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

Methanol-free secretory overproduction of glucoamylase in Komagataella phaffii using an improved transcriptional signal amplification device (iTSAD)

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

BSEG2 is highly thermostable, tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM, suggesting potential to reduce freshwater demand in biomass processing and a strong candidate for simplified, cost-effective enzyme formulations for glucose production from lignocellulosic feedstocks.

Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.

Tri-valorization of methanol in a single bioreactor: co-production of enzyme, chemical, and single-cell protein using engineered Pichia pastoris (Komagataella phaffii)

The economic viability of methanol-based biomanufacturing, particularly with green methanol as feedstock, is often limited by the low value of single-product processes. Here, we developed an integrated co-production strategy in the methylotrophic yeast Pichia pastoris (Komagataella phaffii) for the simultaneous conversion of methanol into three products: the sweetener erythritol, the industrial biocatalyst β-mannanase, and single-cell protein (SCP) biomass. This new strategy explores the inherent spatial and functional separation between the ER-Golgi secretory pathway for enzyme production and the cytosolic pathway for chemical synthesis, thereby reducing interference between the two pathways. The engineered co-production strain achieved β-mannanase and erythritol titers comparable to those of the corresponding β-mannanase- and erythritol-producing reference strains in both shake-flask and fed-batch fermentor cultures. Furthermore, transcriptomic analysis revealed distinct regulatory responses related to enzyme production and erythritol synthesis, supporting the limited cross-pathway interference between the two pathways. In addition, we showed that ultrafiltration enabled efficient downstream separation of the small-molecule erythritol from the secreted β-mannanase, with recovery efficiencies exceeding 89%. These results demonstrate a feasible strategy for methanol valorization into multiple value-added products and expand the potential of methylotrophic yeasts for integrated biomanufacturing.

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

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.

Efficient high-temperature simultaneous saccharification and fermentation of lignocellulosic substrates enabled by engineering thermotolerance in an enzyme-secreting industrial Saccharomyces cerevisiae strain.

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.