Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Myco-remediation of plastic pollution: current knowledge and future prospects.

To date, enumerable fungi have been reported to participate in the biodegradation of several notorious plastic materials following their isolation from soil of plastic-dumping sites, marine water, waste of mulch films, landfills, plant parts and gut of wax moth. The general mechanism begins with formation of hydrophobin and biofilm proceding to secretion of specific plastic degarding enzymes (peroxidase, hydrolase, protease and urease), penetration of three dimensional substrates and mineralization of plastic polymers into harmless products. As a result, several synthetic polymers including polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyurethane and/or bio-degradable plastics have been validated to deteriorate within months through the action of a wide variety of fungal strains predominantly Ascomycota (Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium spp.). Understanding the potential and mode of operation of these organisms is thus of prime importance inspiring us to furnish an up to date view on all the presently known fungal strains claimed to mitigate the plastic waste problem. Future research henceforth needs to be directed towards metagenomic approach to distinguish polymer degrading microbial diversity followed by bio-augmentation to build fascinating future of waste disposal.

Fungal composition, quantification of mycotoxins, and enzyme activity in processed Solanum tuberosum Linn (potato) products stored at different relative humidity.

AIM: Postharvest loss of potatoes at the peak of harvest is of global concern. This study aimed to determine the quality of stored processed potato products based on fungal composition, mycotoxin contamination, and fungal enzyme activity. MATERIALS AND METHODS: Potato products from three cultivars (Caruso, Marabel, and Nicola) were grouped as peeled or unpeeled, oven- or sun-dried, and all samples were in flour form. Samples were incubated separately for 6 weeks at 25%, 74%, and 87% relative humidities (RH) at 25°C. The pH, moisture content (MC), visible deterioration, mycotoxin, fungal identity by DNA sequencing, and enzyme activity were determined. RESULTS: Results of grouped products (based on variety, drying, and peeling method) revealed that MC increased in the oven-dried samples and the pH value reduced after incubation. About 26% of the products at 87% RH showed visible deterioration, low amounts of fumonisin were detected in fermented potato product and nine fungal genera were identified across the three RH levels. Enzyme activities by Aspergillus niger, Fusarium circinatum, and Rhizopus stolonifer isolates were confirmed. CONCLUSION: RH influenced deterioration and fungal activities in some stored processed potato products. Low levels of fumonisin were detected.

Bioprospecting for fungal enzymes for applications in microalgal biomass biorefineries.

Microalgal biomass is a promising feedstock for biofuels, feed/food, and biomaterials. However, while production and commercialization of single-product commodities are still not economically viable, obtaining multiple products in a biomass biorefinery faces several techno-economic challenges. The aim of this study was to identify a suitable source of hydrolytic enzymes for algal biomass saccharification. Screening of twenty-six fungal isolates for secreted enzymes activity on Chlamydomonas reinhardtii biomass resulted in the identification of Aspergillus niger IB-34 as a candidate strain. Solid-state fermentation on wheat bran produced the most active enzyme preparations. From sixty-five proteins identified by liquid chromatography coupled to mass spectrometry (LC-MS) (ProteomeXchange, identifier PXD034998) from A. niger IB-34, the majority corresponded to predicted secreted proteins belonging to the Gene Ontology categories of catalytic activity/hydrolase activity on glycosyl and O-glycosyl compounds. Skimmed biomass of biotechnologically relevant strains towards the production of commodities, Chlorella sorokiniana and Scenedesmus obliquus, was fully saccharified after a mild pretreatment at 80 °C for 10 min, at a high biomass load of 10% (w/v). The soluble liquid stream, after skimming and saccharification of biomass of both strains, was further converted into ethanol by fermentation with Saccharomyces cerevisiae at a theoretical maximum efficiency, in a separated saccharification and fermentation assays. The resulting insoluble protein, after biomass skimming with an organic solvent and enzymatic saccharification, was highly digestible in an in vitro digestion assay. Proof of concept is presented for an enzyme-assisted biomass biorefinery recovering 81% of the main biomass fractions in a likely suitable form for the conversion of lipids and carbohydrates into biofuels and proteins into feed/food. KEY POINTS: • Twenty-six fungal extracts were analyzed for saccharification of microalgal biomass. • Skimmed biomass was fully enzymatically saccharified and fermented into ethanol. • Up to 81% recovery of biomass fractions suitable for biofuels and feed/food.

