Agrocybe Aegerita Research Articles

Cultivation of black poplar mushroom, Cyclocybe aegerita, on woody and non-woody lignocellulosic substrates with a high biological efficiency

The Black Poplar mushroom, Cyclocybe aegerita, is considered a high-quality mushroom due to its high nutritional value, delicious taste, and unique aroma. This fungus is commonly grown in East Asia and Europe. It has shown low biological efficiency (BE) on a majority of lignocellulosic substrates studied to date. This study aims to investigate the possibility of enhancing the BE of C. aegerita and to evaluate the effect of the carbon to nitrogen (C:N) ratio in the substrate. The performance of C. aegerita was determined using five woody and non-woody lignocellulosic substrates with a precisely determined C:N ratio. The result showed that the highest significant BE was 194%, obtained from a non-woody substrate composed of 78% wheat straw supplemented with 20% wheat bran (p ≤ 0.05), followed by a woody substrate composed of 73% of wood chips, 10% of wheat bran, 10% of cottonseed, and 5% of wheat seed, which generated 123% BE. The remaining substrates were unable to produce more than 100% BE (p ≤ 0.05). The mycelial growth of C. aegerita in the substrate as well as in the substrate-derived media was also significantly improved using a non-woody substrate consisting of 78% wheat straw supplemented with 20% wheat bran compared with other substrates (p ≤ 0.05). Further analyses also revealed that an increased level of the C:N ratio resulted in a decline in the total fresh yield that was obtained from the substrates, so that the optimal C:N ratio was found to be within the range of 48–56%. These findings may have implications for improving the BE of C. aegerita and removing one of its most important barriers in commercial production.

Transformation of Agrocybe cylindracea Galectin into αGalNAc-Specific Lectin.

A multi-specific fungal galectin from the mushroom Agrocybe cylindracea (ACG) binds a broad range of β-galactosides, as well as their derivative GalNAcα1-3Gal. Site-directed mutagenesis of the hydrophilic residues His, Asn, Arg, and Glu, involved in carbohydrate recognition, abolished the binding affinity of the derived mutants to β-galactosides, whereas only N46A caused increased affinity to GalNAcα1-3Gal-containing oligosaccharides and loss of β-galactoside-binding activity. Detailed structural analysis revealed that Pro45, the preceding residue of Asn46 of the wild-type ACG, takes the cis imide conformation to tether Asn46 onto a loop region to make new hydrogen bonds with β-galactosides and to compensate for the lack of evolutionarily conserved Asn. In contrast, in the N46A mutant, Pro45 takes the more stable trans conformation, resulting in "switched" specificity to αGalNAc. Such an altered recognition system in the binding specificity of galectins can be observed in other lectin molecules not only in nature but will also be observed in those engineered in the future.

Extraction, physiological function and application of soluble dietary fiber from edible fungi: a review

Soluble dietary fiber has attracted much attention due to its significant health benefits to consumers. Edible fungi are abundant sources of soluble dietary fiber (SDF). The content and composition of soluble dietary fiber in edible fungi variy greatly according to varieties, growth stages and different parts. This review focuses on the extraction methods of soluble dietary fiber from edible fungi, such as Lentinula edodes, Agrocybe cylindracea. The possible health benefits of mushroom soluble dietary fiber in relationship with hypoglycemic effect, regulatory mechanism of pancreatic lipase, gut microbiota and macrophages are also discussed. Finally, the application and development potential of mushroom soluble dietary fiber are pointed out.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast‐growing and unlike yeast, posttranslational modifications like N‐glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non‐activated carbon‐hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α‐promoter. The success of the heterologous production of full‐length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase‐specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases.

Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by 1H, 13C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

Proteomic Research on the Antitumor Properties of Medicinal Mushrooms.

Medicinal mushrooms are increasingly being recognized as an important therapeutic modality in complementary oncology. Until now, more than 800 mushroom species have been known to possess significant pharmacological properties, of which antitumor and immunomodulatory properties have been the most researched. Besides a number of medicinal mushroom preparations being used as dietary supplements and nutraceuticals, several isolates from mushrooms have been used as official antitumor drugs in clinical settings for several decades. Various proteomic approaches allow for the identification of a large number of differentially regulated proteins serendipitously, thereby providing an important platform for a discovery of new potential therapeutic targets and approaches as well as biomarkers of malignant disease. This review is focused on the current state of proteomic research into antitumor mechanisms of some of the most researched medicinal mushroom species, including Phellinus linteus, Ganoderma lucidum, Auricularia auricula, Agrocybe aegerita, Grifola frondosa, and Lentinus edodes, as whole body extracts or various isolates, as well as of complex extract mixtures.

Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita.

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

Effects of Extraction Methods on Extraction Efficiency and Biological Activity of Agrocybe cylindracea Spent Mushroom Substrate Polysaccharide

Effects of Extraction Methods on Extraction Efficiency and Biological Activity of Agrocybe cylindracea Spent Mushroom Substrate Polysaccharide

Biotransformation of [U-13C]linoleic acid suggests two independent ketonic- and aldehydic cycles within C8-oxylipin biosynthesis in Cyclocybe aegerita (V. Brig.) Vizzini

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.

Established Digital Model of Fruit Body Growth of Agrocybe cylindracea Based on Network Programming

Agricultural information technology is an emerging technology based on the cross-fertilization of information technology and agricultural science, which has caused the rapid development of digital agriculture and smart agriculture. Crop growth models, as one of their core components, can dynamically simulate the crop growth and development process and its relationship with climate factors, soil properties, and management techniques, thus effectively overcoming the strong spatial and temporal limitations in traditional agricultural production management research and providing quantitative tools for early warning and effect assessment of crop productivity prediction under different conditions. This study focuses on the general technical approach, the latest research progress, and future development thinking developed by the authors in the construction and application of the tea mushroom growth model. Based on the fitting method of the data model curve of the Internet laboratory, this study is proposed to use network programming technology to digitally fit the growth curve of cap diameter and stalk diameter of the fruiting body in the process of Agrocybe cylindracea growth. The fitting system adopted the development mode of PHP + MYSQL and took the MYSQL database as the core, which made the curve fitting of the final experimental data fast, convenient, and more intelligent. This study laid a preliminary foundation for the effective establishment and improvement of laboratory Agrocybe cylindracea, fruiting body growth data statistics and analysis, and graphical output network sharing.

Qualitative characteristics of the fungus Cyclocybe aegerita strains 2229, 2230, 2231 IBK under industrial cultivation conditions

Poplar mushroom is characterised by attractive fruiting bodies in terms of appearance and taste. Cyclocybe aegerita mushrooms are also known to contain unique substances, including proteins, polysaccharides, and aromatic substances with proven nutritional value and medicinal effects. However, the lack of a sound cultivation technology using available plant material limits the saturation of the domestic market with mushrooms of this species. Three strains of C. aegerita from the IBK National Collection of Agaricale Cultures of the Institute of Ecology and Natural Resources were studied for their potential introduction into commercial culture. It was determined that strains 2230 and 2231 have a vegetation cycle of 42 days compared to 46-64 days for the previously studied strains. The highest yield in the experiment was obtained for strain 2231, which ranged from 217 to 220 g per kilogram of a substrate over three cultivation cycles. The biological efficiency of the strains according to the results of the first wave of fruiting ranged from 20.2 (2229) to 60.4% (2231), which is consistent with the published data of other researchers. The appearance and parameters of the fruiting bodies of the strains, which differed in both the colour of the pileus and the size, were described in detail. Under microclimatic conditions of the experiment, strains 2230 and 2231 had a rich brown colour of pilei, while the formed fruit bodies of strain 2229 were distinguished by a pleasant milky cream colour. The largest fruiting bodies in terms of weight were obtained by cultivation of strain 2229 (7.5 ± 0.4 g), which is on average 2 g higher than the weight of mushrooms of other studied strains. The highest fruiting bodies were discovered in strain 2230 having a height of 91.0 ± 1.8 mm, which also had the widest pilei with a diameter of 29.5 ± 0.9 mm. The largest stem diameter, which reached an average of 10.8 ± 0.3 mm, was detected in the fruiting bodies of strain 2229. This study investigated the variability of morphological traits and identified trends in the number of fruiting bodies of a certain size in the total volume of collected mushrooms, which facilitates the design of packaging and marketing campaigns

Impact of substrate supplemented with CaCO3 on mycelial growth, yield, morphological features and storability of fruiting bodies of black poplar mushroom Agrocybe cylindracea (DC.) Marie.

