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.
Results revealed that substrate recalcitrance was a major limiting factor for enzyme production and that thermochemical pretreatment significantly improved substrate utilization.
Alicia María Gómez-Julián, Carlos Soltero-Sánchez, M. A. Camacho-Ruiz et al.· Biomass· 0 citations
p-Benzoquinone, a lignin-derived inhibitor generated during lignocellulosic biomass pretreatment, severely restricts microbial fermentation and remains a major obstacle to efficient cellulosic ethanol production. In this study, p-benzoquinone concentrations in different lignocellulosic hydrolysates ranged from 130 to 210 mg/L under high-solids conditions. Growth analysis showed that p-benzoquinone strongly inhibited Saccharomyces cerevisiae DQ1 in a concentration-dependent manner, with 100 mg/L p-benzoquinone causing 68.5% growth inhibition. Adaptive evolution under gradually increasing p-benzoquinone stress generated an evolved strain, S. cerevisiae ZX100. Compared with the parental strain, ZX100 exhibited markedly enhanced tolerance and produced approximately 2.7 times more ethanol in biodetoxified corn stover hydrolysate. In biodetoxified hydrolysate supplemented with 100 mg/L p-benzoquinone, ZX100 achieved an ethanol yield of 0.43 g ethanol/g sugar consumed, a glucose consumption rate of 2.3 g/L/h, and a volumetric ethanol productivity of 1.0 g/L/h. Phenotypic analyses further demonstrated that ZX100 possessed improved membrane integrity and reduced intracellular ROS accumulation under p-benzoquinone stress. Transcriptomic analysis indicated that the improved p-benzoquinone tolerance of the evolved strain may be associated with enhanced antioxidant defense, membrane stabilization, energy metabolism, and sugar transport capacity. Overall, this work deepens our understanding of the physiological and molecular mechanisms underlying p-benzoquinone tolerance in S. cerevisiae and provides potential candidate genes for engineering robust biorefinery strains with enhanced p-benzoquinone tolerance.
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.
Jiayu Fang, Shuxian Wang, Guoxia Liu et al.· Bioresources and Bioprocessi...· 0 citations
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.
Debjyoti Ghosh, Aditi Konar, Yi Gao et al.· Biotechnology Journal· 0 citations
Maize cob is an abundant lignocellulosic agricultural residue with potential as a non-edible feedstock for bioethanol production, although its recalcitrant structure limits enzymatic conversion. This study evaluated different chemical and physicochemical pretreatments, enzymatic hydrolysis and fermentation strategies for bioethanol production from maize cob. Pretreated biomass was hydrolysed using commercial cellulase and fermented through Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) using Saccharomyces cerevisiae and Zymomonas mobilis under controlled conditions. Pretreatment significantly influenced cellulose, hemicellulose, lignin, ash content, solid loss, sugar release and ethanol production. The combination of 4% NaOH and 5% H₂O₂ (N2P1) produced the highest cellulose content (61.84%) and reduced the lignin content to 7.68%. This treatment also achieved the highest ethanol yield of 12.70% under SSF using S. cerevisiae. The highest SSF ethanol yield obtained using Z. mobilis was 12.15% with 4% NaOH and 1% H₂SO₄ (N2H1). SSF generally produced more ethanol than SHF, while combined alkali-based pretreatments performed better than the control and individual treatments. Correlation analysis showed positive associations between cellulose, sugar fractions and ethanol yields, whereas lignin and ash were negatively associated with conversion performance. Overall, combined pretreatment followed by SSF, particularly N2P1 with S. cerevisiae, was the most effective tested approach for bioethanol production from maize cob.
K. C. Kiran, Fasiha, C. Gajendra et al.· International Journal of Env...· 0 citations
