@misc{KoenigKiebistKalmbachetal., author = {K{\"o}nig, Rosalie and Kiebist, Jan and Kalmbach, Johannes and Herzog, Robert and Schmidtke, Kai-Uwe and Kellner, Harald and Ullrich, Ren{\´e} and Jehmlich, Nico and Hofrichter, Martin and Scheibner, Katrin}, title = {Novel unspecific peroxygenase from Truncatella angustata catalyzes the synthesis of bioactive lipid mediators}, series = {Microorganisms}, volume = {10}, journal = {Microorganisms}, number = {7}, issn = {2076-2607}, doi = {10.3390/microorganisms10071267}, pages = {1 -- 18}, abstract = {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}, language = {en} } @misc{KellnerFriedrichSchmidtkeetal., author = {Kellner, Harald and Friedrich, Stephanie and Schmidtke, Kai-Uwe and Ullrich, Ren{\´e} and Kiebist, Jan and Z{\"a}nder, Daniel and Hofrichter, Martin and Scheibner, Katrin}, title = {Draft genome sequence of Truncatella angustata (Anamorph) S358}, series = {Microbiology resource announcement}, volume = {11}, journal = {Microbiology resource announcement}, number = {7}, issn = {2169-8287}, doi = {10.1128/mra.00052-22}, abstract = {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).}, language = {en} } @misc{SchrammCarrilloAvilesKalmbachetal., author = {Schramm, Marina and Carrillo Avil{\´e}s, Carlos Renato and Kalmbach, Johannes and Schmidtke, Kai-Uwe and Kiebist, Jan and Kellner, Harald and Hofrichter, Martin and Scheibner, Katrin}, title = {Rapid screening system to identify unspecific peroxygenase activity}, series = {Clinical hemorheology and microcirculation}, volume = {89}, journal = {Clinical hemorheology and microcirculation}, number = {4}, publisher = {Sage}, address = {London}, issn = {1875-8622}, doi = {10.1177/13860291241306566}, pages = {363 -- 373}, abstract = {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.}, language = {en} } @misc{KarichCaiLinhardtetal., author = {Karich, Alexander and Cai, Hongtao and Linhardt, Anne and Antony, Anila and Liers, Christiane and Ullrich, Ren{\´e} and Schwaderer, Fabian and Kalmbach, Johannes and Scheibner, Katrin and Hofrichter, Martin and Synytska, Alla}, title = {Conductive hairy particles with homogeneous and Janus design as carrier materials for the efficient immobilization of unspecific peroxygenases}, series = {Biotechnology journal : systems \& synthetic biology, nanobiotech, medicine}, volume = {20}, journal = {Biotechnology journal : systems \& synthetic biology, nanobiotech, medicine}, number = {7}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1860-7314}, doi = {10.1002/biot.70078}, pages = {1 -- 12}, abstract = {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.}, language = {en} } @misc{GomezdeSantosGonzalezBenjumeaFernandezGarciaetal., author = {Gomez de Santos, Patricia and Gonz{\´a}lez-Benjumea, Alejandro and Fernandez-Garcia, Angela and Aranda, Carmen and Wu, Yinqi and But, Andrada and Molina-Espeja, Patricia and Mat{\´e}, Diana M. and Gonzalez-Perez, David and Zhang, Wuyuan and Kiebist, Jan and Scheibner, Katrin and Hofrichter, Martin and Swiderek, Katarzyna and Moliner, Vicent and Sanz-Aparicio, Julia and Hollmann, Frank and Guti{\´e}rrez, Ana and Alcalde, Miguel}, title = {Engineering a highly regioselective fungal peroxygenase for the synthesis of hydroxy fatty acids}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker}, volume = {62}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker}, number = {9}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1521-3773}, doi = {10.1002/anie.202217372}, pages = {1 -- 9}, abstract = {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.}, language = {en} }