Enhancing extracellular monascus pigment production in submerged fermentation with engineered microbial consortia.

In this study, we investigated the impact of microbial interactions on Monascus pigment (MP) production. We established diverse microbial consortia involving Monascus purpureus and Lactobacillus fermentum. The addition of Lactobacillus fermentum (4% at 48 h) to the submerged fermentation of M. purpureus resulted in a significantly higher MP production compared to that achieved using the single-fermentation system. Co-cultivation with immobilized L. fermentum led to a remarkable increase of 59.18% in extracellular MP production, while mixed fermentation with free L. fermentum caused a significant decrease of 66.93% in intracellular MPs, contrasting with a marginal increase of 4.52% observed during co-cultivation with immobilized L. fermentum and the control group respectively. The findings indicate an evident enhancement in cell membrane permeability of M. purpureus when co-cultivated with immobilized L. fementum. Moreover, integrated transcriptomic and metabolomic analyses were conducted to elucidate the regulatory mechanisms underlying MP biosynthesis and secretion following inoculation with immobilized L. fementum, with specific emphasis on glycolysis, steroid biosynthesis, fatty acid biosynthesis, and energy metabolism.

The effects of environmental factors on the synthesis of water-soluble Monascus red pigments via submerged fermentation: a review.

Monascus pigments (MPs) have been used as natural food pigments for many years. There is a high demand for Monascus red pigments (MRPs) to enhance color and for antibacterial and cancer prevention therapies in food and medicine. Most MRPs are not water soluble, and the yield of water-soluble MRPs is naturally low. On the other hand, water-soluble MRP is more cost effective for application in industrial mass production. Therefore, it is important to improve the yield of water-soluble MRPs. Environmental factors have a significant influence on the synthesis of water-soluble MRPs, which is crucial for the development of industrial production of water-soluble MRPs. This review introduces the biosynthetic pathways of water-soluble MRPs and summarizes the effects of environmental factors on the yield of water-soluble MRPs. Acetyl coenzyme A (acetyl-CoA) is a precursor for MPs synthesis. Carbon and nitrogen sources and the carbon/nitrogen ratio can impact MP production by regulating the metabolic pathway of acetyl-CoA. Optimization of fermentation conditions to change the morphology of Monascus can stimulate the synthesis of MPs. The appropriate choice of nitrogen sources and pH values can promote the synthesis of MRPs from MPs. Additives such as metal ions and non-ionic surfactants can affect the fluidity of Monascus cell membrane and promote the transformation of MRPs into water-soluble MRPs. This review will lay the foundation for the industrial production of water-soluble MRPs. © 2024 Society of Chemical Industry.

Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica.

This study aimed to investigate the effect of hyphal formation in Yarrowia lipolytica and biochar addition on erythritol production by submerged fermentation. Hyphal formation significantly inhibited erythritol production by Y. lipolytica. Transcriptome analysis suggested that the impaired erythritol synthesis of hyphal cells was associated with the differential expression of genes involved in amino acid metabolism, lipid metabolism, and cell wall stability. Deletion of RAS2 responsible for yeast-to-hypha transition and EYD1 included in erythritol degradation blocked hyphal formation and improved erythritol production. Biochar prepared from corncob, sugarcane bagasse (SB), corn straw, peanut shell, coconut shell, and walnut shell (WS) had a positive effect on erythritol production, of which WS pyrolyzed at 500°C (WSc) performed the best in flask fermentation. In a 3.7 L bioreactor, 220.20 ± 10 g/L erythritol with a productivity of 2.30 ± 0.10 g/L/h was obtained in the presence of 1.4% (w/v) WSc and 0.7% SBc (SB pyrolyzed at 500°C) within 96 h. These results suggest that inhibition of hyphal formation together with biochar addition is an efficient way to promote erythritol production.

From Nature to Design: Tailoring Pure Mycelial Materials for the Needs of Tomorrow.

Modern efforts to influence materials science with principles of biology have allowed fungal mycelial materials to take a foothold and develop novel solutions for the circular bioeconomy of tomorrow. However, recent studies have shown that the value of tomorrow's green materials is not determined simply by their environmental viability, but rather by their ability to make the polluting materials of today obsolete. With an inherently strong structure of chitin and ß-glucan, the ever-adaptable mycelia of fungi can compete at the highest levels with a litany of materials from leather to polyurethane foam to paper to wood. There are significant efforts to optimize pure mycelial materials (PMMs) through the entire process of species and strain selection, mycelial growth, and fabrication. Indeed, the promising investigations of novel species demonstrate how the diversity of fungi can be leveraged to create uniquely specialized materials. This review aims to highlight PMMs' current trajectory, evaluate the successes in technology, and explore how these new materials can help shape a better tomorrow.

