Rapid screening system to identify unspecific peroxygenase activity.

Unspecific peroxygenases (UPO, EC 1.11.2.

Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis.

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.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

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.

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.

Novel Unspecific Peroxygenase from Catalyzes the Synthesis of Bioactive Lipid Mediators.

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.

Draft Genome Sequence of Truncatella angustata (Anamorph) S358.

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.

Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases.

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.

Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.

Biocatalytic Syntheses of Antiplatelet Metabolites of the Thienopyridines Clopidogrel and Prasugrel Using Fungal Peroxygenases.

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 P2Y receptor and thereby prevent atherothrombotic events.

Functional Expression of Two Unusual Acidic Peroxygenases from in Yeasts by Adopting Evolved Secretion Mutations.

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 () (UPO) 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 UPOs, long-type UPOs, and shares 65% sequence identity.

High Epoxidation Yields of Vegetable Oil Hydrolyzates and Methyl Esters by Selected Fungal Peroxygenases.

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.

Singlet-Oxygen Generation by Peroxidases and Peroxygenases for Chemoenzymatic Synthesis.

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.

Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells.

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.

Selective Oxygenation of Ionones and Damascones by Fungal Peroxygenases.

Apocarotenoids are among the most highly valued fragrance constituents, being also appreciated as synthetic building blocks. This work shows the ability of unspecific peroxygenases (UPOs, EC1.11.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone.

Unspecific peroxygenases (UPO, EC 1.11.2.

From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation.

One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.