Aspergillus fungi are a source of low-molecular compounds of various structures possessing biological activities. We investigated the secondary metabolite profile of the soil fungus A. calidoustus VKM F-4916. The strain was found to synthesize new metabolites attributed to furoisocoumarins, which we named asperisocoumarin J and K, and a known siderophore desferritriacetylfusigen. The structure of asperisocoumarin J and K were determined by mass spectrometry and NMR spectroscopy. Asperisocoumarins J, K and desferritriacetylfusigen possessed a phytotoxicity, inhibiting the lettuce root growth. Sow thistle leaf and wheat leaf cuttings were sensitive to the action of asperisocoumarin J and K at a concentration of 5 mg/mL. Analysis of the structures of furoisocoumarins (asperisocoumarins J and K) using the online resource Pesti-DGI-Net showed that compounds had the physico-chemical properties favorable for pesticide development, in particular, fungicides and herbicides. An in-depth study of the phytotoxic properties of furoisocoumarins and their natural analogs is of interest in the context of the search for new herbicide compounds.

Fungal laccases oxidize a wide range of substrates with a diverse spectrum of subsequent non-specific free radical reactions, leading to the production of unwanted byproducts. This work describes a unique recombinant alkaliphilic laccase from Paramyrothecium roridum VKM F-3565 capable of performing specific oligomerization of phenylpropanoids (precursors of natural lignin and lignans) in a neutral environment, thus preventing the reverse reaction of depolymerization which occurs in an acidic environment. The recombinant alkaliphilic laccase from P. roridum VKM F-3565 with a specific enzyme activity of about 154.0 U/mg (in the reaction with 1 mM ABTS) was obtained using a Komagataella phaffii transformant with a yield of 20 ± 1.5 mg/L. The recombinant laccase had an increased degree of N-glycosylation (MW = 97 kDa), higher pH optimum in reaction with phenylpropanoids and a decreased temperature optimum, compared to the wild-type laccase. The enzyme exhibited great resistance to surfactants and the EDTA in the neutral conditions rather than the acidic ones, whereas its tolerance to mono- and divalent-metal ions was high at acidic conditions. This work demonstrates the important role of N-glycosylation of the alkaliphilic laccase of P. roridum VKM F-3565 in its functional activity. The presence of pH-dependent reactions makes the studied laccase attractive for the phenylpropanoid oligomerization with the production of novel oligomeric phenylpropanoid derivatives for industrial and pharmacological purposes.

Strains of Cryphonectria parasitica (Murrill) M.E. Barr were isolated from the bark of Castanea sativa Mill. with visual signs of weakening in Krasnodar Krai and Abkhazia. Strains demonstrated varying degrees of phytopathogenicity in vitro towards two plant test objects. Extensive (on C. sativa shoots) and deep (on Granny Smith apples) necrosis was observed in 67

Ascomycete fungi isolated in Central Siberia from canker lesions of Abies sibirica Ledeb. were assigned to the genus Corinectria Gonzalez Chaverri. Our earlier research has shown four Corinectria Siberian strains to be significantly different genetically from the known Corinectria spp. This work investigated the secondary metabolites of 23 morphologically similar Corinectria Siberian strains and their phytotoxicity. The strains were divided into two types by their morphological and cultural traits, MCT1 and MCT2. The MCT1 strains synthesized the meroterpenoids ilicicolins A and B, ilicicolin A epoxide and chlorocylindrocarpol; the MCT2 strains produced ilicicolins C, D (ascochlorin), F and ascofuranol. The phytotoxicity of the Corinectria strains showed a significant heterogeneity to Picea obovata seeds and was exhibited by 31

Most Fusarium species are known as endophytes and/or phytopathogens of higher plants and have a worldwide distribution. Recently, information discovered with molecular tools has been also published about the presence of these fungi in the microbiome of truffle fruiting bodies. In the present work, we isolated and identified three Fusarium strains from truffle fruiting bodies. All isolates were assigned to the same species, F. commune, and the strains were deposited in the All-Russian Collection of Microorganisms under accession numbers VKM F-5020, VKM F-5021, and VKM F-5022. To check the possible effects of the isolated strains on the plants, the isolates were used to infect sterile seedlings of Sarepta mustard (Brassica juncea L.). This model infection led to a moderate suppression of the photosynthetic apparatus activity and plant growth. Here, we present characteristics of the F. commune isolates: description of the conidial morphology, pigmentation, and composition of the mycelium fatty acids. Overall, this is the first description of the Fusarium cultures isolated from truffle fruiting bodies. Possible symbiosis of the F. commune strains with truffles and their involvement in the cooperative fatty acid production are proposed.