Successive process for efficient biovalorization of Brewers' spent grain to lignocellulolytic enzymes and lactic acid production through simultaneous saccharification and fermentation.

This study aimed to increase the value of brewers' spent grain (BSG) by using it as feedstock to produce lignocellulolytic enzymes and lactic acid (LA). Twenty-two fungal strains were screened for lignocellulolytic enzyme production from BSG. Among them, Trichoderma sp. showed the highest cellulase activity (35.84 ± 0.27 U/g-BSG) and considerably high activities of xylanase (599.61 ± 23.09 U/g-BSG) and ß-glucosidase (16.97 ± 0.77 U/g-BSG) under successive solid-state and submerged fermentation. The processes were successfully scaled up in a bioreactor. The enzyme cocktail was recovered and characterized. The maximum cellulase and xylanase activities were found at pH 5.0 and 50 °C, and the activities were highly stable at pH 4-8 and 30-50 °C. The enzyme cocktail was applied in simultaneous saccharification and fermentation of acid-pretreated BSG for LA production. The maximum LA obtained was 59.3 ± 1.0 g/L. This study has shown the efficient biovalorization of BSG, and this approach may also be applicable to other agro-industrial wastes.

An insight into the nutritional and medicinal value of edible mushrooms: A natural treasury for human health.

Edible mushrooms have been cherished worldwide because of their nutraceutical and medicinal properties. They are recognized as the new superfood for the future due to their low-calorie content, high-protein content, low lipid levels, low cholesterol levels, and abundance of essential vitamins. The fruiting body of edible mushrooms contains a plethora of primary and secondary metabolites. However, submerged cultivation is a more reliable and controlled way of production of mycelium biomass and many bioactive compounds. Several bioactive metabolites present in mushrooms possess a range of beneficial properties, including antioxidant, antimicrobial, anticancer, antidiabetic, anti-inflammatory, antiviral and anti-COVID-19 activities. Consumers have turned more intrigued in mushroom-containing products as the world needs to diversify its protein sources to meet the growing demand for protein. In this context, mushrooms are viewed as a promising source of bioactive chemicals that can be employed as an alternative to meat products. This review aims to summarise the most recent data regarding the beneficial health effects and the development of mushroom-based food products.

ß-glucosidases from Saccharomyces cerevisiae: production, protein precipitation, characterization, and application in the enzymatic hydrolysis of delignified sugarcane bagasse.

ß-glucosidase is an essential enzyme for the enzymatic hydrolysis of lignocellulosic biomass, as it catalyzes the final stage of cellulose breakdown, releasing glucose. This paper aims to produce ß-glucosidase from Saccharomyces cerevisiae and evaluate the enzymatic degradation of delignified sugarcane bagasse. S. cerevisiae was grown in yeast peptone dextrose medium. Partial purification of the enzyme was achieved through precipitating proteins with ethanol, and the optimal activity was measured by optimizing pH and temperature. The effects of ions, glucose tolerance, and heat treatment were evaluated. Delignified sugarcane bagasse was hydrolyzed by the enzyme. ß-glucosidase showed a specific activity of 14.0712 ± 0.0207 U mg-1. Partial purification showed 1.22-fold purification. The optimum pH and temperature were 6.24 and 54 °C, respectively. ß-glucosidase showed tolerance to glucose, with a relative activity of 71.27 ± 0.16%. Thermostability showed a relative activity of 58.84 ± 0.91% at 90 °C. The hydrolysis of delignified sugarcane bagasse showed a conversion rate of 87.97 ± 0.10% in the presence of Zn2+, an ion that promoted the highest increase in enzymatic activity. S. cerevisiae produced an extracellular ß-glucosidase with good stability at pH and temperatures conventionally applied in the hydrolysis of lignocellulosic biomass, showing viability for industrial application.

Exopolysaccharides produced by Antrodia cinnamomea using microparticle-enhanced cultivation: Optimization, primary structure and antibacterial property.

