ABSTRACTBiofilm formation is a key survival strategy among microorganisms and a major factor contributing to chronic and treatment‐resistant infections. Staphylococcus aureus is a key biofilm‐forming pathogen associated with persistent and life‐threatening infections. As part of our ongoing search for novel anti‐biofilm agents from Basidiomycota of tropical rainforests, we investigated the metabolome of the African fungus Neonothopanus nambi. This study led to the isolation of four dimeric aristolane‐type sesquiterpenoids, aurisins D, B, A, and G (1–4), along with two monomeric sesquiterpenoids, nambinone C (5) and axinysone B (6), and methyl 4‐butyramidobenzoate (7). All compounds were assessed for their antimicrobial and cytotoxic activities, as well as their ability to inhibit and eradicate S. aureus biofilms. The dimeric sesquiterpenoids (1–4) exhibited potent antibiofilm effects, with aurisin B (2) inhibiting biofilm formation by over 70% at a concentration of 1 µg/mL, well below its minimum inhibitory concentration. Confocal laser scanning microscopy further confirmed the pronounced anti‐biofilm effects of aurisins D and B (1 and 2). Structure–activity relationship analysis suggests that both dimerization and hydroxylation contribute to enhanced activity. Despite some cytotoxic effects, these dimeric aristolane‐type sesquiterpenoids from N. nambi represent promising leads for the development of novel anti‐infective strategies targeting S. aureus biofilms, with potential applications beyond systemic use.

In vivo studies have revealed that the addition of caffeic acid and a low-molecular-weight compound, a bioluminescence stimulator, discovered in our research, to the mycelium of the luminous fungus Neonothopanus nambi leads to a rapid and significant (by an order of magnitude or more) increase in the intensity of its light emission. It has been suggested that the observed effect of activation of fungal bioluminescence may be mediated by the oxidation of added substances by enzymes of the ligninolytic complex of basidiomycetes (in particular, peroxidases) with the emission of visible light quanta. Parallel in vivo experiments showed that additions of hispidin (a luciferin precursor in the luminous higher fungi) have no effect on the light emission intensity of the mycelium. At the same time, in vitro studies have reported that caffeic acid and the detected lowmolecular luminescence stimulator do not affect the level of light emission of the enzyme luminescent system isolated from the N. nambi mycelium in the presence of NADPH and significantly suppress the emission reaction of the system activated by NADPH and hispidin. A set of data collected demonstrate that different biochemical pathways present in the luminous higher fungi and different enzymes (or enzyme systems) and different substrates are involved in generation of visible light quanta.

An extracellular enzyme with oxidase activity was isolated from the mycelium of the higher fungus Neonothopanus nambi by mild treatment of the biomass with β-glucosidase. A substrate specificity and some properties of the isolated extracellular oxidase were studied in the present work. Experiments revealed that the extracellular oxidase exhibited activity with most phenolic compounds chosen as model substrates. It is important to note that the enzyme exhibited a catalytic function in the reactions without the addition of exogenous hydrogen peroxide and other mediators. The highest catalytic activity of the enzyme was observed with veratryl alcohol and dihydric phenols, hydroquinone and guaiacol. The enzyme showed lower activity with aromatic azo compounds (ABTS, DAB, o-dianisidine). In reactions with dihydric phenol resorcinol and monophenol, the enzyme efficiency was extremely low. The kinetic parameters of the enzymatic reactions with actively oxidized substrates were determined. The addition of a divalent metal ion chelator (EDTA) did not affect the activity of the enzyme, while the addition of the SH reagent (DTT) increased the catalytic efficiency of the studied oxidase. The totality of the data obtained indicates that the extracellular oxidase of the N. nambi fungus catalyzes the oxidation of a wide range of aromatic compounds under slightly acidic and neutral conditions without the addition of additional mediators (in particular, hydrogen peroxide). This creates the prerequisites for studying the applicability of the enzyme in biomedical analytics.