Fungal pectinases: an insight into production, innovations and applications.

The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized.

Impact of environmental phthalate on human health and their bioremediation strategies using fungal cell factory- A review.

Phthalates are utilized as plasticizers in plastic products to enhance their durability, transparency, and elasticity. However, phthalates are not covalently bonded to the polymer matrix of the phthalate-containing products and can be gradually released into the environment through biogeochemical processes. Hence, phthalates are now pervasive in our environment, including our food. Reports suggested that phthalates exposure to the mammalian systems is linked to various health consequences. It has become vital to develop highly efficient strategies to reduce phthalates from the environment. In this context, the utilization of fungi for phthalate bioremediation (mycoremediation) is advantageous due to their highly effective enzyme secretory system. Extracellular and intracellular enzymes of fungi are believed to break down the phthalates by ester hydrolysis to produce phthalic acid and alcohol, and subsequent digestion of the benzene rings of phthalic acid and their metabolites. The present review scrutinizes and highlights the knowledge gap in phthalate prevalence, exposure to mammals, and associated human health challenges. Furthermore, discusses the role of fungi and their secretory enzymes in the biodegradation of phthalates and gives a perspective to better describe and tackle this continuous threat.

Approach to a fungal treatment of a biologically treated landfill leachate.

White-rot fungi (WRF) have the ability to synthetize extracellular enzymes that could degrade recalcitrant pollutants. The aim of this work was to evaluate the use of P. chrysosporium to treat a biologically and physically pre-treated landfill leachate which high load of refractory compounds (COD>1000 mg/L, BOD5<50 mg/L) in order to reduce COD and colour. Batch tests were carried out at 26 °C and 135 rpm for 15 days. The soluble chemical oxygen demand (sCOD), soluble biological oxygen demand (sBOD5) and colour, as well as the lignin peroxidase (LiP) and manganese peroxidase (MnP) enzymatic activities were analysed. Besides, the effects of different operating conditions, i.e., pH control, permeate dilution and supplementation, on treatment efficacy were investigated. The control of pH was shown to be key for fungal treatment. In addition, it was found that the addition of carbon and nitrogen sources improved the enzymatic synthesis and the removals of sCOD and colour. Data here obtained open the possibility of using fungi for reducing the amount of recalcitrant pollutants still present in treated landfill leachates or similar effluents.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and ß-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

Fungal Enzymes for Bio-Products from Sustainable and Waste Biomass.

Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries.

Fungal recovery and characterization from Hindu Kush mountain range, Tirich Mir glacier, and their potential for biotechnological applications.

The Hindu Kush mountains spread over Northern areas of Pakistan having hundreds of glaciers representing a unique ecosystem driven by the specific geochemistry and climate. The current study measured the distribution of culturable fungi in Tirich Mir glacier, Hindu Kush range, and the potential of these isolates to show antimicrobial activity and produce biotechnologically important enzymes. Samples of glacial ice, sediments, and meltwater were collected from Tirich Mir glacier, and 46 fungal strains were isolated and characterized for identity and biotechnological applications. The findings revealed Penicillium (10) as the most common genus, followed by Alternaria (9), Cladosporium (7), Coprinopsis, two isolates each belonging to genus Phoma, Ulocladium, Epicoccum, Onygenales, and Didymella, and one isolate of genus Davidiella, Aspergillus, Geomyces, Dothideomycetes, Pseudogymnoascus, Irpex, Scopulariopsis, Ascochyta, Tomicus, and Davidiellaceae. Davidiella tassiana HTF9 showed growth in the presence of 18% NaCl and pH 2, 3, 5, 7, 9, and 11. The isolates Ulocladium sp. and Onygenales sp. inhibited the growth of test fungi, Gram-negative and positive bacteria. Fungal strains were capable of producing cold-active enzymes, including cellulase, lipase, amylase, and deoxyribonuclease. The isolate Penicillium chrysogenum HTF24 was an efficient producer of amylase, deoxyribonuclease, and cellulase. The fungi of high-altitude glaciers are potent candidates for biotechnological applications; however, studies using more sensitive techniques are needed for further exploration.