Black poplar mushroom, Agrocybe cylindracea deserves special attention, due to its medicinal properties. Water and alcohol extracts from fruiting bodies of the fungus have an anti-oxidant, anti-cancer, anti-fungal, cholesterol and triglycerides blood level lowering abilities. This mushroom is rich in proteins and vitamins, mineral elements and low in fat. The aim of the experiments was to determine effect of a substrate supplementation with CaCO3 on mycelial growth, yield, morphological features and storability of fruiting bodies of four strains of A. cylindracea (DC.) Marie. The amount of additive to sawdust substrate affects rate of mycelial growth and yield of investigated strains. A. cylindracea mycelial growth was not affected by addition of CaCO3 to substrate, however a significant effect of this additive was found on yield, which was the highest with CaCO3 addition in an amount of 8 g/100 g of substrate. Carpophores characterized with the largest caps diameter, and the largest individual mass obtained of substrate enriched with CaCO3 addition of 8 g/100 g of substrate. In addition, it was found that supplementation with CaCO3 affect storability of A. cylindracea. The lowest weight loss of fruiting bodies after 3 and 7 days of storage was found with addition of CaCO3 to substrate in an amount of 4 g/100 g of substrate.

Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis 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 Candolleomyces (Psathyrella) aberdarensis (PabUPO) 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 PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with 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/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 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. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

Molecular identification and antimicrobial activities of some wild Egyptian mushrooms: Bjerkandera adusta as a promising source of bioactive antimicrobial phenolic compounds

BackgroundThe discovery of potential, new cost-effective drug resources in the form of bioactive compounds from mushrooms is one way to control the resistant pathogens. In the present research, the fruiting bodies of five wild mushrooms were collected from Egypt and identified using internal transcribed spacer region (ITS) of the rRNA encoding gene and their phylogenetic relationships, antimicrobial activities, and biochemical and phenolic compounds were evaluated. ResultsThe sequences revealed identity to Bjerkandera adusta, Cyclocybe cylindracea, Agrocybe aegerita, Chlorophyllum molybdites, and Lentinus squarrosulus in which Cyclocybe cylindracea and Agrocybe aegerita were closely related, while Chlorophyllum molybdites was far distant. Cyclocybe cylindracea and Agrocybe aegerita showed 100% similarity based on the sequenced ITS-rDNA fragment and dissimilar antimicrobial activities and chemical composition were detected. Bjerkandera adusta and Cyclocybe cylindracea showed strong antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Streptococcus pneumoniae, and Candida albicans. This activity could be attributed to the detected phenolic and related compounds’ contents. ConclusionOur finding provides a quick and robust implement for mushroom identification that would facilitate mushroom domestication and characterization for human benefit.

Influence of the substrate composition on the yield and nutritional value of the fruiting bodies of the edible mushrooms Pleurotus citrinopileatus and Cyclocybe aegerita

Purpose. To reveal the influence of the substrate compositions on technical indicators and the chemical composition of the fruiting bodies of the golden oyster mushroom and poplar mushroom. Methods. The experimental design included cultivation of two species of wood-decay fungi Pleurotus citrinopileatus Singer (strain 2161 IVK) and Cyclocybe aegerita (V.Brig.) Vizzini (strain 2230 IVK) on three variants of substrate composition. Laboratory, laboratory-production methods for evaluating the effectiveness of growing technology, chemical composition of the obtained raw materials, statistical methods of analysis were applied. Results. The structure and composition of substrates affect the technological characteristics of the culture, physical and chemical properties of fruiting bodies. The shortest fruiting cycle of 35.2 ± 1.7 days was determined for C. aegerita under growing conditions on SC1 substrate which formula included «straw, husks, pellet, rapeseed, corn, and CaCO3» in the ratio of 30: 40: 70: 20: 20: 1. The highest yield (170.5 ± 15.2 g per 1 kg of substrate) in the experiment was determined for P. citrinopileatus on the SC1 substrate composed of «straw / pellets / rapeseed / corn / CaCO3» in the ratio 40: 90: 20: 25: 1. Fruiting bodies of P. citrinopileatus obtained from the SC3 substrate composed of «pellets / rapeseed / corn / CaCO3» in the ratio 60: 110: 20: 30: 1 had the highest protein content – 22.47 ± 0.19%, and fruiting bodies from the SC1 substrate had the least amount of proteins – 17.38 ± 2.60%. Fruiting bodies of C. aegerita contained more lipids than those of P. citrinopileatus, but the factor of the influence of the substrate composition on the total amount of lipids for some cultivars was insignificant. The largest amount of endopolysaccharides was isolated from the fruiting bodies of C. aegerita (6.81 ± 0.41%) cultivated on SC3 substrate, and the smallest in the SC1 variant (1.38 ± 0.25%). The content of endopolysaccharides in the fruiting bodies of P. citrinopileatus had less variability from 2.54 ± 0.54 (SC3) to 4.72 ± 0.61% (SC1). Conclusion. Substrate compositions significantly affect the biological efficiency of cultivars and the content of nutrients in fruiting bodies of the studied species. The obtained results enable producers of mushrooms to predict the production efficiency and quality of grown mushrooms in accordance with the use of available raw materials.

