Genetically encodable bioluminescent system from fungi.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado.

Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body.

We previously showed that luminous fungi share a common mechanism in bioluminescence, and identified hispidin as a luciferin precursor in Neonothopanus nambi mycelium. Here we showed the presence of hispidin as a bioluminescent active compound at 25-1000 pmol g-1 in the fruiting bodies of Mycena chlorophos, Omphalotus japonicus, and Neonothopanus gardneri. These results suggest that luminous mushrooms contain hispidin as a luciferin precursor. We also found that non-luminous "young" fruiting bodies exhibited luminescence by hispidin treatment. Furthermore, we observed a gradual luminescence enhancement of the cell-free fruiting body extract by the addition of hispidin biosynthetic components, namely caffeic acid, ATP and malonyl-CoA. These findings suggest that continuous weak glow of luminous mushrooms is regulated by slow recycling biosynthesis of hispidin.

New luminescent mycenoid fungi (Basidiomycota, Agaricales) from São Paulo State, Brazil.

Four species of mycenoid fungi are reported as luminescent (or putatively luminescent) on the basis of specimens collected from São Paulo State, Brazil. Two of them represent new species (Mycena oculisnymphae, Resinomycena petarensis), and two represent new reports of luminescence in previously described species (M. deformis, M. globulispora). Comprehensive descriptions, illustrations, photographs, and comparisons with phenetically similar species are provided. Sequences of nuc rDNA internal transcribed spacer regions were generated for barcoding purposes and for comparisons with similar species.

Mining reactive triplet carbonyls in biological systems.

Aliphatic triplet carbonyls can be treated as short-lived radicals, since both species share similar reactions such as hydrogen atom abstraction, cyclization, addition, and isomerization. Importantly, enzyme-generated triplet carbonyls excite triplet molecular oxygen to the highly reactive, electrophilic singlet state by resonance energy transfer, which can react with proteins, lipids, and DNA. Carbonyl triplets, singlet oxygen, and radicals are endowed with the potential to trigger both normal and pathological responses. In this paper, we present a short review of easy, fast, and inexpensive preliminary tests for the detection of transient triplet carbonyls in chemical and biological systems. This paper covers direct and indirect methods to look for triplet carbonyls based on their spectral distribution of chemiluminescence, photoproduct analysis, quenching of light emission by conjugated dienes, and enhancement of light emission by the sensitizer 9,10-dibromoanthracence-2-sulfonate ion (DBAS).

Eoscyphella luciurceolata gen. and sp. nov. (Agaricomycetes) Shed Light on Cyphellopsidaceae with a New Lineage of Bioluminescent Fungi.

During nocturnal field expeditions in the Brazilian Atlantic Rainforest, an unexpected bioluminescent fungus with reduced form was found. Based on morphological data, the taxon was first identified as belonging to the cyphelloid genus Maireina, but in our phylogenetic analyses, Maireina was recovered and confirmed as a paraphyletic group related to genera Merismodes and Cyphellopsis. Maireina filipendula, Ma. monacha, and Ma. subsphaerospora are herein transferred to Merismodes. Based upon morphological and molecular characters, the bioluminescent cyphelloid taxon is described as the new genus Eoscyphella, characterized by a vasiform to urceolate basidiomata, subglobose to broadly ellipsoid basidiospores, being pigmented, weakly to densely encrusted external hyphae, regularly bi-spored basidia, unclamped hyphae, and an absence of both conspicuous long external hairs and hymenial cystidia. Phylogenetic analyses based on ITS rDNA and LSU rDNA support the proposal of the new genus and confirm its position in Cyphellopsidaceae. Eoscyphella luciurceolata represents a new lineage of bioluminescent basidiomycetes with reduced forms.

Correction: Silva-Filho et al. Eoscyphella luciurceolata gen. and sp. nov. (Agaricomycetes) Shed Light on Cyphellopsidaceae with a New Lineage of Bioluminescent Fungi. J. Fungi 2023, 9, 1004.

In the original publication [...].

Evidence that a single bioluminescent system is shared by all known bioluminescent fungal lineages.

Since the early 20th century, many researchers have attempted to determine how fungi are able to emit light. The first successful experiment was obtained using the classical luciferin-luciferase test that consists of mixing under controlled conditions hot (substrate/luciferin) and cold (enzyme/luciferase) water extracts prepared from bioluminescent fungi. Failures by other researchers to reproduce those experiments using different species of fungi lead to the hypothesis of a non-enzymatic luminescent pathway. Only recently, the involvement of a luciferase in this system was proven, thus confirming its enzymatic nature. Of the 100,000 described species in Kingdom Fungi, only 71 species are known to be luminescent and they are distributed unevenly amongst four distantly related lineages. The question we address is whether the mechanism of bioluminescence is the same in all four evolutionary lineages suggesting a single origin of luminescence in the Fungi, or whether each lineage has a unique mechanism for light emission implying independent origins. We prepared hot and cold extracts of numerous species representing the four bioluminescent fungal lineages and performed cross-reactions (luciferin × luciferase) in all possible combinations using closely related non-luminescent species as controls. All cross-reactions with extracts from luminescent species yielded positive results, independent of lineage, whereas no light was emitted in cross-reactions with extracts from non-luminescent species. These results support the hypothesis that all four lineages of luminescent fungi share the same type of luciferin and luciferase, that there is a single luminescent mechanism in the Fungi, and that fungal luciferin is not a ubiquitous molecule in fungal metabolism.

Metabolomics of the wild mushroom Gymnopilus imperialis (Agaricomycetes, Basidiomycota) by UHPLC-HRMS/MS analysis and molecular network.

Gymnopilus consists in a widely distributed genus of mushroom-forming fungi, especially in tropical regions of the world. Literature on Gymnopilus representatives reports the presence of oligoisoprenoids, and styrylpyrones. Considering the large number of secondary metabolites that basidiomycetes might contain, dereplication tools such as GNPS (Global Natural Products Social Molecular Networking), has become important in prospecting metabolites, saving time and work on isolation and characterization of natural products. Thus, this work identified the wild mushroom Gymnopilus imperialis and dereplicated their extracts with the aid of GNPS to annotate oligoisoprenoids. It was possible to annotate 24 oligoisoprenoids from methanol, dichloromethaneand ethyl acetate extracts of G. imperialis, 4 of them from GNPS spectral library match, and 20 from prediction based on molecular network. Moreover HRMS-ESI-(+) dereplication of the acetate extract annotated bisnoryangonin and hispidin. To the best of our knowledge, this is the first report on the annotation of a series of gymnopilins analogues based on GNPS molecular network. Our findings suggest that GNPS might be an effective, rapid, and open-source device to identify compounds and predict analogues.

Reannotation of Fly Amanita l-DOPA Dioxygenase Gene Enables Its Cloning and Heterologous Expression.

The l-DOPA dioxygenase of Amanita muscaria (AmDODA) participates in the biosynthesis of betalain- and hygroaurin-type natural pigments. AmDODA is encoded by the dodA gene, whose DNA sequence was inferred from cDNA and gDNA libraries almost 30 years ago. However, reports on its heterologous expression rely on either the original 5'-truncated cDNA plasmid or artificial gene synthesis. We provide unequivocal evidence that the heterologous expression of AmDODA from A. muscaria specimens is not possible by using the coding sequence previously inferred for dodA. Here, we rectify and reannotate the full-length coding sequence for AmDODA and express a 205-aa His-tagged active enzyme, which was used to produce the l-DOPA hygroaurin, a rare fungal pigment. Moreover, AmDODA and other isozymes from bacteria were submitted to de novo folding using deep learning algorithms, and their putative active sites were inferred and compared. The wide catalytic pocket of AmDODA and the presence of the His-His-His and His-His-Asp motifs can provide insight into the dual cleavage of l-DOPA at positions 2,3 and 4,5 as per the mechanism proposed for nonheme dioxygenases.

Metaprofiling of the Bacterial Community in Colonized Compost Extracts by Agaricus subrufescens.

It is well-known that bacteria and fungi play important roles in the relationships between mycelium growth and the formation of fruiting bodies. The sun mushroom, Agaricus subrufescens, was discovered in Brazil ca. 1960 and it has become known worldwide due to its medicinal and nutritional properties. This work evaluated the bacterial community present in mushroom-colonized compost extract (MCCE) prepared from cultivation of A. subrufescens, its dynamics with two different soaking times and the influence of the application of those extracts on the casing layer of a new compost block for A. subrufescens cultivation. MCCEs were prepared through initial submersion of the colonized compost for 1 h or 24 h in water followed by application on casing under semi-controlled conditions. Full-length 16S rRNA genes of 1 h and 24 h soaked MCCE were amplified and sequenced using nanopore technology. Proteobacteria, followed by Firmicutes and Planctomycetes, were found to be the most abundant phyla in both the 1 h and 24 h soaked MCCE. A total of 275 different bacterial species were classified from 1 h soaked MCCE samples and 166 species from 24 h soaked MCCE, indicating a decrease in the bacterial diversity with longer soaking time during the preparation of MCCE. The application of 24 h soaked MCCE provided increases of 25% in biological efficiency, 16% in precociousness, 53% in the number of mushrooms and 40% in mushroom weight compared to control. Further investigation is required to determine strategies to enhance the yield and quality of the agronomic traits in commercial mushroom cultivation.

Fungal bioassays for environmental monitoring.

Environmental pollutants are today a major concern and an intensely discussed topic on the global agenda for sustainable development. They include a wide range of organic compounds, such as pharmaceutical waste, pesticides, plastics, and volatile organic compounds that can be found in air, soil, water bodies, sewage, and industrial wastewater. In addition to impacting fauna, flora, and fungi, skin absorption, inhalation, and ingestion of some pollutants can also negatively affect human health. Fungi play a crucial role in the decomposition and cycle of natural and synthetic substances. They exhibit a variety of growth, metabolic, morphological, and reproductive strategies and can be found in association with animals, plants, algae, and cyanobacteria. There are fungal strains that occur naturally in soil, sediment, and water that have inherent abilities to survive with contaminants, making the organism important for bioassay applications. In this context, we reviewed the applications of fungal-based bioassays as a versatile tool for environmental monitoring.

Immunomodulatory Effect of Polysaccharides from the Mushroom-Forming Basidiomycete Gymnopilus imperialis (Agaricomycetes, Basidiomycota).

Gymnopilus consists of a widely distributed genus of basidiomycetes, especially in tropical regions of the world, such as Japan, Australia, Paraguay, and Brazil. This genus biosynthesizes interesting bioactive compounds, such as sesquiterpenoids, oligoisoprenoids, styrylpyrones, and lectins. In the present study, the aqueous extract of the basidiomata of Gymnopilus imperialis (Basidiomycota, Agaricomycetes, Agaricales, Hymenogastraceae) was obtained by using the accelerated solvent extraction (ASE) technique, followed by the precipitation of polysaccharide fraction with ethanol. Further purification by freeze-thawing processes, Fehling solution precipitation, and membrane dialysis with different pore sizes yield three main polysaccharide fractions (Gi-MRSW, Gi-PFME, and Gi-SFME). According to monosaccharide composition and 13C-NMR data, the Gi-MRSW and Gi-SFME fractions showed to be composed mainly of ß-glucans and Gi-PFME by a heterogalactan. Moreover, the immunomodulatory potential of Gi-MRSW was evaluated using RAW 264.7 murine macrophage as a study model. The nitric oxide production was significantly increased in treated samples, and the expression of inducible nitric oxide synthase (iNOS) showed that the fraction Gi-MRSW from G. imperialis induces the M1 polarization phenotype.

Neonothopanus gardneri: a new combination for a bioluminescent agaric from Brazil.

The bioluminescent agaric, Agaricus gardneri Berk., was rediscovered recently in central Brazil. The new combination, Neonothopanus gardneri, is proposed for this long-forgotten taxon supported by morphological and molecular data.

Optimized methodology for obtention of high-yield and -quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri.

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna-like vegetation) in Northeast Brazil, especially in Piauí State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and -quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy® kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of ~130% in comparison to the RNeasy® method and of ~40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy® method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and -yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes.

Aerobic co-oxidation of hemoglobin and aminoacetone, a putative source of methylglyoxal.

Aminoacetone (1-aminopropan-2-one), a putative minor biological source of methylglyoxal, reacts like other α-aminoketones such as 6-aminolevulinic acid (first heme precursor) and 1,4-diaminobutanone (a microbicide) yielding electrophilic α-oxoaldehydes, ammonium ion and reactive oxygen species by metal- and hemeprotein-catalyzed aerobic oxidation. A plethora of recent reports implicates triose phosphate-generated methylglyoxal in protein crosslinking and DNA addition, leading to age-related disorders, including diabetes. Importantly, methylglyoxal-treated hemoglobin adds four water-exposed arginine residues, which may compromise its physiological role and potentially serve as biomarkers for diabetes. This paper reports on the co-oxidation of aminoacetone and oxyhemoglobin in normally aerated phosphate buffer, leading to structural changes in hemoglobin, which can be attributed to the addition of aminoacetone-produced methylglyoxal to the protein. Hydroxyl radical-promoted chemical damage to hemoglobin may also occur in parallel, which is suggested by EPR-spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide and ethanol. Concomitantly, oxyhemoglobin is oxidized to methemoglobin, as indicated by characteristic CD spectral changes in the Soret and visible regions. Overall, these findings may contribute to elucidate the molecular mechanisms underlying human diseases associated with hemoglobin dysfunctions and with aminoacetone in metabolic alterations related to excess glycine and threonine.

l-Tryptophan Interactions with the Horseradish Peroxidase-Catalyzed Generation of Triplet Acetone.

Triplet carbonyls generated by chemiexcitation are involved in typical photobiochemical processes in the absence of light. Due to their biradical nature, ultraweak light emission and long lifetime, electronically excited triplet species display typical radical reactions such as isomerization, fragmentation, cycloaddition and hydrogen abstraction. In this paper, we report chemical reactions in a set of amino acid residues induced by the isobutanal/horseradish peroxidase (IBAL/HRP) system, a well-known source of excited triplet acetone (Ac3* ). Accordingly, quenching of Ac3* by tryptophan (Trp) unveiled parallel enzyme damage and inactivation, likely explained by scavenging of IBAL tertiary radical reaction intermediate and Ac3* -derived 2-hydroxy-i-propyl radical. Quenching constants were calculated from Stern-Volmer plots, and the structure of radical adducts was revealed by mass spectrometry. As expected, a concurrent Schiff-type adduct was found to be one of the reaction by-products. These findings draw attention to potential structural and functional changes in enzymes involved in the electronic chemiexcitation of their products.

Oxidative Modification of Proteins: From Damage to Catalysis, Signaling, and Beyond.

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption. Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance. Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions. Antioxid. Redox Signal. 35, 1016-1080.

Vision in click beetles (Coleoptera: Elateridae): pigments and spectral correspondence between visual sensitivity and species bioluminescence emission.

Among lampyrids, intraspecific sexual communication is facilitated by spectral correspondence between visual sensitivity and bioluminescence emission from the single lantern in the tail. Could a similar strategy be utilized by the elaterids (click beetles), which have one ventral abdominal and two dorsal prothoracic lanterns? Spectral sensitivity [S(lambda)] and bioluminescence were investigated in four Brazilian click beetle species Fulgeochlizus bruchii, Pyrearinus termitilluminans, Pyrophorus punctatissimus and P. divergens, representing three genera. In addition, in situ microspectrophotometric absorption spectra were obtained for visual and screening pigments in P. punctatissimus and P. divergens species. In all species, the electroretinographic S(lambda) functions showed broad peaks in the green with a shoulder in the near-ultraviolet, suggesting the presence of short- and long-wavelength receptors in the compound eyes. The long-wavelength receptor in Pyrophorus species is mediated by a P540 rhodopsin in conjunction with a species-specific screening pigment. A correspondence was found between green to yellow bioluminescence emissions and its broad S(lambda) maximum in each of the four species. It is hypothesized that in elaterids, bioluminescence of the abdominal lantern is an optical signal for intraspecifc sexual communication, while the signals from the prothoracic lanterns serve to warn predators and may also provide illumination in flight.

Luminescent Mycena: new and noteworthy species.

Seven species of Mycena are reported as luminescent, representing specimens collected in Belize, Brazil, Dominican Republic, Jamaica, Japan (Bonin Islands), Malaysia (Borneo) and Puerto Rico. Four of them represent new species (Mycena luxaeterna, M. luxarboricola, M. luxperpetua, M. silvaelucens) and three represent new reports of luminescence in previously described species (M. aff. abieticola, M. aspratilis, M. margarita). Mycena subepipterygia is synonymized with M. margarita, and M. chlorinosma is proposed as a possible synonym. Comprehensive descriptions, illustrations, photographs and comparisons with phenetically similar species are provided. A redescription of M. chlorophos, based on analyses of type specimens and recently collected topotypical material, is provided. The addition of these seven new or newly reported luminescent species of Mycena brings the total to 71 known bioluminescent species of fungi.