOs), whose sequences can be found in the genomes of thousands of filamentous fungi, many yeasts and certain fungus-like protists, are fascinating biocatalysts that transfer peroxide-borne oxygen (from H2O2 or R-OOH) with high efficiency to a wide range of organic substrates, including less or unactivated carbons and heteroatoms. A twice-proline-flanked cysteine (PCP motif) typically ligates the heme that forms the heart of the active site of UPOs and enables various types of relevant oxygenation reactions (hydroxylation, epoxidation, subsequent dealkylations, deacylation, or aromatization) together with less specific one-electron oxidations (e.g., phenoxy radical formation). In consequence, the substrate portfolio of a UPO enzyme always combines prototypical monooxygenase and peroxidase activities. Here, we briefly review nearly 20 years of peroxygenase research, considering basic mechanistic, molecular, phylogenetic, and biotechnological aspects. KW - unspecific peroxygenase KW - monooxygenase KW - peroxidases KW - hydroxylation KW - epoxidation KW - dealkylation Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/1/163 U6 - https://doi.org/10.3390/antiox11010163 SN - 2076-3921 VL - 11 IS - 1 SP - 1 EP - 21 ER - TY - GEN A1 - König, Rosalie A1 - Kiebist, Jan A1 - Kalmbach, Johannes A1 - Herzog, Robert A1 - Schmidtke, Kai-Uwe A1 - Kellner, Harald A1 - Ullrich, René A1 - Jehmlich, Nico A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Novel unspecific peroxygenase from Truncatella angustata catalyzes the synthesis of bioactive lipid mediators T2 - Microorganisms N2 - Lipid mediators, such as epoxidized or hydroxylated eicosanoids (EETs, HETEs) of arachidonic acid (AA), are important signaling molecules and play diverse roles at different physiological and pathophysiological levels. The EETs and HETEs formed by the cytochrome P450 enzymes are still not fully explored, but show interesting anti-inflammatory properties, which make them attractive as potential therapeutic target or even as therapeutic agents. Conventional methods of chemical synthesis require several steps and complex separation techniques and lead only to low yields. Using the newly discovered unspecific peroxygenase TanUPO from the ascomycetous fungus Truncatella angustata, 90% regioselective conversion of AA to 14,15-EET could be achieved. Selective conversion of AA to 18-HETE, 19-HETE as well as to 11,12-EET and 14,15-EET was also demonstrated with known peroxygenases, i.e., AaeUPO, CraUPO, MroUPO, MweUPO and CglUPO. The metabolites were confirmed by HPLC-ELSD, MS1 and MS2 spectrometry as well as by comparing their analytical data with authentic standards. Protein structure simulations of TanUPO provided insights into its substrate access channel and give an explanation for the selective oxyfunctionalization of AA. The present study expands the scope of UPOs as they can now be used for selective syntheses of AA metabolites that serve as reference material for diagnostics, for structure-function elucidation as well as for therapeutic and pharmacological purposes KW - eicosanoids KW - lipid mediators KW - EETs KW - HETEs KW - unspecific peroxygenases KW - human drug KW - metabolites KW - biocatalysis KW - TanUPO Y1 - 2022 UR - https://www.mdpi.com/2076-2607/10/7/1267 U6 - https://doi.org/10.3390/microorganisms10071267 SN - 2076-2607 VL - 10 IS - 7 SP - 1 EP - 18 ER - TY - GEN A1 - Kellner, Harald A1 - Friedrich, Stephanie A1 - Schmidtke, Kai-Uwe A1 - Ullrich, René A1 - Kiebist, Jan A1 - Zänder, Daniel A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Draft genome sequence of Truncatella angustata (Anamorph) S358 T2 - Microbiology resource announcement N2 - The ascomycete Truncatella angustata has a worldwide distribution. Commonly, it is associated with plants as an endophyte, pathogen, or saprotroph. The genome assembly comprises 44.9 Mbp, a G+C content of 49.2%, and 12,353 predicted genes, among them 12 unspecific peroxygenases (EC 1.11.2.1). KW - unspecific peroxygenase KW - GENOME SEQUENCES KW - Truncatella angustata Y1 - 2022 UR - https://journals.asm.org/doi/epub/10.1128/mra.00052-22 U6 - https://doi.org/10.1128/mra.00052-22 SN - 2169-8287 SN - 2576-098X VL - 11 IS - 7 ER - TY - GEN A1 - Schramm, Marina A1 - Carrillo Avilés, Carlos Renato A1 - Kalmbach, Johannes A1 - Schmidtke, Kai-Uwe A1 - Kiebist, Jan A1 - Kellner, Harald A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Rapid screening system to identify unspecific peroxygenase activity T2 - Clinical hemorheology and microcirculation N2 - Unspecific peroxygenases (UPO, EC 1.11.2.1) are a valuable tool for the biocatalytic synthesis of specialty chemicals such as pharmaceutical metabolites. However, the search for new UPOs that are recombinantly expressible can be tedious and dependent on expensive equipment, especially when a large number of clones has to be examined. In this study, we present a simple agar plate-based method for the screening of active, secreted UPOs heterologously expressed in Saccharomyces cerevisiae. This allows a real high-throughput of several thousand clones at once. The approach was successfully tested with a small gene library comprising putative UPO genes and resulted in the identification of two clones producing short UPOs from the filamentous fungi Dendrothele bispora (DbiUPO) and Aspergillus niger (AniUPO). Both UPOs were partly purified and characterized with respect to their catalytic properties. With differing efficiencies and product specificities, they catalyzed the formation of human drug metabolites, e.g., lipid mediators from polyunsaturated fatty acids and the active metabolite of the prodrug clopidogrel, respectively. KW - Peroxygenase Y1 - 2025 U6 - https://doi.org/10.1177/13860291241306566 SN - 1875-8622 VL - 89 IS - 4 SP - 363 EP - 373 PB - Sage CY - London ER -