Most of the currently known fungal laccases show their maximum activity under acidic environmental conditions. It is known that a decrease in the activity of a typical laccase at neutral or alkaline pH values is the result of an increase in the binding of the hydroxide anion to the T2/T3 copper center, which prevents the transfer of an electron from the T1 Cu to the trinuclear copper center. However, evolutionary pressure has resolved the existing limitations in the catalytic mechanism of laccase, allowing such enzymes to be functionally active under neutral/alkaline pH conditions, thereby giving fungi an advantage for their survival. Combined molecular and biochemical studies, homological modeling, calculation of the electrostatic potential on the Connolly surface at pH 5.0 and 7.0, and structural analysis of the novel alkaliphilic laccase of Myrothecium roridum VKM F-3565 and alkaliphilic and acidophilic fungal laccases with a known structure allowed a new intramolecular channel near the one of the catalytic aspartate residues at T2-copper atom to be found. The amino acid residues of alkaliphilic laccases forming this channel can presumably serve as proton donors for catalytic aspartates under neutral conditions, thus ensuring proper functioning. For the first time for ascomycetous laccases, the production of new trimeric products of phenylpropanoid condensation under neutral conditions has been shown, which could have a potential for use in pharmacology.

The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

Basidiomycetes Armillaria infect deciduous, coniferous and fruit trees, causing enormous economic damage. The role of secondary metabolites (tricyclic sesquiterpene aryl esters - melleolides) in the life cycle and pathogenesis of Armillaria is under active investigation. To date, not all species of Armillaria have been tested for the biosynthesis of melleolides. We investigated the secondary metabolite profiles of six root-pathogenic species of the genus Armillaria (A. borealis Marxmüller & Korhonen, A. cepistipes Velenovský, A. gallica Marxm, A. mellea (Vahl) P. Kummer, A. sinapina Bérubé & Dessur, A. ostoyae (Romagn.) Herink) distributed in Siberia (South Krasnoyarsk Krai, Republic of Tyva, Republic of Khakassia, Taimyr Peninsula), Russian Far East (Sikhote-Alin) and Crimea (Krymsky National Park, Chatyr-Dag Mountain Lower Plateau). A total of 15 compounds were identified in the metabolome profile. Two compounds (melleolide D and melledonal C) are synthesized by all investigated strains irrespective of their geographic location and host plant. The maximum spectrum of melleolides (7-8 compounds) was found in isolates of A. borealis, A. gallica, A. sinapina, A. ostoyae. In submerged culture, the maximum accumulation of melleolides varied from 2 up to 239 mg l-1. A mixture of melleolide D and melledonal C (1:1) synthesized by the most productive strain A. mellea Cr2-17 was first found to have a phytotoxic action on the growth parameters of the callus culture Populus balsamifera and 10-day-old conifer seedlings. A 0.5% concentration of melleolides caused a credible decrease of P. balsamifera callus raw biomass; a decrease of the viability of Larix sibirica and, which is especially significant, Pinus sylvestris seedlings; inhibition of stem and root growth processes; dechromation of foliage; loss of turgor. The occurrence of a broad range of melleolides in the metabolome profile and two common compounds in all investigated strains, with a phytotoxic action at their sufficiently high concentration, enables considering the synthesis of melleolides by Armillaria fungi as one of the possible mechanisms of their pathogenicity efficiently realized in strains characterized by overproduction of melleolides under natural conditions.

A new unusual biotechnologically valuable laccase from the ascomycete Myrothecium (Paramyrothecium) roridum VKM F-3565 with a molecular weight of about 80 kDa, which exhibits the maximum oxidative activity against a number of phenolic compounds and phenylpropanoids under neutral-alkaline conditions (pH 6.5-7.8), was isolated and characterized. For the first time for laccases of ascomycetes, the production of new trimeric products of ferulic acid and coniferyl alcohol condensation has been shown, which presumably have a potential for use in pharmacology. The gene encoding the unusual alkaliphilic laccase has been isolated for the first time from fungi of the Myrothecium ( Paramyrothecium) genus. The high phylogenetic relationship between the M. roridum VKM F-3565 laccase gene and genes presumably encoding the laccases of ascomycetes of the genera of Colletotrichum, Verticillium, and Fusarium was shown. Homological modeling, calculation of the electrostatic potential on the molecular surface at pH5.0 and 7.0, and structural analysis of the studied laccase as well as the fungal alkaliphilic and acidophilic laccases with a known structure allowed finding a new intramolecular channel, different from the other known internal channels, located near the catalytic aspartate residue at the T2-copper atom. The amino acid residues of alkaliphilic laccases forming the found channel presumably serve as proton donors for the catalytic aspartate, ensuring its proper functioning.

