@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{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{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{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} }