@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{WangWampGibhardtetal., author = {Wang, Mengyi and Wamp, Sabrina and Gibhardt, Johannes and Holland, Gudrun and Schwedt, Inge and Schmidtke, Kai-Uwe and Scheibner, Katrin and Halbedel, Sven and Commichau, Fabian M.}, title = {Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system}, series = {Environmental microbiology}, volume = {24}, journal = {Environmental microbiology}, number = {9}, issn = {1462-2920}, doi = {10.1111/1462-2920.16084}, pages = {4466 -- 4488}, abstract = {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.}, language = {en} } @misc{FriedrichSchrammKiebistetal., author = {Friedrich, Stephanie and Schramm, Marina and Kiebist, Jan and Schmidtke, Kai-Uwe and Scheibner, Katrin}, title = {Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis}, series = {Enzyme and microbial technology}, volume = {185}, journal = {Enzyme and microbial technology}, publisher = {Elsevier BV}, address = {Amsterdam}, doi = {10.1016/j.enzmictec.2025.110588}, pages = {1 -- 10}, abstract = {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.}, 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} }