Due to the ever-increasing demand for healthy and safe food, much attention has been gained by natural food colorants. This study showed the culture fluid extract of the fungus Aspergillus cavernicola VKM F-906 to contain red pigment and monasnicotinic acid (MNA) in predominant amounts. The structure of the pigment corresponded to cis-cavernamine (red pigment, RP). Two tautomers, NH and OH forms, in rapid equilibrium were present in a solution of RP. The critical factors for RP to form were the presence of NH4+ salt and pH 6.3-6.5. In vitro experiments showed that MNA was synthesized from RP as a result of chemical transformations without the participation of enzymes. In this case, the main influence on the reaction rate is exerted by the pH of the medium, which is associated with the keto-enol tautomerism of RP in solution. The culture broth extract and MNA exhibited antifungal activity against Fusarium fungi.

The new ascomycete Microthielavia ovispora VKM F-1735 has been found to produce oxidases with laccase activity capable to decolorize many industrial dyes in the absence of any mediators. The optimal conditions of submerged cultivation of the fungus for production of the oxidases decolorizing dyes were developed. Two unusual homodimeric oxidases with laccase activity and different physicochemical properties could be purified from the fungal culture liquid. The purified oxidases possessed individual selectivity in relation to different laccase substrates. The oxidases I and II (147 and 160 kDa, respectively) were most active under acidic conditions at elevated temperatures of 64 and 66 degrees C, correspondingly. The activation phenomena for the fungal oxidases during incubation in the presence of nonionic surfactants or K+ and Co2+ ions was shown. The dynamics of decolorization of malachite green in various concentrations by the fungus oxidases was studied. Based on the identified intermediates the scheme of the dye transformation by the oxidases has been presented. Being a nontoxic protein component exhibiting the decolorizing activity against a wide range of industrial dyes without additional expensive mediators, the M. ovispora VKM F-1735 oxidase has great potential for the development of new effective "green" technologies for bioremediation of water resources and soils.

Naphthalene, as a component of crude oil, is a common environmental pollutant. Biochemical and genetic aspects of naphthalene catabolism have been examined in most detail in the bacteria of Pseudomonas genus. In pseudomonads, the key intermediate in naphthalene degradation is salicylate. In this study, we investigated the ability of Rhodococcus opacus strain 3D to utilize naphthalene as a sole carbon and energy source. The characteristic feature of this strain is the inability to grow in the mineral medium supplemented with salicylate (typical intermediate of naphthalene degradation in Gram-negative bacteria). The absence of salicylate hydroxylase activity and salicylate accumulation in the course of R. opacus 3D cultivation in the mineral medium supplemented with naphthalene indicated existence of an alternative pathway of naphthalene oxidation. At the same time, R. opacus 3D was able to use monoaromatic compounds (salts of gentisic, ortho-phthalic, and 2-hydroxycinnamic acids and coumarin) as growth substrates. Based on the analysis of enzymatic activities, identification of the reaction intermediates, genetic determinants, and growth substrates, we concluded that R. opacus 3D carries out naphthalene degradation through an alternative pathway via formation of ortho-phthalic acid, which is untypical for pseudomonads. Using mass spectrometry, we showed for the first time that salicylic acid associate formed in trace amounts in the process of naphthalene degradation is not further metabolized and accumulated in the growth medium in a form of a dimer.

The composition of secondary metabolites from strains Heterobasidion genus isolated in central and western Siberia and in South Korea were studied. Morphological–cultural and molecular-genetic methods were used to assign the cultures to the species of H. annosum (Fr.) Bref. (five strains), H. abietinum Niemelä & Korhonen (four strains), and H. ecrustosum Tokuda, T. Hatt. & Y.C. Give (one strain). Fomannoxin predominated in the metabolome profiles of three H. annosum strains and all H. abietinum strains. Two strains of H. annosum synthesized fomannoxin-related compounds: 2-(2-hydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde and 2-(2-hydroxypropan-2-yl)benzofuran-5-carbaldehyde. Fomannosin and its precursors were identified in H. annosum 45-2. It was shown that the composition of the fermentation medium affected the number and range of the synthesized metabolites. Under in vitro conditions, all H. annosum and H. abietinum strains exhibited phytopathogenic effects on Pinus sylvestris L. seedlings, causing necrotic damage to stems of various degrees and plant death. Higher phytopathogenicity is characteristic of H. annosum strains: maximal aggressiveness was observed in H. annosum 45-2 with the greatest diversity of compounds in the metabolome profile and active fomannoxin production.

