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Despite the widespread use of the COVID-19 vaccines, the search for effective antiviral drugs for the treatment of patients infected with SARS-CoV-2 is still relevant. Genetic variability leads to the continued circulation of new variants of concern (VOC). There is a significant decrease in the effectiveness of antibody-based therapy, which raises concerns about the development of new antiviral drugs with a high spectrum of activity against VOCs. We synthesized new analogs of uracil derivatives where uracil was substituted at the N

A novel Aspergillus nidulans lac№4 (argB–) strain, a producer of recombinant laccase A from basidiomycete Trametes hirsuta 072, was obtained. This strain was used for the biocatalytic transformation of progesterone (PG). The major biotransformation products are 11α-hydroxy-PG, 11α-acetoxy-PG and 6β, 11α-dihydroxy-PG. Macronet MN-200 sorbent was used to study the adsorption kinetics of PG, and the main biotransformation products from the transformation medium. The solid-phase extraction of steroids without preliminary removal of the mycelium was shown to be efficient.

Abscess formation is a common complication of severe life-threatening infections caused by obligate anaerobes. Fusobacterium necrophorum is among the frequently detected anaerobic pathogens from clinical specimens associated with liver abscesses, skin and soft tissue infections, or oral abscesses. The antimicrobial therapy for this kind of infection needs to be optimized. Here, we examined the possibility of treating F. necrophorum-induced abscess wound infections with candidate therapeutics based on three endolysins with activity against a broad spectrum of aerobe Gram-negative pathogens. Antibacterial gel containing three Gram-negative bacteria-targeting endolysins, LysAm24, LysAp22, and LysECD7, was formulated for topical use. Abscess formation was induced in rabbits with F. necrophorum and caused systemic infection. The survival and lifespan of the animals, general parameters, and biochemical and hematological blood tests were analyzed to assess the effectiveness of the gel treatment for the wound infection. The administration of the investigated gel twice per day for 5 days resulted in less acute inflammation, with decreased leukocytes and segmented neutrophils in the blood, retardation of infection progression, and an almost two-fold increase in the lifespan of the animals compared to the placebo group. The results indicate that endolysin-based therapy is an effective approach to treat anaerobic bacterial infections. The use of endolysins as independent pharmaceuticals, or their combination with antibiotics, could significantly reduce the development of complications in infectious diseases caused by sensitive bacterial species.

Laccases (EC 1.10.3.2) are multicopper polyphenol oxidases found in various organisms, in particular, in fungi. Fungal laccases are encoded by multigene families, which can contain up to 17 genes. However, not all isozymes can be obtained from native producers. Previous studies have shown that the filamentous fungus Penicillium canescens is a promising object for the heterologous expression of various laccase isozymes of Trametes hirsuta 072. In this work, the cultivation conditions of P. canescens strains, recombinant producers of T. hirsuta heterologous minor laccase, are optimized. The optimization of the rLacD and rLacF purification method increased their yield and specific activity of rLacD. In addition, the melting points of minor laccase isoenzymes are measured and the glycosylation of isoenzymes is studied.

It is well-known that various oxidative enzymes of wood-degrading fungi are widely demanded in various fields of biotechnology. Laccases attract particular attention, since they have different substrate specificities and can be used not only in pulp and paper industry, but also for the degradation of various xenobiotics, including decolorization of dyes. In this work, a comparative analysis of the native major (LacA) and the recombinant minor (rLacC, rLacD, rLacF) Trametes hirsuta 072 laccase isoenzymes applicability for bleaching of four widely used recalcitrant dyes was conducted. It was shown that all isoenzymes decolorized dyes to different extent, and the minor isoenzymes rLacD and rLacF are the most promising for decolorization of congo red and phenol red dyes. Moreover, we provide detailed exoproteome analysis of T. hirsuta 072 growing on the media supplemented with compounds that were previously shown to increase transcription levels of the minor laccase isoenzymes.

