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Gegenstand der vorliegenden Arbeit war die Synthese von enantiomerenreinen α-substituierten β-Hydroxyestern zu neuartigen 4,5-disubstituieren Oxazolidin-2-onen, die auch als EVANS-Auxilare bekannt sind.

Nematodes represent a diverse and ubiquitous group of metazoans in terrestrial environments. They feed on bacteria, fungi, plants, other nematodes or parasitize a variety of animals and hence may be considered as active members of many food webs. Deadwood is a structural component of forest ecosystems which harbors many niches for diverse biota. As fungi and bacteria are among the most prominent decomposing colonizers of deadwood, we anticipated frequent and diverse nematode populations to co‐occur in such ecosystems. However, knowledge about their ability to colonize this habitat is still limited. We applied DNA‐based amplicon sequencing (metabarcoding) of the 18S rRNA gene to analyze nematode communities in sapwood and heartwood of decaying logs from 13 different tree species. We identified 247 nematode ASVs (amplicon sequence variants) from 27 families. Most of these identified families represent bacterial and fungal feeders. Their composition strongly depended on tree species identity in both wood compartments. While pH and water content were the only wood properties that contributed to nematodes’ distribution, co‐occurring fungal and prokaryotic (bacteria and archaea) α‐ and β‐ diversities were significantly related to nematode communities. By exploring thirteen different tree species, which exhibit a broad range of wood characteristics, this study provides first and comprehensive insights into nematode diversity in deadwood of temperate forests and indicates connectivity to other wood‐inhabiting organisms.

[EN] 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 in yeast of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) and their production at large scale in bioreactor. Our strategy was based on adopting secretion mutations from Agrocybe aegerita UPO mutant −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 that shares 65% sequence identity. After replacing the native signal peptides by 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/L in S. cerevisiae. These variants were subsequently transferred to P. pastoris for overproduction in fed-batch bioreactor, boosting expression levels up to 290 mg/L with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/L, with veratryl alcohol as substrate). With a broad pH activity profile, ranging from 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.

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C–H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops., BT/Biocatalysis

Fungi and prokaryotes are dominant colonizers of wood and mediate its decomposition. Much progress has been achieved to unravel these communities and link them to specific wood properties. However, comparative studies considering both groups of organisms and assessing their relationships to wood resources are largely missing. Bipartite interaction networks provide an opportunity to investigate this colonizer-resource relationship more in detail and aim to directly compare results between different biotic groups. The main questions were as follows. Are network structures reflecting the trophic relationship between fungal and prokaryotic colonizers and their resources? If so, do they reflect the critical role of these groups, especially that of fungi, during decomposition? We used amplicon sequencing data to analyze fungal and prokaryotic interaction networks from deadwood of 13 temperate tree species at an early to middle stage of decomposition. Several diversity- and specialization-related indices were determined and the observed network structures were related to intrinsic wood traits. We hypothesized nonrandom bipartite networks for both groups and a higher degree of specialization for fungi, as they are the key players in wood decomposition. The results reveal highly modular and specialized interaction networks for both groups of organisms, demonstrating that many fungi and prokaryotes are resource-specific colonizers. However, as the level of specialization of fungi significantly surpassed that of prokaryotes, our findings reflect the strong association between fungi and their host. Our novel approach shows that the application of bipartite interaction networks is a useful tool to explore, quantify, and compare the deadwood-colonizers relationship based on sequencing data. IMPORTANCE Deadwood is important for our forest ecosystems. It feeds and houses many organisms, e.g., fungi and prokaryotes, with many different species contributing to its decomposition and n

