Efficient one-pot lactic acid production from cellulose via a Trichoderma reesei and Bacillus coagulans cooperation leveraging substrate-induced secretomes.
Jun 2026· Bioresource Technology· pp.
135276
· 0 citations· 42 references
Medicine
TL;DR
This work provides an induction-based strategy for designing efficient microbial consortia and offers insights for improving the efficiency and sustainability of lignocellulosic biorefining.
Abstract
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.
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
The high cost of commercial enzyme cocktails remains a major barrier for lignocellulosic (second-generation) bioethanol production. Simultaneous saccharification and fermentation (SSF) at elevated temperatures using enzyme-secreting yeast can reduce enzyme demand, but is constrained by the limited thermotolerance of industrial strains. In this study, thermotolerant isolates of an inhibitor-tolerant, xylose-utilizing, enzyme-secreting industrial Saccharomyces cerevisiae strain were generated using whole-genome transformation (WGT). Screening in mixed-sugar fermentations at 41 °C identified several improved isolates, of which one isolate, designated Cellusec®4.0, achieved an ethanol titer of 5.45%(v/v), representing an 86% increase compared to the parental strain. This was driven by near-complete utilization of glucose, xylose, and cellobiose. In SSF at 40 °C with sorghum pulp, Cellusec®4.0 reached 5.83%(v/v) ethanol, 24% higher than the parental strain. Fed-batch SSF of pretreated softwood demonstrated the benefit of elevated temperature, with Cellusec®4.0 achieving 4.36%(v/v) ethanol at 40 °C, 29% higher than at 35 °C. In addition, fed-batch SSF of alkali-pretreated sugarcane bagasse at 39 °C using an in-house produced enzyme cocktail resulted in ethanol titers of up to 8.0% (v/v) within 48 h, corresponding to an 83% yield. These results demonstrate that WGT is an effective strategy to introduce thermotolerance into industrial yeast while maintaining key traits. The improved thermotolerance of Cellusec® 4.0 enabled high-temperature SSF, thereby increasing ethanol titers. Combined with retained inhibitor tolerance, enzyme secretion, and mixed-sugar utilization, this supported efficient ethanol production across multiple lignocellulosic substrates under industrially relevant conditions.
Bart Thevelein, Mekonnen M Demeke, Stijn De Graeve et al.· Bioresource Technology· 0 citations
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
Global warming and industrial pollutants pose significant threats to environmental sustainability. In response, enzymes have emerged as promising tools for mitigating pollution. Enzymatic hydrolysis is considered environmentally sustainable due to its biological nature. In Brazil, the estimated agro-industrial waste production is an abundant source of lignocellulosic biomass, which contains cellulose, hemicellulose, and lignin. Its effective degradation requires a synergistic cocktail of enzymes, including cellulases, hemicellulases, and lignin-degrading enzymes. Within this system, β-glucosidases play a key role as a limiting factor in cellulose conversion, acting synergistically with other enzymes but being inhibited by glucose. This study aimed to produce and characterize β-glucosidases from the crude extract of the cyanobacterium Microcystis aeruginosa CACIAM 03. Enzyme production occurred after 10 days of incubation. The crude enzymatic extract exhibited optimal activity at pH 4.5 and 40°C (28.51 U/mL), high thermostability at 50°C (60% activity retained after 480 min), and tolerance to methanol (∼15% loss). The presence of Zn2+, Cl2+, and Mn2+ increased enzymatic activity by 2-3×. Using 300 µg of total protein from the crude enzymatic extract, the enzyme demonstrated high affinity for cellobiose, with K M app of 0.156 ± 0.02 mM and V ma x app of 0.246 ± 0.01 µM/min. These results reinforce the promising enzymatic potential of M. aeruginosa strains isolated from the Amazon region for sustainable biotechnological applications.
G. Serra, M. S. Diniz, E. Gonçalves et al.· Biotechnology and applied bi...· 0 citations
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.
Debjyoti Ghosh, Aditi Konar, Y. Gao et al.· Biotechnology Journal· 0 citations
Fungal cellulases are key biocatalysts for lignocellulosic biomass valorization and the development of sustainable biorefineries. This review examines recent advances in the production of endoglucanase (EGL), exoglucanase (EXG), and β-glucosidase (BGL) by fungi of the genera Trichoderma, Aspergillus, and Penicillium under solid-state fermentation (SSF) and submerged fermentation (SmF). Emphasis is placed on fermentation strategies, substrate selection, process optimization, and emerging chemometric and artificial intelligence-based approaches. The literature reveals a predominance of SSF systems, especially when agri-food residues such as wheat bran, sugarcane bagasse, rice-derived residues, fruit-processing wastes, and cocoa by-products are employed as low-cost substrates. Among the evaluated genera, Aspergillus was among the most frequently investigated genera and exhibited broad substrate versatility, whereas Trichoderma reesei remains the principal industrial production host for cellulase-rich enzyme preparations used mainly in the saccharification of lignocellulosic biomass for cellulosic ethanol and other biorefinery applications. In contrast, Penicillium stands out as an important source of BGL, complementing cellulase systems derived from other fungi. Temperature, pH, moisture content, and fermentation time were consistently identified as the main factors affecting cellulase biosynthesis, with optimal production generally occurring under mildly acidic conditions and mesophilic temperatures. CCD and BBD were the predominant optimization strategies, while artificial neural network-based models are emerging as promising alternatives. The complementary characteristics of these fungi genera support their application in integrated biomass conversion and future lignocellulosic biorefineries.
I. V. L. de Moura, S. Araujo, I. C. F. Sampaio et al.· Biomass· 0 citations