Background First vaccines for prevention of Coronavirus disease 2019 (COVID‐19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti‐SARS‐CoV‐2 effect of siRNA both in vitro and in vivo. Methods To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS‐CoV‐2 genes fused with the firefly luciferase reporter gene and SARS‐CoV‐2‐infected cells was used. The most potent siRNA, siR‐7, was modified by Locked nucleic acids (LNAs) to obtain siR‐7‐EM with increased stability and was formulated with the peptide dendrimer KK‐46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation – siR‐7‐EM/KK‐46. Using the Syrian Hamster model for SARS‐CoV‐2 infection the antiviral capacity of siR‐7‐EM/KK‐46 complex was evaluated. Results We identified the siRNA, siR‐7, targeting SARS‐CoV‐2 RNA‐dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA‐modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR‐7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR‐7‐EM/KK‐46 in vivo. Conclusions Thus, we developed a therapeutic strategy for COVID‐19 based on inhalation of a modified siRNA‐peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID‐19 patients., siR‐7, targeting SARS‐CoV‐2 RdRp inhibits viral replication in vitro. LNA‐modification and complexation with peptide dendrimer KK‐46 enhanced the antiviral capacity of siR‐7 in vitro. Inhalation exposure of SARS‐CoV‐2‐infected Syrian Hamsters with siR‐7‐EM/KK‐46 reduces the virus titer and lung inflammation. ​

The process of kraft lignin modification by the white-rot fungus Trametes hirsuta was investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS), and groups of systematically changing compounds were delineated. In the course of cultivation, fungus tended to degrade progressively more reduced compounds and produced more oxidized ones. However, this process was not gradual – the substantial discontinuity was observed between 6th and 10th days of cultivation. Simultaneously, the secretion of ligninolytic peroxidases by the fungus was changing in a cascade manner – new isoenzymes were added to the mixture of the already secreted ones, and once new isoenzyme appeared both its relative quantity and number of isoforms increased as cultivation proceeded. It was proposed, that the later secreted peroxidases (MnP7 and MnP1) possess higher substrate affinity for some phenolic compounds and act in more specialized manner than the early secreted ones (MnP5 and VP2).

The spread of bacterial strains resistant to commonly used antibiotics urges the development of novel antibacterial compounds. Ideally, these novel antimicrobials should be less prone to the development of resistance. Peptidoglycan-degrading enzymes are a promising class of compounds with a fundamentally different mode of action compared to traditionally used antibiotics. The difference in the mechanism of action implies differences both in the mechanisms of resistance and the chances of its emergence. To critically assess the potential of resistance development to peptidoglycan-degrading enzymes, we review the available evidence for the development of resistance to these enzymes

Surfaces of implanted medical devices are highly susceptible to biofilm formation. Bacteria in biofilms are embedded in a self-produced extracellular matrix that inhibits the penetration of antibiotics and significantly contributes to the mechanical stability of the colonizing community which leads to an increase in morbidity and mortality rate in clinical settings. Therefore, new antibiofilm approaches and substances are urgently needed. In this paper, we test the efficacy of a broad-range recombinant endolysin of the coliphage LysECD7 against forming and mature biofilms. We used a strong biofilm producer&mdash, Klebsiella pneumoniae Ts 141-14 clinical isolate. In vitro investigation of the antibacterial activity was performed using the standard biofilm assay in microtiter plates. We optimized the implantable diffusion chamber approach in order to reach strong biofilm formation in vivo avoiding severe consequences of the pathogen for the animals and to obtain a well-reproducible model of implant-associated infection. Endolysin LysECD7 significantly reduced the biofilm formation and was capable of degrading the preformed biofilm in vitro. The animal trials on the preformed biofilms confirmed these results. Overall, our results show that LysECD7 is a promising substance against clinically relevant biofilms.