Die vorliegende Arbeit befasst sich mit der unspezifischen Peroxygenase (UPO), einer ausschließlich von Pilzen produzierten Oxidoreduktase. Die Dissertation besteht aus drei wesentlichen Teilen: 1.) Untersuchungen zum Substratspektrum zweier ausgewählter UPOs, 2.) Untersuchungen zur Inaktivierung von UPOs, und 3.) die Etablierung einer Kaskadenreaktion von UPOs und pilzlichen Oxidasen zur Oxidation von Hydroxymethylfurfural (HMF) zu 2,5-Furandicarbonsäure (FDCA). 1) Oxidation ausgewählter organischer Schadstoffe Die Mehrheit der getesteten organischen Schadstoffe (35 von 44 chemischen Verbindungen, vgl. Tabelle 6), inklusive verschiedener Xenobiotika wie chlorierte Benzole und deren Derivate, halogenierte Biphenylether, Nitroaromaten, PAK und Phthalate, wurden durch lediglich zwei UPOs (AaeUPO und MroUPO) oxidativ umgewandelt und damit aktiviert. Die von den UPOs katalysierten Reaktionen waren durch drei Substrat-Eigenschaften limitiert: 1.) sterische Hemmung, u.a. infolge einer hohen Zahl an Substituenten (z.B. Hexachlorbenzol) oder aufgrund der grundsätzliche Sperrigkeit (Größe) des Substrat-Moleküls (z.B. 6-Ring-PAK wie Benzo[g,h,i]perylen), 2.) Inaktivierung des aromatischen Ringes durch elektronenziehende Gruppen (z.B. im Nitrobenzol) und 3.) geringe Bioverfügbarkeit und Wasserlöslichkeit. Während die Verhinderung der Oxidation durch geringe Löslichkeit und sterische Hemmung bereits von Peter (2013) und Poraj-Kobielska (2013) beschrieben wurden, stellt die Limitation durch Substrat-Deaktivierung, wie im Fall des Nitrobenzols, einen neuen Befund. 2) Inaktivierung von UPOs Für alle getesteten UPOs konnte eine intrinsische Katalase-Aktivität nachgewiesen werden, wobei deren Ausmaß unter den getesteten Enzymen stark variierte. So unterschieden sich die katalytischen Effizienzen der Katalase-Aktivität von AaeUPO und rCciUPO um eine Größenordnung, während die der MroUPO sich dazwischen einordnete. Im Rahmen der Untersuchungen konnte für die AaeUPO die Bildung eines r

Epoxidation of unsaturated fatty acids by unspecific peroxygenases (UPOs) of the best-known long-UPO subfamily, including the Agrocybe aegerita (AaeUPO) and Coprinopsis cinerea enzymes, is reported here. To understand the different oxygenation patterns by members of the long-UPO and short-UPO subfamilies, the latter represented by the Marasmius rotula enzyme (MroUPO), fatty-acid diffusion into their heme pockets was simulated with the adaptive PELE software. Computational results shed light on the inability of AaeUPO to epoxidize oleic acid (C18:1), opposed to MroUPO, due to steric hindrances to harbor (with a good interaction energy) the substrate with the ¿9 C10 atom at a catalytically relevant distance (<3.5 Å) from the oxo group in simulated heme compound-I. However, effective a-linolenic acid epoxidation is anticipated because the ¿15 C16 atom would attain such a distance in AaeUPO thanks to its more terminal position. The above hypothesis was verified using an engineered MroUPO variant (I153F/S156F) with a narrowed heme access channel mimicking that of AaeUPO. Experimental oxygenation of unsaturated fatty acids by this variant thus resembles that of AaeUPO, including regioselective (from 95% to >99%) formation of cis,cis-15,16-epoxyoctadeca-9,12-dienoic acid. The nearly complete conversion of a-linolenic acid by the two enzymes was transferred to a small preparative scale, the yield of purified product was estimated, its chemical structure analyzed by NMR, and more interestingly, stereoselective production of the 15(R),16(S) enantiomer (80–83% ee) assessed by chiral HPLC. This enzymatic synthesis overcomes the unspecificity of chemical epoxidation where the reaction cannot be restricted to the formation of monoepoxides as found during m-perchlorobenzoic acid oxidation of a-linolenic acid. Moreover, the variant was able to produce the unsaturated dicarboxylic fatty acid, together with subterminal oxygenation products, during partial conversion of oleic acid. Th, This work has received funding from the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No 792063 (“Development and pilot production of sustainable binder systems for wood based panels”, https://susbind.eu), the CTQ2016-79138-R and BIO2017-86559-R projects of Spanish MINECO, the Secretaria d’Universitats i Recerca of Generalitat de Catalunya, and the European Social Fund (ESF2019-FI-B-00154). The authors thank Novozymes A/S for supplying rCciUPO. MM acknowledges a Catalan Government doctoral grant., Peer Reviewed, Postprint (published version)