Crystal Structure of a GH3 ß-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

Beta-glucosidases (ß-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 ß-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other ß-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family ß-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in ß-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Genetic Bases of Fungal White Rot Wood Decay Predicted by Phylogenomic Analysis of Correlated Gene-Phenotype Evolution.

Fungal decomposition of plant cell walls (PCW) is a complex process that has diverse industrial applications and huge impacts on the carbon cycle. White rot (WR) is a powerful mode of PCW decay in which lignin and carbohydrates are both degraded. Mechanistic studies of decay coupled with comparative genomic analyses have provided clues to the enzymatic components of WR systems and their evolutionary origins, but the complete suite of genes necessary for WR remains undetermined. Here, we use phylogenomic comparative methods, which we validate through simulations, to identify shifts in gene family diversification rates that are correlated with evolution of WR, using data from 62 fungal genomes. We detected 409 gene families that appear to be evolutionarily correlated with WR. The identified gene families encode well-characterized decay enzymes, e.g., fungal class II peroxidases and cellobiohydrolases, and enzymes involved in import and detoxification pathways, as well as 73 gene families that have no functional annotation. About 310 of the 409 identified gene families are present in the genome of the model WR fungus Phanerochaete chrysosporium and 192 of these (62%) have been shown to be upregulated under ligninolytic culture conditions, which corroborates the phylogeny-based functional inferences. These results illuminate the complexity of WR and suggest that its evolution has involved a general elaboration of the decay apparatus, including numerous gene families with as-yet unknown exact functions.

Polyporales Brown Rot Species Fomitopsis pinicola: Enzyme Activity Profiles, Oxalic Acid Production, and Fe3+-Reducing Metabolite Secretion.

Basidiomycota fungi in the order Polyporales are specified to decomposition of dead wood and woody debris and thereby are crucial players in the degradation of organic matter and cycling of carbon in the forest ecosystems. Polyporales wood-decaying species comprise both white rot and brown rot fungi, based on their mode of wood decay. While the white rot fungi are able to attack and decompose all the lignocellulose biopolymers, the brown rot species mainly cause the destruction of wood polysaccharides, with minor modification of the lignin units. The biochemical mechanism of brown rot decay of wood is still unclear and has been proposed to include a combination of nonenzymatic oxidation reactions and carbohydrate-active enzymes. Therefore, a linking approach is needed to dissect the fungal brown rot processes. We studied the brown rot Polyporales species Fomitopsis pinicola by following mycelial growth and enzyme activity patterns and generating metabolites together with Fenton-promoting Fe3+-reducing activity for 3 months in submerged cultures supplemented with spruce wood. Enzyme activities to degrade hemicellulose, cellulose, proteins, and chitin were produced by three Finnish isolates of F. pinicola Substantial secretion of oxalic acid and a decrease in pH were notable. Aromatic compounds and metabolites were observed to accumulate in the fungal cultures, with some metabolites having Fe3+-reducing activity. Thus, F. pinicola demonstrates a pattern of strong mycelial growth leading to the active production of carbohydrate- and protein-active enzymes, together with the promotion of Fenton biochemistry. Our findings point to fungal species-level "fine-tuning" and variations in the biochemical reactions leading to the brown rot type of wood decay.IMPORTANCEFomitopsis pinicola is a common fungal species in boreal and temperate forests in the Northern Hemisphere encountered as a wood-colonizing saprotroph and tree pathogen, causing a severe brown rot type of wood degradation. However, its lignocellulose-decomposing mechanisms have remained undiscovered. Our approach was to explore both the enzymatic activities and nonenzymatic Fenton reaction-promoting activities (Fe3+ reduction and metabolite production) by cultivating three isolates of F. pinicola in wood-supplemented cultures. Our findings on the simultaneous production of versatile enzyme activities, including those of endoglucanase, xylanase, ß-glucosidase, chitinase, and acid peptidase, together with generation of low pH, accumulation of oxalic acid, and Fe3+-reducing metabolites, increase the variations of fungal brown rot decay mechanisms. Furthermore, these findings will aid us in revealing the wood decay proteomic, transcriptomic, and metabolic activities of this ecologically important forest fungal species.

Biodegradation of pesticide in agricultural soil employing entomopathogenic fungi: Current state of the art and future perspectives.