Microparticle-enhanced cultivation was used to enhance the production of exopolysaccharides (EPSs) from Antrodia cinnamomea. The structure and antibacterial activity of two EPSs produced by A. cinnamomea treated with Al2O3 [EPS-Al (crude) and EPS-Al-p (purified)] and without Al2O3 [EPS-C (crude) and EPS-C-p (purified)] were compared. It was observed that the addition of 4 g/L Al2O3 at 0 h resulted in the highest EPS yield of 1.46 g/L, possible attributed to the enhanced permeability of the cell membrane. The structural analysis revealed that EPS-C-p and EPS-Al-p had different structures. EPS-C-p was hyperbranched and spherical with a Mw of 10.8 kDa, while EPS-Al-p was irregular and linear with a Mw of 12.5 kDa. The proportion of Man in EPS-Al-p decreased, while those of Gal and Glc increased when compared to EPS-C-p. The total molar ratios of 6-Glcp and 4-Glcp in EPS-Al-p are 1.45 times that of EPS-C-p. Moreover, EPSs could alter bacterial cell morphology, causing intracellular substance leakage and growth inhibition, with EPS-Al having a stronger antibacterial activity than EPS-C. In conclusion, A. cinnamomea treated with Al2O3 could produce more EPSs, changing monosaccharide composition and glycosidic linkage profile, which could exert stronger antibacterial activity than that produced by untreated A. cinnamomea.

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform.

BACKGROUND: Cordycepin (3'-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study. RESULTS: An engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions. CONCLUSIONS: This study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling.

Analysis of Aureobasidium pullulans LB83 secretome reveals distinct carbohydrate active enzymes for biomass saccharification.

Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83.

Comparative Transcriptomic Analyses Propose the Molecular Regulatory Mechanisms Underlying 1,8-Cineole from Cinnamomum kanehirae Hay and Promote the Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation.

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

Recovery and partial purification of fibrinolytic protease from Pleurotus ostreatus and P. eryngii and cytotoxic and antioxidant activity of their extracts.

Mushrooms are a source of primary and secondary metabolites. Little is known about the most suitable conditions for production of mushrooms by submerged fermentation. This article reports antioxidant and cytotoxic assays, in addition to quantitatively evaluating the content of proteases with fibrinolytic action in the crude extracts of two species of edible mushrooms produced in different formulations, as well as evaluating the recovery of these enzymes by aqueous two-phase systems (ATPS). The mushrooms Pleurotus ostreatus and Pleurotus eryngii, at concentration of 100 µg/mL, displayed inhibition of DPPH and ABTS radicals below 50%. In the cytotoxicity test, the cells human fibroblast cell lines (MRC-5) showed cell viability greater than 80%. Concerning fibrinolytic activity, P. eryngii presented 226.47 ± 7.26 U/mL, therefore being more efficient than P. ostreatus (71.5 ± 0.56 U/mL). In the recovery of the P. eryngii extract by ATPS, the fibrinolytic protease was partitioned in the salt phase (30.25 U/mL). The molecular mass of the proteases was between 75 and 100 kDa. These results prove the low cytotoxicity of the extracts produced and that fermentation in supplemented malt broth favored the excretion of fibrinolytic proteases compared to the other evaluated media.

Harvesting Mycelial Biomass of Selected Basidiomycetes for Chitosan Biopolymer Extraction.

This study investigates the mycelial biomass production and chitosan extraction potential of various Basidiomycota strains, including Heterobasidion annosum, Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor, and Lentinus lepideus. Both submerged fermentation (SF) and solid-state fermentation (SSF) methods were employed. The chitosan yield in basidiocarps of Pleurotus ostreatus, Agaricus bisporus, and Ganoderma applanatum was also evaluated as a reference material. The chitosan extracted from fungal cells was characterized using elemental analyses and FTIR spectroscopy. Among the cultivated strains, P. chrysosporium exhibited the highest mycelial biomass concentration in SF (1.03 g 100 mL-1) after 14 days, while T. versicolor achieved the highest biomass concentration in SSF (3.65 g 100 mL-1). The highest chitosan yield was obtained from the mycelium of P. chrysosporium (0.38%) and T. versicolor (0.37%) in shaken SF. Additionally, commercially cultivated A. bisporus demonstrated the highest chitosan yield in fungal fruiting bodies (1.7%). The extracted chitosan holds potential as a functional biopolymer additive for eco-friendly materials, serving as an alternative to synthetic wet and dry strength agents in packaging materials.

