Oxylipins are metabolites with a variety of biological functions. However, the biosynthetic pathway is widely unknown. It is considered that the first step is the oxygenation of polyunsaturated fatty acids like linoleic acid. Therefore, a lipoxygenase (LOX) from the edible basidiomycete Agrocybe aegerita was investigated. The Aae LOX4 was heterologously expressed in E . coli and purified via affinity chromatography and gel filtration. Biochemical properties and kinetic parameters of the purified Aae LOX4 were determined with linoleic acid and linolenic acid as substrates. The obtained K m , v max and k cat values for linoleic acid were 295.5 μM, 16.5 μM · min -1 · mg -1 and 103.9 s -1 , respectively. For linolenic acid K m , v max and k cat values of 634.2 μM, 19.5 μM · min -1 · mg -1 and 18.3 s -1 were calculated. Maximum activities were observed at pH 7.5 and 25 °C. The main product of linoleic acid conversion was identified with normal-phase HPLC. This analysis revealed an explicit production of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The experimental regio specificity is underpinned by the amino acid residues W384, F450, R594 and V635 considered relevant for regio specificity in LOX. In conclusion, HPLC-analysis and alignments revealed that Aae LOX4 is a 13-LOX.

This study introduces a new type of ene/yne-reductase from Cyclocybe aegerita with a broad substrate scope including aliphatic and aromatic alkenes/alkynes from which aliphatic C8alkenones, C8-alkenals and aromatic nitroalkenes were the preferred substrates. By comparing alkenes and alkynes, a~2fold lower conversion towards alkynes was observed. Furthermore, it could be shown that the alkyne reduction proceeds via a slow reduction of the alkyne to the alkene followed by a rapid reduction to the corresponding alkane. An accumulation of the alkene was not observed. Moreover, a regioselective reduction of the double bond in α,β-position of α,β,γ,δ-unsaturated alkenals took place. This as well as the first biocatalytic reduction of different aliphatic and aromatic alkynes to alkanes underlines the novelty of this biocatalyst. Thus with this study on the new ene-reductase CaeEnR1, a promising substrate scope is disclosed that describes conceivably a broad occurrence of such reactions within the chemical landscape.

Although the typical aroma contributing compounds in fungi of the phylum Basidiomycota are known for decades, their biosynthetic pathways are still unclear. Amongst these volatiles, C8-compounds are probably the most important ones as they function, in addition to their specific perception of fungal odour, as oxylipins. Previous studies focused on C8-oxylipin production either in fruiting bodies or mycelia. However, comparisons of the C8-oxylipin biosynthesis at different developmental stages are scarce, and the biosynthesis in basidiospores was completely neglected. In this study, we addressed this gap and were able to show that the biosynthesis of C8-oxylipins differs strongly between different developmental stages. The comparison of mycelium, primordia, young fruiting bodies, mature fruiting bodies, post sporulation fruiting bodies and basidiospores revealed that the occurance of the two main C8-oxylipins octan-3-one and oct-1-en-3-ol distinguished in different stages. Whereas oct-1-en-3-ol levels peaked in the mycelium and decreased with ongoing maturation, octan-3-one levels increased during maturation. Furthermore, oct-2-en-1-ol, octan-1-ol, oct-2-enal, octan-3-ol, oct-1-en-3-one and octanal contributed to the C8-oxylipins but with drastically lower levels. Biotransformations with [U-13C]linoleic acid revealed that early developmental stages produced various [U-13C]oxylipins, whereas maturated developmental stages like post sporulation fruiting bodies and basidiospores produced predominantly [U-13C]octan-3-one. Based on the distribution of certain C8-oxylipins and biotransformations with putative precursors at different developmental stages, two distinct biosynthetic cycles were deduced with oct-2-enal (aldehydic-cycle) and oct-1-en-3-one (ketonic-cycle) as precursors.

The basidiomycetous lipoxygenase Lox1 from Cyclocybe aegerita catalyzes the oxygenation of polyunsaturated fatty acids (PUFAs) with a high preference towards the C18-PUFA linoleic acid (C18:2 (ω-6)). In contrast, longer PUFAs, generally not present in the fungal cell such as eicosatrienoic acid (C20:3(ω-3)) and docosatrienoic acid (C22:3 (ω-3)), are converted with drastically lower activities. With site-directed mutagenesis, we were able to create two variants with enhanced activities towards longer chain PUFAs. The W330L variant showed a ~ 20 % increased specific activity towards C20:3(ω-3), while a ~ 2.5-fold increased activity against C22:3 (ω-3) was accomplished by the V581 variant.

Based on transcriptomic, volatilomic and metabolomic data of the model organism Cyclocybe aegerita, the endogenous and enzymatic production of the C8‐oxylipins oct‐2‐en‐1‐ol, octan‐ 1‐ol, oct‐2‐enal, octanal, octan‐3‐ol, oct‐1‐en‐3‐ol, oct‐1‐en‐3‐one and octan‐3‐ one via biotransformations with [U‐13C]linoleic acid was compared to the developmental state depending gene expression patterns of putative oxylipin‐associated enzyme classes like lipoxygenases, dioxygenases, ene‐reductases and alcohol dehydrogenases. On closer consideration of these data sets, the C8‐oxylipin production seemed to occur cluster‐like.While one cluster (ketonic‐cycle) consisted of octan‐3‐ol, oct‐1‐en‐3‐one, octan‐3‐one and oct‐1‐en‐3‐ol, which were detected through out all developmental stages, a second cluster (aldehydic‐cycle) primarily active at early developmental stages involved oct‐2‐enal, octanal, oct‐2‐en‐1‐ol and octan‐1‐ol. Studying these clusters revealed that all C8‐oxylipins of the aldehydic‐cycle and ketonic‐cycle can be derived from oct‐2‐enal and oct‐1‐en‐3‐one, respectively. A detailled consideration of the gene expression patterns revealed several enzyme classes putatively involved in the C8‐oxylipin biosynthesis. Especially the two genes AAE3 04864 and AAE3 13549 encoding the lox4‐ and enr1‐gene showed great similarities between their strongly increasing transcript counts with maturation. Subsequent recombinant production of CaeLOX4 and CaeEnR1 revealed that CaeLOX4 showed a preference for C18‐ fatty acids with the highest affinity towards linoleic acid. Analysis of the reaction product revealed that this LOX selectively oxygenated linoleic acid at position 13 to 13‐ hydroperoxyoctadecadienoic acid (13‐HPOD). CaeEnR1 showed high affinities towards the reduction of oct‐1‐en‐3‐one and oct‐2‐enal to their saturated counterparts octan‐3‐one and octanal, respectively. Due to the strong similarities between the expression patterns of CaeLOX4 and CaeEnR1 a coherence between their main reaction products 13‐HPOD and the precursor of the ketonic‐cycle oct‐1‐en‐3‐one seems very likely. This suggests a biosynthetic route from linoleic acid to 13‐HPOD, followed by a subsequent cleavage step to oct‐1‐en‐3‐ one and therefore to C8‐oxylipins of the ketonic‐cycle.