The present study reports experimental data on substrate specificity and some properties of the extracellular enzyme with oxidase activity isolated from the mycelium of the higher fungus Neonothopanus nambi IBSO 2391 by treating the biomass with \(\beta\) -glucosidase. Gel-filtration chromatography showed that molecular weight of the isolated enzyme was 80 kDa. Spectral analysis did not reveal any chromophore components in the enzyme. The extracellular oxidase of the basidiomycete N. nambi IBSO 2391 was active with most of the aromatic compounds chosen as model substrates. An important fact is that the enzyme exhibited catalytic activity with no hydrogen peroxide or any other mediators added to the reaction mixture. The highest activity of the enzyme was observed in reactions with veratryl alcohol and hydroquinone. In reactions with guaiacol and aromatic amines (diaminobenzidine, o-dianisidine), the level of activity of the extracellular oxidase was considerably lower – by a factor of 2.5–3.5. In reactions with resorcinol, phenol, and caffeic acid, the catalytic efficiency of the enzyme was no greater than 6% of its activity with veratryl alcohol. Kinetic parameters of enzymatic reactions were determined for the most efficiently oxidized substrates. The addition of the chelating agent of divalent metal ions (EDTA) did not affect the activity of the extracellular oxidase from the fungus N. nambi IBSO 2391, indicating the absence of divalent metal ions in the molecule of the enzyme. At the same time, addition of the SH reagent (DTT) increased catalytic efficiency of the enzyme. The study showed that the extracellular oxidase of the fungus N. nambi IBSO 2391 functions in wide ranges of temperature and pH of the reaction medium, showing the highest catalytic activity at temperatures between 22 and 35 °C and pH 6.0. Results obtained in the current study provide the basis for studying potential uses of the isolated enzyme in biomedical analytics and bioremediation.

Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus Neonothopanusnambi

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.

Some fungi are capable of bioluminescence. One of the intriguing bioluminescent mushrooms is Neonothopanus. This mushroom has large fruity body and can produce light throughout their entire body. Light is produced by the reaction of luciferase enzyme to its substrate, luciferin. The information of Neonothopanus luciferase gene is still limited. The aim of this study is to characterize Neonothopanus sp. luciferase cDNA. Samples were collected in Pesisir Selatan West Sumatera. Total RNA and LuzF and LuzR primers were used to amplify 830 bp Neonothopanus sp luciferase cDNA. It was found that the query cover of the luciferase cDNA of Neonothopanus sp. to N. nambi is 93%, and 37% for N. gardnery. Neonothopanus sp and N. nambi have different nucleotides at position 320 – 369, while Neonothopanus sp and N. gardnery just have similar nucleotides at position 9 - 322 pb. Phylogenetic analysis shows bootstrap value of Neonothopanus sp cDNA sequences and Neonothopanus nambi 100% and 99.5% with N. gardneri. This suggests that Neonothopanus sp. luciferase cDNA is closely related to Neonothopanus nambi and N. gardneri.

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self-sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red-light switch for tight regulation of metabolic pathways.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.

Activity of aurisin A isolated from Neonothopanus nambi against methicillin-resistant Staphylococcus aureus strains

Aurisin A (AA), an aristolane dimer sesquiterpene isolated from the luminescent mushroom Neonothopanus nambi, exhibits various biological and pharmacological effects. However, its poor solubility limits its use for further medicinal applications. This study aimed to improve the water solubility of AA via complexation with β-cyclodextrin (βCD) and its derivatives (2,6-di-O-methyl-βCD (DMβCD) and 2-hydroxypropyl-βCD (HPβCD). A phase solubility analysis demonstrated that the solubility of AA linearly enhanced with increasing concentrations of βCDs (ranked in the order of AA/DMβCD > AA/HPβCD > AA/βCD). Notably, βCDs, especially DMβCD, increased the thermal stability of the inclusion complexes. The thermodynamic study indicated that the complexation between AA and βCD(s) was a spontaneous endothermic reaction, and AA/DMβCD possesses the highest binding strength. The complex formation between AA and DMβCD was confirmed by means of FT-IR, DSC, and SEM. Molecular dynamics simulations revealed that the stability and compactness of the AA/DMβCD complex were higher than those of the DMβCD alone. The encapsulation of AA led to increased intramolecular H-bond formations on the wider rim of DMβCD, enhancing the complex stability. The antiproliferative activity of AA against A549 and H1975 lung cancer cells was significantly improved by complexation with DMβCD. Altogether, the satisfactory water solubility, high thermal stability, and enhanced antitumor potential of the AA/DMβCD inclusion complex would be useful for its application as healthcare products or herbal medicines.

A stimulator of light emission of the fungus was found in an aqueous extract from mycelium of the luminous basidiomycete Neonothopanus nambi after its treatment with β-glucosidase. The addition of the extract to the luminous mycelium increases the level of light emission from several times to 1.5 orders of magnitude or more. The luminescence stimulator is a low-molecular-weight thermostable compound: it is detected in the permeate after filtering the extract through a 10-kDa cutoff membrane and it retains the stimulating effect after heat treatment at 100°C for 5 min. In the absorption spectrum of the aqueous sample of the stimulator, two main peaks are observed in the shortwave region (205 and 260 nm) and a shoulder in the range of 350-370 nm can be seen. The luminescence stimulator exhibits blue fluorescence with an emission maximum at 440 nm when excited at 360 nm. It was established that the luminescence-stimulating component is not a substrate (or its precursor) of the luminescent system of the N. nambi fungus.

Seven undescribed scalarane sesterterpenoids, nambiscalaranes B–H (1–7), together with two known compounds, nambiscalarane (8) and aurisin A (9) were isolated from the cultured mycelium of the luminescent mushroom Neonothopanus nambi. Their structures were elucidated by thorough analysis of their 1D and 2D NMR spectroscopic data. The absolute configurations of 1–8 were determined by electronic circular dichroism (ECD) calculations and optical rotation measurements. The isolated sesterterpenoids were evaluated against A549, HT29, HeLa, and HCT-116 cancer cell lines, and against five bacterial strains. Compounds 3, 5, and 7 showed strong cytotoxicity against HCT-116 cell line, with IC50 values ranging from 13.41 to 16.53 µM, and showed no cytotoxicity towards Vero cells. Moreover, compound 8 inhibited the growth of Bacillus subtilis with a MIC value of 8 µg/mL, which was equivalent to the MIC value of the standard kanamycin.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

The extracellular enzyme with oxidase function was extracted from the Neonothopanus nambi luminescent fungus by using mild processing of mycelium with β-glucosidase and then isolated by gel-filtration chromatography. The extracted enzyme is found to be a FAD-containing protein, catalyzing phenol co-oxidation with 4-aminoantipyrine without addition of H2O2, which distinguishes it from peroxidases. This fact allowed us to assume that this enzyme may be a mixed-function oxidase. According to gel-filtration chromatography and SDS-PAGE, the oxidase has molecular weight of 60kDa. The enzyme exhibits maximum activity at 55-70°C and pH 5.0. Kinetic parameters Km and Vmax of the oxidase for phenol were 0.21mM and 0.40µMmin-1. We suggest that the extracted enzyme can be useful to develop a simplified biosensor for colorimetric detection of phenol in aqueous media, which does not require using hydrogen peroxide.

The recently described bioluminescent system from fungi has great potential for developing highly efficient tools for biomedical research. Luciferase enzyme is one of the most crucial components of this system. The luciferase from Neonothopanus nambi fungus belongs to the novel still undescribed protein family. The structure data for this protein is almost absent. A detailed study of the N. nambi luciferase properties is necessary for the improvement of analytical methods based on the fungal bioluminescent system. Here we present the positions of key amino acid residues and their effect on enzyme function described using bioinformatic and experimental approaches. These results are useful for further fungal luciferase structure determination.

Anticancer effects of aurisin A extracts from Neonothopanus nambi on human papillomavirus-infected cervical cancer cells

A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.

Using the original technique of treating biomass with β-glucosidase, a pool of extracellular fungal enzymes was obtained for the first time from the mycelium of basidiomycete Neonothopanus nambi. Two protein fractions containing enzymes with oxidase activity were isolated from the extract by gel-filtration chromatography and conventionally called F1 and F2. Enzyme F1 has a native molecular weight of 80-85 kDa and does not contain chromophore components; however, it catalyzes the oxidation of veratryl alcohol with Km = 0.52 mM. Probably, this enzyme is an alcohol oxidase. Enzyme F2 with a native molecular weight of approximately 60 kDa is a FAD-containing protein. It catalyzes the cooxidation of phenol with 4-aminoantipyrine without the addition of exogenous hydrogen peroxide, which distinguishes it from the known peroxidases. It was assumed that this enzyme may be a mixed-function oxidase. F2 oxidase has Km value 0.27 mM for phenol. The temperature optimums for oxidases F1 and F2 are 22-35 and 55-70°C, and pH optimums are 6 and 5, respectively.

Development of BRET-sensors based on the luciferase from bioluminescent fungus Neonothopanus nambi