Probiotic growth-stimulating capacity and antimicrobial activities of aqueous extracts of Lentinus crinitus (L.) Fr (polyporales, basidiomycota).

Lentinus crinitus (L.) Fr is a wild macrofungus that is popular as antimicrobial and various biological activities. This study aims to determine the capacity growth stimulation of Lactobacillus paracasei and antimicrobial activity of aqueous extracts of L. crinitus obtained from wild basidiomata, mycelial biomass by liquid fermentation and spent mushroom substrate obtained by solid-state fermentation. The antimicrobial activity was investigated against bacterial and fungal pathogens and growth stimulation L. paracasei probiotic bacterium. The total carbohydrate and ß-glucan contents of the extracts were determined using colorimetric analysis. The aqueous extracts obtained showed inhibition against Fusarium oxysporum., Penicillium sp., Rhizopus oryzae, Aspergillus niger, Escherichia coli and Salmonella typhimurium. The aqueous extract obtained from wild basidiomata, and mycelial biomass showed the highest percentage of stimulation of L. paracasei growth in 48 h. The extracts obtained from L. crinitus have antimicrobial potential and stimulating capacity of the probiotic Lactobacillus paracasei. Additionally, different biotechnological techniques such as liquid and solid-state fermentation can be used to obtain aqueous extracts.

Valorization of lignin for the production of vanillin by Bacillus aryabhattai NCIM 5503.

Coconut coir waste is a rich lignocellulosic biomass. The coconut coir waste generated from temples is resistant to natural degradation, and its accumulation causes environmental pollution. Ferulic acid, a vanillin precursor, was extracted from the coconut coir waste by hydro-distillation extraction. The extracted ferulic acid was used for vanillin synthesis by Bacillus aryabhattai NCIM 5503 under submerged fermentation. In the present study, the Taguchi DOE (design of experiment) software was used to optimize the fermentation process, which resulted in a 1.3 fold increase in vanillin yield (640.96 ± 0.02 mg/L), as compared to the unoptimized yield of 495.96 ± 0.01 mg/L. The optimized media for enhanced vanillin production comprised; fructose 0.75 % (w/v), beef extract 1 % (w/v), pH 9, temperature 30℃, agitation speed 100 rpm, trace metal solution 1 % (v/v), and ferulic acid 2 % (v/v). The results show that the commercial production of vanillin can be envisioned using coconut coir waste.

Statistical Optimization of Biosurfactant Production from Aspergillus niger SA1 Fermentation Process and Mathematical Modeling.

In this study, we sought to investigate the production and optimization of biosurfactants by soil fungi isolated from petroleum oil-contaminated soil in Saudi Arabia. Forty-four fungal isolates were isolated from ten petroleum oil-contaminated soil samples. All isolates were identified using the internal transcribed spacer (ITS) region, and biosurfactant screening showed that thirty-nine of the isolates were positive. Aspergillus niger SA1 was the highest biosurfactant producer, demonstrating surface tension, drop collapsing, oil displacement, and an emulsification index (E24) of 35.8 mN/m, 0.55 cm, 6.7 cm, and 70%, respectively. This isolate was therefore selected for biosurfactant optimization using the Fit Group model. The biosurfactant yield was increased 1.22 times higher than in the nonoptimized medium (8.02 g/l) under conditions of pH 6, temperature 35°C, waste frying oil (5.5 g), agitation rate of 200 rpm, and an incubation period of 7 days. Model significance and fitness analysis had an RMSE score of 0.852 and a p-value of 0.0016. The biosurfactant activities were surface tension (35.8 mN/m), drop collapsing (0.7 cm), oil displacement (4.5 cm), and E24 (65.0%). The time course of biosurfactant production was a growth-associated phase. The main outputs of the mathematical model for biomass yield were Yx/s (1.18), and µmax (0.0306) for biosurfactant yield was Yp/s (1.87) and Yp/x (2.51); for waste frying oil consumption the So was 55 g/l, and Ke was 2.56. To verify the model's accuracy, percentage errors between biomass and biosurfactant yields were determined by experimental work and calculated using model equations. The average error of biomass yield was 2.68%, and the average error percentage of biosurfactant yield was 3.39%.

