Jul 2026· Biotechnology for Biofuels and Bioproducts· 0 citations
Medicine
TL;DR
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.
Abstract
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.
Efficient saccharification of lignocellulose, the most abundant renewable carbon reservoir resource, is of great industrial importance. Trichoderma reesei is a premier cellulase producer, but its fermentation efficiency is often constrained by dual challenges: dissolved oxygen limitation and intrinsic oxidative stress. To address this, we engineered T. reesei to heterologously express a robust catalase gene (cat-3) from Neurospora crassa. The recombinant strain Tr-cNcat3 exhibited a 7.4-fold increase in extracellular catalase activity. Tr-cNcat3 showed an increase in total extracellular protein, resulting in markedly enhanced filter paper activity (FPA) and β-glucosidase activity compared to the control. Strikingly, this intervention specifically triggered a significantly higher expression of β-glucosidase, a known bottleneck in T. reesei’s cellulase system, particularly on bagasse and straw as the carbon source. Moreover, the ability of the supernatant to degrade cellulose substrates was improved. Our results reveal that overexpression of cat-3 in T. reesei could modify the cellulase cocktail by triggering a higher level of β-glucosidase. This study provides a novel and effective genetic engineering strategy to unlock the full industrial potential of T. reesei for cost-effective lignocellulosic biorefining.
Haowen Sun, Chang-Bin Tang, Yifan Chen et al.· Journal of Fungi· 0 citations
Lignocellulosic biomass is an attractive renewable feedstock for sustainable biomanufacturing, but inhibitors generated during pretreatment and saccharification severely limit microbial growth and productivity. Among various strategies to overcome this, we proposed a novel strategy to control a morphology-related transcriptional regulator and compared the wild-type Cupriavidus necator H16 with its mraZ deletion mutant H16 ΔmraZ, in which mraZ functions as a transcriptional regulator influencing cell size, nutrient utilization, and polyhydroxybutyrate (PHB) synthesis under lignocellulose-derived inhibitors such as furfural, vanillin, acetate, and formate. The H16 ΔmraZ strain mostly exhibited higher growth and PHB production than the wild type across all tested inhibitors. Scanning electron microscopy (SEM) revealed that ΔmraZ maintained cell morphology and length after furfural treatment, whereas the wild type displayed significantly decreased cell size. Consistent with these observations, viability and IC50 analyses demonstrated a 3.5-fold increase in viability and a 1.7-fold increase in IC50 in H16 ΔmraZ. When the xylA and xylB genes from Bacillus subtilis 168 were introduced into both H16 and H16 ΔmraZ for cultivation with barley straw- and pine-derived hydrolysates, H16 ΔmraZ showed 1.18-fold higher biomass accumulation and 1.41-fold higher PHB synthesis than the wild type. H16 ΔmraZ showed a higher cyclopropane index in phospholipid fatty acid analysis and increased cfa and H16_A0706 (groEL) expression under furfural stress, suggesting that membrane fatty acid remodeling and chaperone-associated stress responses contributed to improved tolerance. These findings indicate that deletion of the transcriptional regulator mraZ is an effective strategy to enhance stress tolerance and improve bioproduction.
Taeyun Kim, Yuni Shin, Geonwoo Doh et al.· Journal of Biotechnology· 0 citations
Auxiliary activity family 7 (AA7) oxidoreductases are fungal flavoenzymes that catalyze the C1 oxidation of diverse oligosaccharides coupled to the reduction of molecular oxygen (oxidase activity) or organic molecules (dehydrogenase activity). These enzymes are predominantly derived from Ascomycota, with a smaller, less-explored fraction from Basidiomycota and Oomycota plant pathogens. Moreover, AA7 members are promising biocatalysts for the selective oxidation of carbohydrates, which is an enduring challenge in catalysis. However, the sequence space of AA7 enzymes remains largely uncharted, particularly within basidiomycota members, and the molecular determinants shaping substrate selectivity remain ill-defined. Focusing on the basidiomycete maize pathogen Ustilago maydis, we explored the boundaries of the AA7 sequence space by identifying and characterizing two enzymes, UmAA7A and UmAA7B, that are divergent from hitherto described members. We found that UmAA7s oxidize both chitooligosaccharides (CHOS) and their deacetylated forms (dCHOS) in an acetylation-site-dependent manner. The X-ray crystal structure of UmAA7A, sequence, and structural comparisons with a model of UmAA7B and other known CHOS-active AA7s, combined with docking analyses on CHOS and dCHOS, revealed specific active-site residues behind this unprecedented substrate specificity. Moreover, a previously not reported combination of a bicovalently tethered FAD and an atypical arrangement of residues at re-side of the FAD cofactor was associated with a mainly dehydrogenase activity profile, contrasting the majority of oxidases in AA7. Altogether, this work reveals the enzymatic oxidation of dCHOS, extending the substrate scope of AA7s and laying the biochemical foundation for uncovering their biological functions during plant infection.
