TY - GEN A1 - Aranda, Carmen A1 - Olmedo, Andrés A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Río, José C. del A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters by Fungal Peroxygenases T2 - CHEMCATCHEM N2 - Recently discovered fungal unspecific peroxygenases from Marasmius rotula and Chaetomium globosum catalyze the epoxidation of unsaturated fatty acids (FA) and FA methyl esters (FAME), unlike the well‐known peroxygenases from Agrocybe aegerita and Coprinopsis cinerea. Reactions of a series of unsaturated FA and FAME with cis‐configuration revealed high (up to 100 %) substrate conversion and selectivity towards epoxidation, although some significant differences were observed between enzymes and substrates with the best results being obtained with the C. globosum enzyme. This and the M. rotula peroxygenase appear as promising biocatalysts for the environmentally‐friendly production of reactive FA epoxides given their self‐sufficient monooxygenase activity and the high conversion rate and epoxidation selectivity. KW - Peroxygenase Y1 - 2018 U6 - https://doi.org/10.1002/cctc.201800849 SN - 1867-3899 VL - 10 IS - 18 SP - 3964 EP - 3968 ER - TY - GEN A1 - Steinbrecht, Susanne A1 - Kiebist, Jan A1 - König, Rosalie A1 - Thiessen, Markus A1 - Schmidtke, Kai-Uwe A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Scheibner, Katrin T1 - Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells T2 - AMB Express N2 - Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment. KW - Biocatalysis KW - Cyclophosphamide KW - Human drug metabolites KW - Peroxygenase KW - Toxicity Y1 - 2020 UR - https://amb-express.springeropen.com/articles/10.1186/s13568-020-01064-w U6 - https://doi.org/10.1186/s13568-020-01064-w SN - 2191-0855 VL - 10 ER - TY - GEN A1 - Ingenbosch, Kim N. A1 - Quint, Stephan A1 - Dyllick-Brenzinger, Melanie A1 - Wunschik, Dennis S. A1 - Kiebist, Jan A1 - Süss, Philipp A1 - Liebelt, Ute A1 - Zuhse, Ralf A1 - Menyes, Ulf A1 - Scheibner, Katrin A1 - Mayer, Christian A1 - Opwis, Klaus A1 - Gutmann, Jochen S. A1 - Hoffmann-Jacobsen, Kerstin T1 - Singlet oxygen generation by peroxidases and peroxygenases for chemo-enzymatic synthesis T2 - ChemBioChem N2 - Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singlet‐oxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet‐oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet‐oxygen formation. All peroxidases and peroxygenases show singlet‐oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye‐decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet‐oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen. KW - Peroxygenase Y1 - 2021 U6 - https://doi.org/10.1002/cbic.202000326 SN - 1439-7633 SN - 1439-4227 VL - 22 IS - 2 SP - 398 EP - 407 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 - CHAP A1 - Kiebist, Jan A1 - Hofrichter, Martin A1 - Zuhse, Ralf A1 - Scheibner, Katrin ED - Grunwald, Peter T1 - Oxyfunctionalization of Pharmaceuticals by Fungal Peroxygenases T2 - Pharmaceutical biocatalysis : chemoenzymatic synthesis of active pharmaceutical ingredients N2 - Throughout drug discovery and development, metabolic studies are driven by an increased interest to understand the potential for side effects and drug-drug interactions. Peroxygenases are a subclass of peroxide-dependent enzymes that catalyze the transfer of a peroxide-borne oxygen to diverse substrates. The selective oxyfunctionalization of organic molecules is one of the major challenges for the chemical community. Benzylic hydroxylation is one of the most frequently observed reactions of unspecific peroxygenases due to the activated nature of benzylic C–H bonds. The hydroxylation of aromatic rings is a common reaction in the formation of drug metabolites by P450s in mammals including humans. In the liver, P450s facilely metabolize secondary and tertiary amines as well as ethers to the corresponding dealkylated metabolites. The regio- and stereoselective direct introduction of oxygen functionalities into complex pharmaceuticals is a great challenge for organic chemists. KW - Peroxygenase Y1 - 2019 SN - 978-981-4800-80-8 SN - 978-1-00-070757-1 SP - 643 EP - 673 PB - Jenny Stanford Publishing Pte. Ltd. CY - Singapore ET - 1. Auflage ER - TY - GEN A1 - Kiebist, Jan A1 - Koncz, Tino A1 - Friedrich, Stephanie A1 - Scheibner, Katrin T1 - Oxidative biocatalysts to design new oxyfunctionalization tools for drugs and added value bio-based products T2 - International Biotech Innovation Days 2020 (IBID), 28th – 29th October 2020 KW - Peroxygenase Y1 - 2020 UR - https://www.b-tu.de/ibid/program#c243720 CY - Senftenberg 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 