Pesticides play a pivotal role in agriculture for the effective production of various crops. The indiscriminate use of pesticides results in the significant bioaccumulation of pesticide residues in vegetables. This situation is beyond the control of consumers and poses a serious health issue for human beings. Occupational exposure to pesticides may occur for farmers, agricultural workers, and industrial producers of pesticides. This occupational exposure primarily causes food and water contamination that gets into humans and environmental pollution. Depending on the toxicity of pesticides, the causes and effects differ in the environment and in human health. The number of criteria used and the method of implementation employed to assess the effect of pesticides on humans and the environment have been increasing, as they may provide characterization of pesticides that are already on the market as well as those that are on the way. The biological control of pests has been increasing nowadays to combat all these effects caused by synthetic pesticides. Myco-biocontrol has received great attention in research because it has no negative impact on humans, the environment, or non-target species. Entomopathogenic fungi are microbes that have the ability to kill insect pests. Fungi also make enzymes like the lytic enzymes, esterase, oxidoreductase, and cytochrome P450, which react with chemical residues in the field and break them down into nontoxic substances. In this review, the authors looked at how entomopathogenic fungi break down insecticides in the environment and how their enzymes break down insecticides on farms.

Exploring the Synergistic Secretome: Insights from Co-Cultivation of Aspergillus brasiliensis and Trichoderma reesei RUT-C30.

The spectrum of enzymes required for complete lignocellulosic waste hydrolysis is too diverse to be secreted by a single organism. An alternative is to employ fungal co-cultures to obtain more diverse and complete enzymatic cocktails without the need to mix enzymes during downstream processing. This study evaluated the co-cultivation of Aspergillus brasiliensis and Trichoderma reesei RUT-C30 in different conditions using sugarcane bagasse as the carbon source. The resulting enzymatic cocktails were characterized according to the impact of strain inoculation time on enzymatic activities and proteome composition. Data revealed that the profile of each enzymatic extract was highly dependent on the order in which the participating fungi were inoculated. Some of the co-cultures exhibited higher enzyme activities compared to their respective monocultures for enzymes such as CMCase, pectinase, ß-glucosidase, and ß-xylosidase. Analysis of the T. reesei RUT-C30 and A. brasiliensis co-culture secretome resulted in the identification of 167 proteins, with 78 from T. reesei and 89 from A. brasiliensis. In agreement with the enzymatic results, proteome analysis also revealed that the timing of inoculation greatly influences the overall secretome, with a predominance of T. reesei RUT-C30 proteins when first inoculated or in simultaneous inoculation.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Enzymatic Treatments for Biosolids: An Outlook and Recent Trends.

Wastewaters are nutrient-rich organic materials containing significant concentrations of different nutrients, dissolved and particulate matter, microorganisms, solids, heavy metals, and organic pollutants, including aromatic xenobiotics. This variety makes wastewater treatment a technological challenge. As a result of wastewater treatment, biosolids are generated. Biosolids, commonly called sewage sludge, result from treating and processing wastewater residuals. Increased biosolids, or activated sludge, from wastewater treatment is a major environmental and social problem. Therefore, sustainable and energy-efficient wastewater treatment systems must address the water crisis and environmental deterioration. Although research on wastewater has received increasing attention worldwide, the significance of biosolids treatments and valorization is still poorly understood in terms of obtaining value-added products. Hence, in this review, we established some leading technologies (physical, chemical, and biological) for biosolids pretreatment. Later, the research focuses on natural treatment by fungal enzymes to end with lignocellulosic materials and xenobiotic compounds (polyaromatic hydrocarbons) as a carbon source to obtain biobased chemicals. Finally, this review discussed some recent trends and promising renewable resources within the biorefinery approach for bio-waste conversion to value-added by-products.

Fucose as a nutrient ligand for Dikarya and a building block of early diverging lineages.