In the present article, we report data on the whole-genome sequencing of wood-rotting (white-rot) fungus Steccherinum ochraceum LE-BIN 3174. The S. ochraceum LE-BIN 3174 genome consists of 770 scaffolds (N50 = 62,812 bp) with the total length of assembly ∼35 Mb. The structural annotation of the genome resulted in the prediction of 12,441 gene models, among which 181 were models of tRNA-coding genes, and 12,260 – protein-coding genes. The protein-coding genes were annotated with different databases (Pfam, InterPro, eggNOG, dbCAN, and MEROPS). The whole genome sequence and functional annotation provide an important information for the deep investigation of biochemical processes that take place during the late stages of wood decomposition by Basidiomycetes. The Whole Genome project of S. ochraceum LE-BIN 3174 had been deposited at DDBJ/ENA/GenBank under the accession RWJN00000000. The version described in this work is version RWJN00000000.1. For further interpretation of the data provided in this article, please refer to the research article “Fungal Adaptation to the Advanced Stages of Wood Decomposition: Insights from the Steccherinum ochraceum” [1]. Keywords: Steccherinum ochraceum, Draft genome sequence, White-rot, Wood decay

The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall.

Approximately 1/6 of humanity is at high risk of experiencing cholera epidemics. The development of effective and safe vaccines against Vibrio cholerae, the primary cause of cholera, is part of the public health measures to prevent cholera epidemics. Natural nontoxigenic V. cholerae isolates represent a source of new genetically improved and relatively safe vaccine strains. However, the genomic engineering of wild-type V. cholerae strains is difficult, and these strains are genetically unstable due to their high homologous recombination activity. We comprehensively characterized two V. cholerae isolates using genome sequencing, bioinformatic analysis, and microscopic, physiological, and biochemical tests. Genetic constructs were Gibson assembled and electrotransformed into V. cholerae. Bacterial colonies were assessed using standard microbiological and immunological techniques. As a result, we created a synthetic chromoprotein-expressing reporter operon. This operon was used to improve the V. cholerae genome engineering approach and monitor the stability of the genetic constructs. Finally, we created a stable candidate V. cholerae vaccine strain bearing a recA deletion and expressing the β-subunit of cholera toxin. Thus, we developed a strategy for the rapid creation of genetically stable and relatively safe candidate vaccine strains. This strategy can be applied not only to V. cholerae but also to other important human bacterial pathogens.

The SARS-CoV-2 pandemic remains a global health issue for several reasons, such as the low vaccination rates and a lack of developed herd immunity to the evolution of SARS-CoV-2, as well as its potential inclination to elude neutralizing antibodies. It should be noted that the severity of the COVID-19 disease is significantly affected by the presence of co-infections. Comorbid conditions are caused not only by pathogenic and opportunistic microorganisms but also by some representatives of the environmental microbiome. The presence of patients with moderate and severe forms of the disease in hospitals indicates the need for epidemiological monitoring of (1) bacterial pathogens circulating in hospitals, especially the ESKAPE group pathogens, and (2) the microbiome of various surfaces in hospitals. In our study, we used combined methods based on PCR and NGS sequencing, which are widely used for epidemiological monitoring. Through this approach, we identified the DNA of pathogenic bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, CoNS, and Achromobacter spp.) on various surfaces. We also estimated the microbiome diversity of surfaces and identified the potential reservoirs of infections using 16S rRNA profiling. Although we did not assess the viability of identified microorganisms, our results indicate the possible risks of insufficient regular disinfection of surfaces, regardless of department, at the Infectious Diseases Hospital. Controlling the transmission of nosocomial diseases is critical to the successful treatment of COVID-19 patients, the rational use of antimicrobial drugs, and timely decontamination measures.

Utilization of laccases in biotechnology and bioremediation has created a strong demand for the characterization of new enzymes and an increase in production of known laccases. Thus, additional research into these enzymes is critically needed. In this study, we report a comparative study of the biochemical and transcriptional properties of two different laccase isozymes from Trametes hirsuta 072 – the constitutive and inducible forms. A recombinant LacC enzyme was expressed in Penicillium canescens to characterize its properties. LacC is single-purpose enzyme, unlike LacA, which can operate efficiently under a wide range of temperatures and pHs (55–70 °C and pH 3–5, respectively). LacC has a lower RedOx potential than LacA and does not oxidize substrates containing amine groups. Expression of the lacC gene was selective compared to that of the lacA gene and increased significantly in the presence of complex synthetic compounds such as dyes and xenobiotics. This study shows that laccases from the multigene families of basidiomycetes differ significantly in their properties, thus providing a complementary effect during lignin degradation.

Steccherinum ochraceum is a white rot basidiomycete with wide ecological amplitude. It occurs in different regions of Russia and throughout the world, occupying different climatic zones. S. ochraceum colonizes stumps, trunks, and branches of various deciduous (seldom coniferous) trees. As a secondary colonizing fungus, S. ochraceum is mainly observed at the late decay stages. Here, we present the de novo assembly and annotation of the genome of S. ochraceum, LE-BIN 3174. This is the 8th published genome of fungus from the residual polyporoid clade and the first from the Steccherinaceae family. The obtained genome provides a first glimpse into the genetic and enzymatic mechanisms governing adaptation of S. ochraceum to an ecological niche of pre-degraded wood. It is proposed that increased number of carbohydrate-active enzymes (CAZymes) belonging to the AA superfamily and decreased number of CAZymes belonging to the GH superfamily reflects substrate preferences of S. ochraceum. This proposition is further substantiated by the results of the biochemical plate tests and exoproteomic study, which demonstrates that S. ochraceum assumes the intermediate position between typical primary colonizing fungi and litter decomposers or humus saprotrophs. Phylogenetic analysis of S. ochraceum laccase and class II peroxidase genes revealed the distinct evolutional origin of these genes in the Steccherinaceae family.

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy - AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family.

Here we present the results of the exploration of laccase multigene families (MGFs) in basidiomycetous fungi from different taxonomic groups using a next generation sequencing (NGS) technology. In our study, multiple laccase genes were identified in all of the investigated fungi (13 species) from Polyporaceae, Phanerochaetaceae, Meruliaceae, Pleurotaceae, Physalacriaceae, and Peniophoraceae families. It was shown that phylogenetic positioning of the newly identified sequences exhibit patterns of clusterization with respect to enzyme properties. This can be a potentially useful tool for selecting naturally existing laccases with different physicochemical characteristics relevant to different biotechnological applications. Moreover, the method developed in this study can be used in the screening of environmental samples and fast characterization of laccase MGFs in newly identified fungal species.

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5–7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; β-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of β-carotene and quercetin by 18–24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of β-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of β-carotene and quercetin.

GamTBvac is a candidate tuberculosis vaccine with two fusion proteins, containing Ag85a, ESAT6, CFP10, and a dextran-binding domain (DBD). Phase II of a double-blind, randomized, multicenter, placebo-controlled study in parallel groups in healthy adults to evaluate the safety and immunogenicity of GamTBvac in 180 previously-vaccinated with Bacillus Calmette&ndash, Gu&eacute, rin vaccine (BCG) healthy volunteers without Mycobacterium tuberculosis (MTB) infection was conducted. The dose (0.5 mL) of either the study drug or a placebo was administered subcutaneously twice with an 8-week interval. At eight timepoints from 14 to 150 days, whole blood and sera were assayed. Antigen-specific T-cell responses were measured by an in-house interferon-gamma release assay (IGRA-test), the QuantiFERON (QTF) test, and intracellular cytokine staining (ICS). For antibody response detection, the bead-based multiplex immunoassay (MIA) was applied. The vaccine confirmed an acceptable safety profile previously shown in a first-in-human clinical study. After stimulation with both fusions, the highest median level of INF-&gamma, was detected on day 21. The GamTBvac vaccine induced antigen-specific interferon-gamma release, Th1 cytokine-expressing CD4+ T-cells, and IgG responses and results support further clinical testing of GamTBvac.