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Schramm, Marina A1 - Hofrichter, Martin A1 - König, Rosalie A1 - Quint, Stephan A1 - Kohlmann, Johannes A1 - Zuhse, Ralf A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases T2 - Journal of Fungi N2 - 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. KW - Peroxygenase KW - clopidogrel Y1 - 2021 UR - https://www.mdpi.com/2309-608X/7/9/752 U6 - https://doi.org/10.3390/jof7090752 SN - 2309-608X VL - 7 IS - 9 SP - 1 EP - 17 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 - PAT A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner A1 - Schmidtke, Kai-Uwe A1 - Miethbauer, Sebastian A1 - Herzog, Natalie T1 - Verfahren zur Herstellung von biogenen Stoffen T1 - Method for producing biogenic substances Y1 - 2020 UR - https://patents.google.com/patent/EP3068868B1/de ER - TY - PAT A1 - Scheibner, Katrin A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Küpper, Jan-Heiner T1 - Verfahren zur enzymatischen Herstellung von zytostatischen 4-Hydroxy-Oxazaphosphorinen“ Y1 - 2020 UR - https://www.b-tu.de/enzymtechnologie/publikationen/patente ER - TY - PAT A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner A1 - Jung, Friedrich T1 - In vitro Arzneimittel aus Prodrugs und deren Verwendung Y1 - 2021 UR - https://www.b-tu.de/enzymtechnologie/publikationen/patente ER - TY - GEN A1 - Babot, Esteban D. A1 - Aranda, Carmen A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutierrez, Ana T1 - Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases T2 - Antioxidants N2 - 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. KW - Peroxygenase KW - oxyfunctionalization KW - epoxidation KW - terminal alkenes KW - epoxides Y1 - 2022 UR - https://www.mdpi.com/2076-3921/11/3/522 U6 - https://doi.org/10.3390/antiox11030522 SN - 2076-3921 VL - 11 IS - 3 SP - 1 EP - 12 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 - Wang, Mengyi A1 - Wamp, Sabrina A1 - Gibhardt, Johannes A1 - Holland, Gudrun A1 - Schwedt, Inge A1 - Schmidtke, Kai-Uwe A1 - Scheibner, Katrin A1 - Halbedel, Sven A1 - Commichau, Fabian M. T1 - Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system T2 - Environmental microbiology N2 - The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes. KW - Listeria monocytogenes KW - c-di-AMP metabolism Y1 - 2022 UR - https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.16084?af=R U6 - https://doi.org/10.1111/1462-2920.16084 SN - 1462-2920 VL - 24 IS - 9 SP - 4466 EP - 4488 ER - TY - GEN A1 - Friedrich, Stephanie A1 - Schramm, Marina A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Scheibner, Katrin T1 - Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis T2 - Enzyme and microbial technology N2 - There is an enormous potential for cell-free protein synthesis (CFPS) systems based on filamentous fungi in view of their simple, fast and mostly inexpensive cultivation with high biomass space-time yields and in view of their catalytic capacity. In 12 of the 22 different filamentous fungi examined, in vitro translation of at least one of the two reporter proteins GFP and firefly luciferase was detected. The lysates showing translation of a reporter protein usually were able to synthesize a functional cell-free expressed unspecific peroxygenase (UPO) from the basidiomycete Cyclocybe (Agrocybe) aegerita. For the most promising candidate Neurospora crassa, the influence of different conditions of cultivation and lysate preparation on in vitro translation of the reporter proteins was investigated and optimized. In general, the greatest improvements in the translational activity were achieved by the choice of the growth medium, the addition of organic nitrogen being most beneficial. Optimizing the culture and preparation conditions of the N. crassa platform improved protein yield of the original lysate by a factor of 25 for firefly luciferase and 17 for GFP, respectively. In addition to the reporter proteins, the aforementioned UPO as well as a functional UPO from Aspergillus niger were cell-free expressed using the different lysates from N. crassa. CFPS with fungal lysates opens the door to expressing UPOs in high throughput and in parallel, for example to optimize synthesis conditions or adapt catalyst properties. The presented method proves the general potential of fungal lysates for application in cell-free syntheses. KW - Cell-free syntheses KW - Cell-free protein synthesis (CFPS) KW - Fungal lysates KW - Neurospora crassa KW - Unspecific peroxygenase KW - In vitro translation Y1 - 2025 U6 - https://doi.org/10.1016/j.enzmictec.2025.110588 VL - 185 SP - 1 EP - 10 PB - Elsevier BV CY - Amsterdam 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 -