@incollection{HofrichterKellnerHerzogetal., author = {Hofrichter, Martin and Kellner, Harald and Herzog, Robert and Karich, Alexander and Liers, Christiane and Scheibner, Katrin and Kimani, Virginia Wambui and Ullrich, Ren{\´e}}, title = {Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions.}, series = {Grand Challenges in Fungal Biotechnology}, booktitle = {Grand Challenges in Fungal Biotechnology}, editor = {Nevalainen, Helena}, edition = {1. Auflage}, publisher = {Springer Nature}, address = {Cham}, isbn = {978-3-030-29540-0}, issn = {2367-1017}, doi = {10.1007/978-3-030-29541-7}, pages = {369 -- 403}, language = {en} } @misc{KiebistSchmidtkeSchrammetal., author = {Kiebist, Jan and Schmidtke, Kai-Uwe and Schramm, Marina and Hofrichter, Martin and K{\"o}nig, Rosalie and Quint, Stephan and Kohlmann, Johannes and Zuhse, Ralf and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin}, title = {Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases}, series = {Journal of Fungi}, volume = {7}, journal = {Journal of Fungi}, number = {9}, issn = {2309-608X}, doi = {10.3390/jof7090752}, pages = {1 -- 17}, abstract = {Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL-1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h-1 H2O2 and 20\% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25\%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44\%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays.}, language = {en} } @misc{GomesdeSantosHoangKiebistetal., author = {Gomes de Santos, Patricia and Hoang, Manh Dat and Kiebist, Jan and Kellner, Harald and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin and Liers, Christiane and Alcalde, Miguel}, title = {Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations}, series = {Applied and environmental microbiology}, volume = {87}, journal = {Applied and environmental microbiology}, number = {19}, issn = {1098-5336}, doi = {10.1128/AEM.00878-21}, abstract = {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.}, language = {en} } @misc{SchrammFriedrichSchmidtkeetal., author = {Schramm, Marina and Friedrich, Stephanie and Schmidtke, Kai-Uwe and Panzer, Paul and Kellner, Harald and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin}, title = {Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases}, series = {Antioxidants}, volume = {11}, journal = {Antioxidants}, number = {2}, issn = {2076-3921}, doi = {10.3390/antiox11020284}, pages = {1 -- 15}, abstract = {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.}, language = {en} } @misc{HofrichterKellnerHerzogetal., author = {Hofrichter, Martin and Kellner, Harald and Herzog, Robert and Karich, Alexander and Kiebist, Jan and Scheibner, Katrin and Ullrich, Ren{\´e}}, title = {Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases}, series = {Antioxidants}, volume = {11}, journal = {Antioxidants}, number = {1}, issn = {2076-3921}, doi = {10.3390/antiox11010163}, pages = {1 -- 21}, abstract = {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.}, language = {en} } @misc{BabotArandaKiebistetal., author = {Babot, Esteban D. and Aranda, Carmen and Kiebist, Jan and Scheibner, Katrin and Ullrich, Ren{\´e} and Hofrichter, Martin and Mart{\´i}nez, Angel T. and Gutierrez, Ana}, title = {Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases}, series = {Antioxidants}, volume = {11}, journal = {Antioxidants}, number = {3}, issn = {2076-3921}, doi = {10.3390/antiox11030522}, pages = {1 -- 12}, abstract = {Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, flavoring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosubstrate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosubstrate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, notable differences were observed between them, with rCciUPO being responsible for the highest substrate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies.}, language = {en} } @misc{PiontekStrittmatterUllrichetal., author = {Piontek, Klaus and Strittmatter, Eric and Ullrich, Ren{\´e} and Gr{\"o}be, Glenn and Pecyna, Marek J. and Kluge, Martin and Scheibner, Katrin and Hofrichter, Martin and Plattner, Dietmar A.}, title = {Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits}, series = {The Journal of Biological Chemistry}, journal = {The Journal of Biological Chemistry}, number = {288}, issn = {1083-351X}, doi = {10.1074/jbc.M113.514521}, pages = {34767 -- 34776}, abstract = {Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.}, language = {en} } @misc{ArandaUllrichKiebistetal., author = {Aranda, Carmen and Ullrich, Ren{\´e} and Kiebist, Jan and Scheibner, Katrin and R{\´i}o, Jos{\´e} C. del and Hofrichter, Martin and Mart{\´i}nez, Angel T. and Guti{\´e}rrez, Ana}, title = {Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases}, series = {Catalysis Science \& Technology}, volume = {9}, journal = {Catalysis Science \& Technology}, number = {8}, issn = {2044-4761}, doi = {10.1039/C8CY00272J}, pages = {2394 -- 2401}, abstract = {This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000) than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63\% of products) and oxyRSV (78\%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV.}, 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{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} }