Identification and Expression of New Unspecific Peroxygenases - Recent Advances, Challenges and Opportunities.

In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

3'-Sulfo-TF Antigen Determined by GAL3ST2/ST3GAL1 Is Essential for Antitumor Activity of Fungal Galectin AAL/AAGL.

Many lectins have been reported to have antitumor activities; identifying the glycan ligands in tumor cells of lectins is crucial for lectin clinical application. An edible mushroom galectin, Agrocybe aegerita lectin (AAL/AAGL), that has a high antitumor activity has been reported. In this paper, based on the glycan array data, it is showed that the Thomsen-Friedenreich antigen (TF antigen)-related O-glycans were found to be highly correlated with the antitumor activity of AAL/AAGL. Further glycosyltransferase quantification suggested that the ratio between GAL3ST2 and ST3GAL1 (GAL3ST2/ST3GAL1), which determined the 3′-sulfo-TF expression level, was highly correlated with the antitumor activity of AAL/AAGL. Overexpressing the enzyme of GAL3ST2 in HL60 and HeLa cell lines could increase the growth inhibition ratio of AAL/AAGL from 22.7 to 43.9% and 27.8 to 39.1%, respectively. However, ST3GAL1 in Jurkat cells could decrease the growth inhibition ratio from 44.7 to 35.6%. All the data suggested that the 3′-sulfo-TF antigen is one of the main glycan ligands that AAL/AAGL recognizes in tumor cells. AAL/AAGL may potentially serve as a reagent for cancer diagnosis and a targeted therapy for the 3′-sulfo-TF antigen.

Pilot-Scale Production of Peroxygenase from Agrocybe aegerita.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

The Pacific Tree-Parasitic Fungus Cyclocybe parasitica Exhibits Monokaryotic Fruiting, Showing Phenotypes Known from Bracket Fungi and from Cyclocybe aegerita.

Cyclocybe parasitica is a wood-destroying parasitic edible mushroom growing on diverse broad-leafed trees in New Zealand and other Pacific areas. Recent molecular systematics of European Cyclocybe aegerita, a newly delimited Asian phylum and of related species, corroborated the distinction of the chiefly saprobic cultivated edible mushroom C. aegerita from C. parasitica. Here, we show that C. parasitica exhibits a morpho-physiological trait characteristic to its European cousin, i.e., monokaryotic fruiting sensu stricto (basidiome formation without mating). Monokaryotic fruiting structures formed by C. parasitica ICMP 11668-derived monokaryons were categorized into four phenotypes. One of them displays ulcer-like structures previously reported from bracket fungi. Histology of dikaryotic and monokaryotic C. parasitica fruiting structures revealed anatomical commonalities and differences between them, and towards monokaryotic fruiting structures of C. aegerita. Mating experiments with C. parasitica strains representative of each fruiting phenotype identified compatible sibling monokaryons. Given reports on hypothetically monokaryotic basidiome field populations of ‘C. aegerita sensu lato’, it seems worthwhile to prospectively investigate whether monokaryotic fruiting s.str. occurs in nature. Sampling from such populations including karyotyping, comparative -omics, and competition assays may help to answer this question and provide evidence whether this trait may confer competitive advantages to a species capable of it.

Directed evolution of unspecific peroxygenase in organic solvents.

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 DNAshuffling 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.