Heterologous Expression of a Neurospora crassa Catalase Reprograms the Cellulase System and Enhances β-Glucosidase Production in Trichoderma reesei

High-cell-density fermentation of Komagataella phaffii enables the overproduction of a novel, broad-substrate endoglucanase, WcEG394 from Fuling (Wolfiporia cocos), for lignocellulose biorefining.

Previous transcriptomic analysis of W. cocos strain "Xiangjing 28" grown on filter paper as the sole carbon source identified a highly abundant endoglucanase, WcEG394, implicating its role in efficient cellulose degradation. To investigate the structural and functional basis of the enzyme and evaluate its potential for application, we employed an integrated approach involving bioinformatic analysis, high-cell-density fermentation, and enzymatic characterization. Sequence analysis and structural modeling classified WcEG394 into glycoside hydrolase family 5 (GH5), revealing its canonical (α/β)8 TIM barrel architecture. High-cell-density fed-batch fermentation in Komagataella phaffii enabled the secretory production of recombinant WcEG394 at a yield of 23 g/L. The crude enzyme exhibited carboxymethyl cellulase activity of 2217.6 U/mL and filter paper activity of 110.4 U/mL. The enzyme showed optimal activity at pH 4.0 and 50 °C, maintained stability under acidic and mesothermal conditions, and displayed notable tolerance to various metal ions. Using carboxymethyl cellulose as the substrate, the Km and specific activity were determined as 2.8 mg/mL and 113.7 U/mg, respectively. Recombinant WcEG394 efficiently hydrolyzed carboxymethyl cellulose, microcrystalline cellulose, and filter paper, and demonstrated broad substrate specificity toward xylans, pectin, chitin, agarose and agricultural residues such as corn cob and sugarcane bagasse. Scanning electron microscopy further confirmed that treatment with rWcEG394 resulted in smoother surface morphology of filter paper and cotton fibers while preserving their structural integrity. The combined properties of rWcEG394 demonstrate its potential as a viable biocatalyst for the scalable processing of cellulosic biomass.

Efficient one-pot lactic acid production from cellulose via a Trichoderma reesei and Bacillus coagulans cooperation leveraging substrate-induced secretomes.

The high cost of cellulase production presents a major bottleneck in lignocellulosic biorefining. Herein, we investigated the role of secretomes induced by different substrates in mediating the synergistic interplay of Trichoderma reesei (T. reesei) and Bacillus coagulans (B. coagulans) for efficient lactic acid production. Substrate crystallinity directly affects the expression levels and enzymatic profiles in T. reesei, thereby influencing its hydrolytic performance. High-crystalline substrate induced a more balanced cellulase system, whereas low-crystallinity substrate favored the production of a β-glucosidase-enriched cocktail. This "substrate-enzyme-function" relationship was leveraged to construct a CBP system in which B. coagulans compensates for the intrinsic β-glucosidase deficiency of T. reesei through its efficient cellobiose metabolism. The synergistic cooperation achieved a near-theoretical lactic acid yield of 96.41% from cellulose without external enzyme supplementation. This work provides an induction-based strategy for designing efficient microbial consortia and offers insights for improving the efficiency and sustainability of lignocellulosic biorefining.

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass

Abstract Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis–degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol production

TrLPMO9A drives oxidative cellulose depolymerization in Trichoderma reesei and is enhanced by heterologous cellobiose dehydrogenase expression.

BACKGROUND One of the most prominent mechanisms for plant cell wall deconstruction in nature, and widely employed in industry, relies on the coordinated action of hydrolytic and oxidative enzymes. However, how redox networks sustain synergistic biomass deconstruction remains incompletely understood, particularly in the industrial workhorse Trichoderma reesei. This fungus lacks a cellobiose dehydrogenase (CDH), a pivotal redox partner for lytic polysaccharide monooxygenases (LPMOs) in many fungal systems. Here, we investigated the oxidative machinery of T. reesei and the contribution of key redox-active enzymes to lignocellulose deconstruction. RESULTS We demonstrate that the oxidative capacity of the T. reesei secretome is largely driven by a single enzyme, TrLPMO9A, the most abundant oxidoreductase in the secretome. Proteomic analyses also revealed a lower abundance of other redox-active enzymes, including TrLPMO9B and AA5 oxidase. Although deletion of TrLPMO9B and TrAA5 had a less pronounced impact on saccharification efficiency compared with TrLPMO9A, the secretome remodeling triggered by their deletion, along with the associated decrease in saccharification performance, indicates that these redox enzymes play distinct, non-redundant roles. They likely play a system-level role within a cooperative redox network that fuels oxidative cellulose deconstruction, potentially extending beyond direct catalysis to processes associated with redox balance or protein secretion. Finally, we challenged the CDH-lacking paradigm by heterologously expressing a CDH in T. reesei. In vivo reconstitution of this redox duet increased biomass saccharification by 13-19%, demonstrating a strong synergistic relationship between LPMOs and CDHs even in a native CDH-lacking host. CONCLUSION These findings define the core oxidative machinery underlying biomass deconstruction in T. reesei, revealing the major cellulose-oxidative role of TrLPMO9A and the importance of a cooperative redox network for efficient lignocellulose depolymerization. Moreover, successful reconstitution of CDH activity in a naturally CDH-deficient host establishes redox engineering as a promising strategy to enhance industrial biomass conversion.

A Thermostable Multifunctional GH5 Endoglucanase From Bacillus sp. for Lignocellulosic Biomass Saccharification.

The enzymatic conversion of lignocellulosic biomass to glucose remains a bottleneck for cost‑effective biofuel production, largely due to the complexity and expense of enzymes. Here we describe BsEG2, a multifunctional GH5‑like endoglucanase cloned from Bacillus sp. strain BS and heterologously expressed. BsEG2 displays optimal activity at pH 6.0 and 55°C and efficiently hydrolyzes both amorphous and crystalline cellulose. Kinetic analysis on carboxymethyl cellulose (CMC) indicates high substrate affinity and catalytic efficiency. Unlike typical endoglucanases, BsEG2 combines processive endoglucanase activity with broad substrate specificity. It degrades long cellulose chains and oligosaccharides and also shows β‑glucosidase‑like activity on cellobiose, yielding glucose across a wide range of chain lengths. BsEG2 is highly thermostable (a half-life of ~15 days at 55°C), tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM. It remains active in seawater, suggesting potential to reduce freshwater demand in biomass processing. These properties, namely, multifunctionality, stability, inhibitor tolerance, and halotolerance, make BsEG2 a strong candidate for simplified, cost‑effective enzyme formulations for glucose production from lignocellulosic feedstocks.