Cutinase production from Fusarium verticillioides using response surface methodology and its application as potential insecticide degrader.

Cutinase, a multifunctional enzyme, has shown great potential in environmental applications such as degradation of plastics and some commonly used insecticides. To overcome these environmental threatening problems, attempts should be made to enhance enzyme production. In the present study, a cutinolytic fungus was isolated from the soil. Based on 18 s rDNA sequencing, it was found that isolate AR08 belongs to the genus Fusarium and clades with Fusarium verticillioides. Optimization of medium composition for enhancement in cutinase production was done using. classical and statistical methods. Firstly, key factors were selected by one variable at a time (OVAT) method, then by Plackett- Burman design. Concentration of these important factors was optimized by Central Composite design. A total of 30 experiments were conducted and the optimized concentration of sodium nitrate, dipotassium hydrogen phosphate, flaxseed oil and zinc sulphate were found to be 0.455%, 0.305%, 2% and 0.0355% respectively. The result of ANOVA (analysis of variance) test revealed that p value was significant for the model. Interaction between flaxseed oil and sodium nitrate was found to have a positive effect on cutinase production. A 14.57 fold increase in enzyme activity was found under optimized conditions with the maximum cutinase activity of 626.6 IUml-1.

Utilization of xylose enriched extract from spent lemongrass hydrolysate for clavulanic acid production using Streptomyces clavuligerus (MTCC 1142).

The main goal of this study was to provide possible alternative production medium containing xylose enriched spent lemongrass hydrolysate with glycerol as a feedstock and corn gluten meal as a nitrogen source for their ability to support the cell growth of the Streptomyces clavuligerus MTCC 1142 for the production of clavulanic acid. The xylose was extracted from spent lemongrass by using 0.25% dilute nitric acid and further partial purification of acid spent hydrolysate was performed using ion exchange resin. The method was optimized using xylose enriched hydrolysate as feed stock combined with glycerol at ratio 1:1 and growing the selected strain aerobically in media at neutral pH containing 5 mM phosphate ion concentration and using corn gluten meal as a nitrogen source, fermenting at a temperature of 28-30 °C for 96 h and 0.59 g/L clavulanic acid was effectively produced. These results demonstrate the feasibility of using spent lemongrass as feedstock for the cultivation of S. clavuligerus to produce clavulanic acid.

Valorization of Using Agro-Wastes for Levan through Submerged Fermentation and Statistical Optimization of the Process Variables Applying Response Surface Methodology (RSM) Design.

Levan is a homopolysaccharide of fructose units that repeat as its structural core. As an exopolysaccharide (EPS), it is produced by a great variety of microorganisms and a small number of plant species. The principal substrate used for levan production in industries, i.e., sucrose, is expensive and, hence, the manufacturing process requires an inexpensive substrate. As a result, the current research was designed to evaluate the potential of sucrose-rich fruit peels, i.e., mango peels, banana peels, apple peels, and sugarcane bagasse, to produce levan using Bacillus subtilis via submerged fermentation. After screening, the highest levan-producing substrate, mango peel, was used to optimize several process parameters (temperature, incubation time, pH, inoculum volume, and agitation speed) employing the central composite design (CCD) of response surface methodology (RSM), and their impact on levan production was assessed. After incubation for 64 h at 35 °C and pH 7.5, the addition of 2 mL of inoculum, and agitation at 180 rpm, the highest production of levan was 0.717 g/L of mango peel hydrolysate (obtained from 50 g of mango peels/liter of distilled water). The F-value of 50.53 and p-value 0.001 were calculated using the RSM statistical tool to verify that the planned model was highly significant. The selected model's accuracy was proven by a high value (98.92%) of the coefficient of determination (R2). The results obtained from ANOVA made it clear that the influence of agitation speed alone on levan biosynthesis was statistically significant (p-value = 0.0001). The functional groups of levan produced were identified using FTIR (Fourier-transform ionization radiation). The sugars present in the levan were measured using HPLC and the levan was found to contain only fructose. The average molecular weight of the levan was 7.6 × 106 KDa. The findings revealed that levan can be efficiently produced by submerged fermentation using inexpensive substrate, i.e., fruit peels. Furthermore, these optimized cultural conditions can be applied on a commercial scale for industrial production and commercialization of levan.

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives.

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.