Relation between lignin molecular profile and fungal exo-proteome during kraft lignin modification by Trametes hirsuta LE-BIN 072.

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).

Fungal Adaptation to the Advanced Stages of Wood Decomposition: Insights from the Steccherinum ochraceum.

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.

The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism.

Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X-ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X-ray diffraction data sets were collected with increasing doses of absorbed X-ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccase crystal confirmed the previously deduced enzymatic mechanism of molecular oxygen reduction. The structures of the complexes allowed the localization of chloride and fluoride ions in the channel near the T2 copper ion. These ions replace the oxygen ligand of the T2 copper ion in this channel and can play the role of this ligand in the enzymatic reaction. As follows from analysis of the structures from the increasing dose series, the inhibition of laccases by chloride and fluoride anions can be explained by the fact that the binding of these negatively charged ions at the position of the oxygen ligand of the T2 copper ion impedes the reduction of the T2 copper ion.

Biotransformation of progesterone by Aspergillus nidulans VKPM F-1069 (wild type).

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 6ß,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 6ß,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.

Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta.

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.

White-rot basidiomycetes Junghuhnia nitida and Steccherinum bourdotii: Oxidative potential and laccase properties in comparison with Trametes hirsuta and Coriolopsis caperata.

White-rot basidiomycetes from the poorly studied residual polyporoid clade of Polyporales order Junghuhnia nitida (Pers.) Ryvarden and Steccherinum bourdotii Saliba & A. David grow as secondary xylotrohps on well decomposed woody materials. The main objective of the current study was to compare oxidative potential, growth, production of oxidative enzymes and laccase properties of J. nitida and S. bourdotii with that of typical primary xylotrohps Trametes hirsuta (Wulfen) Lloyd and Coriolopsis caperata (Berk.) Murrill, belonging to the core polyporoid clade. For the first time we report species J. nitida and S. bourdotii as active laccase producers. New laccases from J. nitida and S. bourdotii were purified and characterized. They had an identical molecular weight of 63 kDa and isoelectric points of 3.4 and 3.1, respectively. However, the redox potential of the T1 copper site for both J. nitida (610 mV) and S. bourdotii (640 mV) laccases was lower than those for T. hirsuta and C. caperata laccases. The new laccases showed higher temperature optima and better thermal stability than T. hirsuta and C. caperata laccases. Their half-lives were more than 40 min at 70 °C. The laccases from J. nitida and S. bourdotii showed higher affinity to syringyl-type phenolic compounds than T. hirsuta and C. caperata laccases. The oxidative potential of studied fungi as well as the properties of their laccases are discussed in terms of the fungal life-style.

Orchestration of the expression of the laccase multigene family in white-rot basidiomycete Trametes hirsuta 072: Evidences of transcription level subfunctionalization.

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.

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family.

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.

Draft Genome Sequence of the Fungus Trametes hirsuta 072.

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).

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

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.

Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes.

Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.