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.
Hongbo Li, Wenling Yan, Lingzhi Tang et al.· International Journal of Bio...· 0 citations
Trichothecium roseum is a highly destructive postharvest pathogenic fungus that causes pink mold rot in various fruit and leads to significant agricultural and economic losses. Phytoene is crucial for phytopathogens, but the molecular mechanism by which the phytoene synthase gene crtB regulates fungal pathogenicity remains largely unclear. In this study, we evaluated the functions of TrcrtB a phytoene synthase, in T. roseum via in vivo and in vitro assays. Our results showed that knock-out of TrcrtB showed inhibition of production of phytoene and its relevant downstream metabolites, such as lycopene, carotenes and carotenal, resulting in colorless colony and branching at the mycelial edges in the knockout mutant ΔTrcrtB. Compared with the wild type (WT) strain, the colony expansion was significantly reduced 35% at 5 days post-inoculation (dpi), and conidiation was notably decreased to 45%, 36%, and 70% at 3, 5, and 7 dpi in ΔTrcrtB in vitro. Scanning electron microscope observation revealed similar results. Moreover, the ΔTrcrtB showed higher sensitivity to abiotic stresses than WT, as evidenced by inhibition rates of ΔTrcrtB colony expansion up to 71.76% (menadione), 47.73% (Congo red), 23.01% (SDS), and 17.13% (KCl). The pathogenicity of ΔTrcrtB was dramatically impaired by decreasing the rotten area up to 85.26% on apple fruit and 70.30% on pears fruit. These results suggest that TrcrtB and phytoene are critical for development, stress tolerance and pathogenicity of T. roseum. Collectively, our study highlights the roles of TrcrtB and phytoene in the pathogenic fungus T. roseum, providing new insights into the molecular mechanisms of pink rot pathogenesis.
Lignin-rich industrial streams represent an abundant but underutilized source of renewable aromatic carbon. Efficient biological conversion requires microbial hosts capable of metabolizing chemically diverse lignin-derived aromatic compounds; however, such capabilities are typically found in environmental bacteria, which are constrained by physiological and metabolic limitations. Sphingobium lignivorans SYK-6 harbors extensive aromatic catabolic pathways, but its inability to utilize glucose and its methionine auxotrophy have limited its use as a production host. Here, we identified the metabolic basis of these constraints and systematically rewired the underlying pathways to overcome them. Introduction of a heterologous glucose transporter, reconstruction of methionine biosynthesis, chromosomal integration of pathway genes, and adaptive laboratory evolution collectively enabled robust growth on glucose while eliminating methionine auxotrophy. The engineered strain converted lignin-derived aromatics in oxygen-soda-anthraquinone pulping black liquor derived from Japanese cedar, achieving high-yield production of the polymer precursor 2-pyrone-4,6-dicarboxylic acid (PDC) (2.71 g/L and >130 mol% conversion relative to major quantified aromatics). We further show that gluconolactonase can substitute for 6-phosphogluconolactonase in the Entner-Doudoroff pathway, demonstrating that central carbon metabolism can accommodate functionally analogous enzymes. Together, these results provide a metabolically rewired SYK-6 strain as a platform for the valorization of industrial lignin streams and suggest that overcoming physiological and metabolic constraints can enable non-model aromatic-degrading bacteria to function as industrial production hosts.