Three laccase isoforms with different physicochemical properties could be purified from culture liquid of basidiomycete Lentinus strigosus 1566 obtained during submerged cultivation. The purified laccases possessed individual selectivity in relation to different phenolic compounds. Laccases I, II, and III (59, 65, and 61 kDa respectively) were more active in acidic conditions at around 70 degrees C. However, in contrast to laccases I and II, laccase III retained its activity (8-30%) and stability during at least one week of incubation at neutral conditions that allows its biotechnological application carried out at neutral environment. The activation phenomena for some of the purified laccases from L. strigosus 1566 during incubation at high temperature, different pH, and sulfates is shown and discussed. According to MALDI-TOF analysis, laccases I and II are most closely related to the laccase of Panus rudis (AAR13230). Transformation of phenylpropanoids by the predominant laccases of L. strigosus 1566 to different polymers was demonstrated, indicating a great potential for producing novel pharmaceutical valuable analogues of lignans, stilbenes, flavonoids, and etc.. The studied laccases, which are products of the same strain, can become a convenient model for further studies of the structural mechanisms of the shift of T-/pH-optima, activation, and T-/pH-stability. (C) 2019 Elsevier B.V. All rights reserved.

Secondary metabolites of the basidiomycetes of Armillaria borealis Marxm. and Korhonen, A. cepistipes Velen., A. gallica Marxm., A. ostoyae (Romagn.) Herink, and A. sinapina Bérubé and Dessur isolated in Southern Siberia (Krasnoyarsk region and Tyva Republic) and in the Far East (Sikhote-Alin) were studied. Metabolites belonging to the class of protoilludene sesquiterpene aryl esters of the melleolides group have been identified in the species A. borealis, A. cepistipes, and A. sinapina. The strains differ in the spectrum of synthesized melleolides. A. borealis strain 74g synthesized a wider range of melleolides than other strains of the species: melleolides B, C, D and H, melledonals B and C, 5'-O-methylmelledonal, 13-hydroxy-5'-O-methylmelledonal, and armillarinin. It was shown that the composition of the medium influenced the amount of synthesized metabolites. All of the studied strains synthesizing melleolides exhibited a toxicogenic and phytopathogenic effect on seeds and seedlings of Picea abies (L.) H. Karst. and Abies sibirica Ledeb. A significant decrease in seed germination energy, laboratory and ground seed germination, the development of the root system of conifer seedlings was revealed. The maximum inhibitory effect was shown by the culture fluid of A. borealis 74g with the greatest amounts and variety of melleolides in the metabolome profile.

Mass spectrometry was applied to identify metabolites and estimate the efficiency of phenanthrene biodegradation and transformation by rhizosphere bacteria Pseudomonas aureofaciens BS1393. Strains P. aureofaciens BS1393(pOV17) and P. aureofaciens BS1393(NPL-41) bearing various naphthalene biodegradation plasmids were used in the work. The strain BS1393(pOV17) contains the pOV17 wild type naphthalene biodegradation plasmid that determines the oxidation of naphthalene to Krebs cycle metabolites. The strain BS1393(NPL-41) contains the mutant plasmid NPL-41 governing the initial stages of naphthalene oxidation into salicylic acid. The limiting stages of phenanthrene biodegradation in bacteria with various plasmids have been identified according to the accumulation of intermediates. When bacteria were grown on phenanthrene, the main metabolites were as follows: (a) 2-hydroxy-2H-benzo[h]chromene-2-carboxylic acid/trans-4-(1-hydroxynaph-2-yl)-2-oxobut-3-enoic acid, (b) 1-hydroxy-2-naphthoic acid, and (c) salicylic acid. In the strain BS1393(pOV17), metabolite (а) was observed during 1–14 days of cultivation. Unlike it, in the strain BS1393(NPL-41), an insignificant amount of this metabolite was found after only 14 days. The availability of metabolite (b) in the growth of both strains was an evidence of the limited rate of its further decarboxylation. Metabolite (c) as a final product was found in the growth of the strain BS1393(NPL-41). Contrastingly, in the strain BS1393(pOV17), this metabolite was not found, which indicates the complete oxidation of phenanthrene.