European seabass (Dicentrarchus labrax, Linnaeus, 1758) (L) and gilthead seabream (Sparus aurata, Linnaeus, 1758) (C) muscles were hydrolysated by Alcalase (Lalc, Calc) and Chymotrypsin (Lch, Cch) then hydrolysates were examined and their peptide profiles obtained. A total of 765, 794, 132 and 232 peptides were identified in Calc, Lalc, Cch and Lch, respectively. Although, Lch and Cch were expected to have more antioxidant capacity because of their peptide profiles, Alcalase hydrolysates observed in vitro, were slightly higher (TEAC assay for Calc: 848.11 ± 60.78 μmol TE/g protein). Maximum inhibition of oxidative stress was determined for Lalc (12.8% ± 4.5%) in MDCK1 cell lines. Highest proliferative capacity observed for Calc (147.0% ± 3.1%) at MTT assay in MDCK1 cell culture. Lch showed the highest chemopreventive effect with a 40–60% decrease for human colon adenocarcinoma cell line HT-29. This research points out the importance of aquatic sources as raw materials for peptide researches.

The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6β,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6β-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

Laccases, blue copper-containing oxidases, ≿an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is an enzyme that has been studied for over 100 y and is present in virtually all fungi. As increasing numbers of fungal genomes have been sequenced, it has become apparent that the laccase genes in white-rot fungi commonly form multigene families consisting of many nonallelic genes. Although a number of reports focussing on laccase gene expression in different fungal species were published over the decades, the fundamental questions of why fungi need such a redundant array of genes and how they manage this array to perform biological function(s) remain far from answered. In this article, we present a comprehensive study of the transcription of the whole Trametes hirsuta laccase multigene family under different conditions, including exposure to different nutritional factors such as nitrogen sources (organic and inorganic) and concentrations of nitrogen and carbon in the culture medium; in different growth phases (lag phase and stationary phase); and in the presence of different inducer agents (water-soluble lignin, bromocresol green dye, p-coumaric acid, ferulic acid, guaiacol, vanillin, veratryl alcohol, vanillic acid and syringic acid). Our findings are discussed in the context of the evolution of the laccase multigene family, and the presence of transcription-level subfunctionalization is highlighted.

Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli. Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross-reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase-producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed.

Biotechnological transformation of steroids using enzyme systems of microorganisms is often the only possible method to modify the molecule in the industrial production of steroid drugs. Filamentous fungus Aspergillus nidulans has been little studied as a steroid-transforming microorganism. We studied the ability of the A. nidulans VKPM F-1069 strain to transform progesterone (PG) for the first time. This strain converts PG into 3 main products: 11α-hydroxy-PG, 11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It has been established that in the first stage, the hydroxylation of PG occurs into C11α position, then the formed 11α-hydroxy-PG is modified into 11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It was found that changes in the composition of the growth medium, aeration and the duration of the mycelium cultivation do not affect the qualitative composition of PG transformation products, but their ratios have changed. Under conditions of limited aeration, the direction of secondary modification of 11α-hydroxy-PG is shifted towards the formation of 11α-acetoxy-PG.

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.

Background Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. Results This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus. The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-β-1,3-glucanase and α-amylase and turned on secretion of endo-β-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with lignocellulose, thus indicating a specific role of these proteins in degradation of the lignocellulose substrates. Conclusions Our results suggest a sequential mechanism of natural substrate degradation by T. hirsuta, in which the fungus produces different sets of enzymes to digest all main components of the substrate during cultivation. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0729-0) contains supplementary material, which is available to authorized users.

Here we present the results of the exploration of laccase multigene families (MGFs) in basidiomycetous fungi from different taxonomic groups using a next generation sequencing (NGS) technology. In our study, multiple laccase genes were identified in all of the investigated fungi (13 species) from Polyporaceae, Phanerochaetaceae, Meruliaceae, Pleurotaceae, Physalacriaceae, and Peniophoraceae families. It was shown that phylogenetic positioning of the newly identified sequences exhibit patterns of clusterization with respect to enzyme properties. This can be a potentially useful tool for selecting naturally existing laccases with different physicochemical characteristics relevant to different biotechnological applications. Moreover, the method developed in this study can be used in the screening of environmental samples and fast characterization of laccase MGFs in newly identified fungal species.

A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 ( Basidiomycota , Polyporales ) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.

Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO₄ were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO₄ as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (β-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.

Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli. Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross-reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase-producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed.