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Abstract Background There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/feruloyl esterase A family, whose functional diversification makes it a very promising candidate. Results The results suggested functional shift following a duplication event where neofunctionalisation of feruloyl esterase A had occurred with conservation of the ancestral lipase function. Evolutionary shift was detected using the branch-site model for testing positive selection on individual codons along specific lineages. Positively selected amino acids were detected. Furthermore, biological data obtained from site-directed mutagenesis experiments clearly demonstrated that certain amino acids under positive selection were involved in the functional shift. We reassessed evolutionary history in terms of environmental response, and hypothesized that environmental changes such as colonisation by terrestrial plants might have driven adaptation by functional diversification in Euascomycetes (Aspergilli), thus conferring a selective advantage on this group. Conclusion The results reported here illustrate a rare example of connection between fundamental events in molecular evolution. We demonstrated an unequivocal connection between evolutionary and functional shifts, which led us to conclude that these events were probably linked to environmental change.

Antibiotics play a crucial role in human and animal medical healthcare, but widespread use and overuse of antibiotics poses alarming health and environmental issues. Fluoroquinolones constitute a class of antibiotics that has already become ubiquitous in the environment, and their increasing use and high persistence prompt growing concern. Here we investigated a fungal secretome prepared from the white-rot fungus Coriolopsis gallica , which is able to effectively degrade the environmentally persistent fluoroquinolone, levofloxacin. We tested various physical-chemical factors such as concentrations of 1-hydroxybenzotriazol (HBT), of enzyme, and of antibiotic, and pH and temperature of the reaction for biotransformation of the antibiotic. We compared the free with the immobilized Coriolopsis gallica secretome proteins, and analyzed the collective reaction products for residual activity against E. coli (growth inhibition test). We also performed HPLC analysis. The results show that treatment with the free secretome yielded a highest removal efficiency of 50 mg L -1 levofloxacin in the presence of 2.5 mM HBT, whereas the immobilized secretome was only able to degrade 10 mg L -1 levofloxacin with the same concentration of mediator, but presenting the advantage of being reusable.

Plant metabolites have a great potential for limiting the spread of harmful fungi. However, a better understanding of the mode-of-action of these molecules and the defense systems developed by fungi to resist them, is needed to assess the benefits/risks of using them as antifungal treatment. White-rot fungi are excellent models in this respect, as they have adapted to the hostile habitat that is wood. In fact, wood is a source of putative antifungal compounds that can be derived using extraction techniques. In this study, we demonstrated that esculin and esculetin, which are coumarins found in plants and wood, reduce the growth of the wood-rotting fungi Fomitiporia mediterranea, Phanerochaete chrysosporium, Pycnoporus cinnabarinus and Trametes versicolor. We have shown that extracellular strategies are developed by the fungi to deal with esculin, through the involvement of laccases, peroxidases and glycoside hydrolases, and intracellular strategies, mainly via upregulated protein translation. Comparative proteomic and metabolomic approaches revealed that, despite the fact that the species analysed are closely related (they all belong to the Agaricomycetes, and have the same trophic mode), their defense responses to esculin differ., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Coriolopsis gallica ( Cga ) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that Cga DyP1 has high similarity with other fungal DyPs. The recombinant Cga DyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H 2 O 2 . Cga DyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. Cga DyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications.

Continued widespread use of antibiotics, especially fluoroquinolones, raises environmental concerns, as its driving bacterial resistance and disrupts microbial ecosystems. Here we investigate the biodegradation of ten fluoroquinolone antibiotics (six for medical use and four for veterinary use) by ligninolytic fungi, including Trametes versicolor , Bjerkandera adusta , Porosterum spadiceum , Irpex lacteus , Pleuroteus ostreatus , Phanerochaete chrysosporium , Pycnoporus cinnabarinus , Ganoderma lucidum , and Gloeophyllum trabeum . The results show significant variations between strains in the efficiency of antibiotic transformation. B. adusta and P. spadiceum were the fungi that most efficiently reduced antibiotic concentrations and were able to totally degrade eight and six antibiotics, respectively, within a 15-day period. T. versicolor and P. ostreatus also showed the ability to effectively degrade antibiotics. Specifically , T. versicolor degraded six out of the ten fluoroquinolone antibiotics by more than 70 %, while P. ostreatus degraded the tested antibiotics between 43 % and 100 %. The remaining antibiotic activity did not always correlate with a reduction in antibiotic concentrations, which points to the presence of post-transformation antimicrobial metabolites. This study also explores the potential mechanisms used by these fungi to remove selected models of fluroquinolones via enzymatic routes, such as oxidation by laccases, heme-peroxidases, and cytochrome P450, or via adsorption on fungal biomass., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Rapeseed meal (RSM) is a cheap, abundant and renewable feedstock, whose biorefinery is a current challenge for the sustainability of the oilseed sector. RSM is rich in sinapic acid (SA), a p -hydroxycinnamic acid that can be decarboxylated into canolol (2,6-dimethoxy-4-vinylphenol), a valuable bioactive compound. Microbial phenolic acid decarboxylases (PADs), mainly described for the non-oxidative decarboxylation of ferulic and p -coumaric acids, remain very poorly documented to date, for SA decarboxylation. The species Neolentinus lepideus has previously been shown to biotransform SA into canolol in vivo, but the enzyme responsible for bioconversion of the acid has never been characterized. In this study, we purified and characterized a new PAD from the canolol-overproducing strain N. lepideus BRFM15. Proteomic analysis highlighted a sole PAD-type protein sequence in the intracellular proteome of the strain. The native enzyme ( Nle PAD) displayed an unusual outstanding activity for decarboxylating SA (V max of 600 U.mg -1 , k cat of 6.3 s -1 and k cat /K M of 1.6 s -1 .mM -1 ). We showed that Nle PAD (a homodimer of 2 × 22 kDa) is fully active in a pH range of 5.5-7.5 and a temperature range of 30-55 °C, with optima of pH 6-6.5 and 37-45 °C, and is highly stable at 4 °C and pH 6-8. Relative ratios of specific activities on ferulic, sinapic, p -coumaric and caffeic acids, respectively, were 100:24.9:13.4:3.9. The enzyme demonstrated in vitro effectiveness as a biocatalyst for the synthesis of canolol in aqueous medium from commercial SA, with a molar yield of 92%. Then, we developed processes to biotransform naturally-occurring SA from RSM into canolol by combining the complementary potentialities of an Aspergillus niger feruloyl esterase type-A, which is able to release free SA from the raw meal by hydrolyzing its conjugated forms, and Nle PAD, in aqueous medium and mild conditions. Nle PAD decarboxylation of biobased SA led to an overall yield of 1.6-3.8 mg canolol per gram of initial meal. Besides being the first characterization of a fungal PAD able to decarboxylate SA, this report shows that Nle PAD is very promising as new biotechnological tool to generate biobased vinylphenols of industrial interest (especially canolol) as valuable platform chemicals for health, nutrition, cosmetics and green chemistry.

Despite being one of the first enzymes discovered in 1883, the determination of laccase activity remains a scientific challenge, and a barrier to the full use of laccase as a biocatalyst. Indeed, laccase, an oxidase of the blue multi-copper oxidases family, has a wide range of substrates including substituted phenols, aromatic amines and lignin-related compounds. Its one-electron mechanism requires only oxygen and releases water as a reaction product. These characteristics make laccase a biocatalyst of interest in many fields of applications including pulp and paper industry, biorefineries, food, textile, and pharmaceutical industries. But to fully envisage the use of laccase at an industrial scale, its activity must be reliably quantifiable on complex substrates and in complex matrices. This review aims to describe current and emerging methods for laccase activity assays and place them in the context of a potential industrial use of the enzyme., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H 2 O 2 -producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS 2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ben Ayed Amal reports financial support was provided by Programme Hubert Curien - Utique. Grigoriev Igor V reports financial support was provided by US Department of Energy Office of Science., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of Pci Lac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm -1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions., Competing Interests: The authors declare that they have no conflict of interest.

Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called Um GH16_1-A and Um AA3_2-A. We show that Um GH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and Um AA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by Um GH16_1-A are further oxidized by Um AA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both Um GH16_1-A and Um AA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. IMPORTANCE Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.

The textile industry generates huge volumes of colored wastewater that require multiple treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from Coriolopsis gallica. Decolorization was optimized in a 1 mL reaction mixture using the response surface methodology (RSM) to test the influence of five variables, i.e., laccase-like activity, dye concentration, redox mediator (HBT) concentration, pH, and temperature, on dye decolorization. Statistical tests were used to determine regression coefficients and the quality of the models used, as well as significant factors and/or factor interactions. Maximum decolorization was achieved at 120 min (82 ± 0.6%) with the optimized protocol, i.e., laccase-like activity at 0.5 U mL−1, dye at 25 mg L−1, HBT at 4.5 mM, pH at 4.2 and temperature at 55 °C. The model proved significant (ANOVA test with p < 0.001): coefficient of determination (R²) was 89.78%, adjusted coefficient of determination (R²A) was 87.85%, and root mean square error (RMSE) was 10.48%. The reaction conditions yielding maximum decolorization were tested in a larger volume of 500 mL reaction mixture. Under these conditions, the decolorization rate reached 77.6 ± 0.4%, which was in good agreement with the value found on the 1 mL scale. RB5 decolorization was further evaluated using the UV-visible spectra of the treated and untreated dyes.

The ability of Trichoderma reesei , a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the β-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei . This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5&#95;1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ββ, SS βO4 and GG β5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase ( Tr LOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by Tr LOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the "Auxiliary Activity" family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3&#95;2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3&#95;2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin., Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario., Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals., (© 2021. The Author(s).)

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris . The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40-50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5-6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed.