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 - TY - GEN A1 - Karich, Alexander A1 - Cai, Hongtao A1 - Linhardt, Anne A1 - Antony, Anila A1 - Liers, Christiane A1 - Ullrich, René A1 - Schwaderer, Fabian A1 - Kalmbach, Johannes A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Synytska, Alla T1 - Conductive hairy particles with homogeneous and Janus design as carrier materials for the efficient immobilization of unspecific peroxygenases T2 - Biotechnology journal : systems & synthetic biology, nanobiotech, medicine N2 - Efficient immobilization of unspecific peroxygenases (UPOs) on hairy particles with homogeneous and Janus design possessingconductive core and polymeric shell was demonstrated. PDMAEMA brushes (hairs) with controlled chain lengths weresuccessfully grown from the conductive silver particles allowing further immobilization of enzymes and keeping their activity.The Janus design of the synthesized particles maintained the conductivity of the core material. Enzyme immobilization onbrush-modified particles was first carried out with two laccases as model enzymes [Trametes versicolor (TveLac) and Pycnoporuscinnabarinus (PciLac)] and then successfully extended to UPOs [two wild-type and one recombinant UPO from Marasmius rotula(MroUPO) and Agrocybe aegerita (AaeUPO, rAaeUPO)]. The most efficient immobilization was achieved for MroUPO on Janusparticles. The enzyme loading is reversible and could be successfully repeated after cleaning of the particles. Thus, the successfulimmobilization of an MroUPO on conductive hairy Janus particles was demonstrated for the first time. The major advantage ofthe proposed approach lies in the reusability of the enzyme and its carrier, as well as in the use of conductive core materials, whichcould be promising, for example, as material for electrochemical biosensors in future. KW - Ag particles KW - Core shell particles KW - Enzyme immobilization KW - Janus particles KW - PDMAEMA KW - Polymer brushes KW - Unspecific peroxygenases Y1 - 2025 U6 - https://doi.org/10.1002/biot.70078 SN - 1860-7314 VL - 20 IS - 7 SP - 1 EP - 12 PB - Wiley-VCH CY - Weinheim ER - TY - GEN A1 - Gomez de Santos, Patricia A1 - González-Benjumea, Alejandro A1 - Fernandez-Garcia, Angela A1 - Aranda, Carmen A1 - Wu, Yinqi A1 - But, Andrada A1 - Molina-Espeja, Patricia A1 - Maté, Diana M. A1 - Gonzalez-Perez, David A1 - Zhang, Wuyuan A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Swiderek, Katarzyna A1 - Moliner, Vicent A1 - Sanz-Aparicio, Julia A1 - Hollmann, Frank A1 - Gutiérrez, Ana A1 - Alcalde, Miguel T1 - Engineering a highly regioselective fungal peroxygenase for the synthesis of hydroxy fatty acids T2 - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker N2 - The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1) hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer. KW - Peroxygenase Y1 - 2023 U6 - https://doi.org/10.1002/anie.202217372 SN - 1521-3773 VL - 62 IS - 9 SP - 1 EP - 9 PB - Wiley-VCH CY - Weinheim ER -