Fucose is a deoxyhexose sugar present and studied in mammals. The process of fucosylation has been the primary focus in studies relating to fucose in animals due to the presence of fucose in Lewis antigens. Very few studies have reported its presence in Fungi, mostly in Mucoromycotina. The constitution of 25% and 12% of this sugar in the carbohydrates of cell wall in the respective Umbelopsis and Mucorales strains boosts the need to bridge the gap of knowledge on fucose metabolism across the fungal tree of life. In the absence of a network map involving fucose proteins, we carried out an in-silico approach to construct the fucose metabolic map in Fungi. We analyzed the taxonomic distribution of 85 protein families in Fungi including diverse early diverging fungal lineages. The expression of fucose-related protein-coding genes proteins was validated with the help of transcriptomic data originating from representatives of early diverging fungi. We found proteins involved in several metabolic activities apart from fucosylation such as synthesis, transport and binding. Most of the identified protein families are shared with Metazoa suggesting an ancestral origin in Opisthokonta. However, the overall complexity of fucose metabolism is greater in Metazoa than in Fungi. Massive gene loss has shaped the evolutionary history of these metabolic pathways, leading to a repeated reduction of these pathways in most yeast-forming lineages. Our results point to a distinctive mode of utilization of fucose among fungi belonging to Dikarya and the early diverging lineages. We speculate that, while Dikarya used fucose as a source of nutrients for metabolism, the early diverging group of fungi depended on fucose as a building block and signaling compound.

Glycosylation of Epigallocatechin Gallate by Engineered Glycoside Hydrolases from Talaromyces amestolkiae: Potential Antiproliferative and Neuroprotective Effect of These Molecules.

Glycoside hydrolases (GHs) are enzymes that hydrolyze glycosidic bonds, but some of them can also catalyze the synthesis of glycosides by transglycosylation. However, the yields of this reaction are generally low since the glycosides formed end up being hydrolyzed by these same enzymes. For this reason, mutagenic variants with null or drastically reduced hydrolytic activity have been developed, thus enhancing their synthetic ability. Two mutagenic variants, a glycosynthase engineered from a ß-glucosidase (BGL-1-E521G) and a thioglycoligase from a ß-xylosidase (BxTW1-E495A), both from the ascomycete Talaromyces amestolkiae, were used to synthesize three novel epigallocatechin gallate (EGCG) glycosides. EGCG is a phenolic compound from green tea known for its antioxidant effects and therapeutic benefits, whose glycosylation could increase its bioavailability and improve its bioactive properties. The glycosynthase BGL-1-E521G produced a ß-glucoside and a ß-sophoroside of EGCG, while the thioglycoligase BxTW1-E495A formed the ß-xyloside of EGCG. Glycosylation occurred in the 5″ and 4″ positions of EGCG, respectively. In this work, the reaction conditions for glycosides' production were optimized, achieving around 90% conversion of EGCG with BGL-1-E521G and 60% with BxTW1-E495A. The glycosylation of EGCG caused a slight loss of its antioxidant capacity but notably increased its solubility (between 23 and 44 times) and, in the case of glucoside, also improved its thermal stability. All three glycosides showed better antiproliferative properties on breast adenocarcinoma cell line MDA-MB-231 than EGCG, and the glucosylated and sophorylated derivatives induced higher neuroprotection, increasing the viability of SH-S5Y5 neurons exposed to okadaic acid.

Fungal Proteases as Emerging Biocatalysts to Meet the Current Challenges and Recent Developments in Biomedical Therapies: An Updated Review.

With the increasing world population, demand for industrialization has also increased to fulfill humans' living standards. Fungi are considered a source of essential constituents to produce the biocatalytic enzymes, including amylases, proteases, lipases, and cellulases that contain broad-spectrum industrial and emerging applications. The present review discussed the origin, nature, mechanism of action, emerging aspects of genetic engineering for designing novel proteases, genome editing of fungal strains through CRISPR technology, present challenges and future recommendations of fungal proteases. The emerging evidence revealed that fungal proteases show a protective role to many environmental exposures and discovered that an imbalance of protease inhibitors and proteases in the epithelial barriers leads to the protection of chronic eosinophilic airway inflammation. Moreover, mitoproteases recently were found to execute intense proteolytic processes that are crucial for mitochondrial integrity and homeostasis function, including mitochondrial biogenesis, protein synthesis, and apoptosis. The emerging evidence revealed that CRISPR/Cas9 technology had been successfully developed in various filamentous fungi and higher fungi for editing of specific genes. In addition to medical importance, fungal proteases are extensively used in different industries such as foods to prepare butter, fruits, juices, and cheese, and to increase their shelf life. It is concluded that hydrolysis of proteins in industries is one of the most significant applications of fungal enzymes that led to massive usage of proteomics.