Comparative Genomic and Transcriptomic Analyses of 60Co-Mutagenized Scheffersomyces stipitis Strains: Identification of Candidate Genes Associated with High-Ethanol-Producing Xylose-to-Ethanol Fermentation
60 candidate key genes associated with high xylose-to-ethanol yield in S. stipitis are identified, predominantly involved in the cell cycle pathway, including CDC15 and PHO81.
Abstract
Sugarcane bagasse is an important renewable lignocellulosic resource, yet its bioconversion efficiency remains low, primarily because wild-type Saccharomyces cerevisiae cannot utilize xylose, which limits the industrial production of cellulosic ethanol. In this study, a high-ethanol-yielding strain 31.1 was obtained from Scheffersomyces stipitis (formerly known as Pichia stipitis) 1960 through 60Co mutagenesis and long-term domestication. Strain 31.1 exhibited an ethanol productivity of 0.78 g/(L·h), a sugar-to-ethanol conversion rate of 0.38 g/g, and a fermentation efficiency of 82.61%. Using the wild-type strain 1960 and a low-yielding strain 12.1 as controls, comparative genomics and transcriptomics were employed to elucidate the mechanism underlying the high ethanol production. Our findings are as follows: at the genomic level, there were 271 genomic structural variations. At the transcriptomic level, most genes involved in secondary metabolite synthesis, antibiotic synthesis, ribosomal pathways, amino acid biosynthesis, and oxidative phosphorylation pathways were down-regulated. Additionally, two key genes—XYL1 (xylose reductase gene) and XUT4 (high-affinity xylose transporter gene)—were significantly up-regulated. Through comprehensive integration of phenotypic comparison (e.g., fermentation performance of the high-yield strain 31.1 in yeast propagation and ethanol fermentation), comparative genomics, transcriptomics, and bioinformatics analyses of pathways involved in oxidative phosphorylation and the cell cycle (related to yeast cell growth), we identified 60 candidate key genes associated with high xylose-to-ethanol yield in S. stipitis. These genes are predominantly involved in the cell cycle pathway, including CDC15 and PHO81. In conclusion, our study preliminarily reveals the mechanisms underlying the high xylose ethanol production of the high-yield strain at the genomic and transcriptomic levels.
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
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.
Jing Zeng, Jianjun Guo, Shuaiwen Zhang et al.· Journal of Industrial Microb...· 0 citations
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
In this paper, we reported a wild strain Z68, isolated from Tibet, was identified as Flammulina yunnanensis based on ITS sequence and phylogenetic analysis. Optimal condition for its mycelial growth (maltose, yeast extract, magnesium sulfate, 20 °C and pH 7.0–8.0) and fruiting ability was confirmed. Its fruiting bodies contained 25.1 g/100 g protein and 19.80 g/100 g of 16 amino acids, with iron, potassium and zinc, contents significantly higher than these in the controls F. fennae and F. velutipes. Significantly, whole-genome sequencing and comparative analysis of iron-metabolism gene families were performed between F. yunnanensis, F. fennae and F. velutipes to elucidate the basis of its notably elevated iron content, revealing most single copied and conserved iron-related gene families and two-fold expanded iron permease FTR1 in F. yunnanensi, which is the iron–sulfur cluster assembly protein family. Sequence, topology (DeepTMHMM), and structural-confidence (ColabFold/AlphaFold2) analyses of FTR1 identified six species-specified physicochemical substitutions, of which a charge reverse at the intracellular channel (Glu→Lys) and a polar-to-nonpolar change in the TM3 pore (Ser→Ala) were predicted to enhance iron transport efficiency. These suggested a possible two-tier mechanism underlying the elevated iron content of F. yunnanensis, combining gene-family expansion and predicted functional fine-tuning of the FTR1 permease. This study provided insight for biological characterization, domestication, and the molecular basis of iron enrichment in a F. yunnanensis Z68.
Yuanchao Liu, Xinyu Shi, Yifan Li et al.· Journal of Fungi· 0 citations
Background and Objectives: Second-generation bioethanol, a sustainable and environmentally friendly alternative to fossil fuels, can significantly contribute to the economy. However, saccharification accounts for approximately 20–25% of total bioethanol production costs, which can be reduced by using the improved enzyme cocktails. Therefore, the present study aimed to isolate and characterize potent cellulolytic Streptomyces strains capable of enhancing the hydrolytic conversion of lignocellulosic biomass. Materials and Methods: Degraded wood and soil samples were collected and enriched with paddy straw. The isolates were obtained using standard methods and characterized by 16S rRNA sequencing. The best isolate was subjected to whole-genome sequencing to identify hydrolytic genes involved in agro-waste degradation. Saccharification assays were performed using crude culture filtrate in combination with the commercial cellulase cocktail Celluclast® to evaluate sugar release from alkali-pretreated paddy straw. Results: Among the 38 isolates obtained, five were identified as potential cellulolytic Streptomyces strains based on 16S rRNA gene sequencing: Streptomyces tunisiensis SSPJ1, Streptomyces griseoincarnatus SSPJ4, Streptomyces werraensis SSPJ14, Streptomyces ardesiacus SSPJ32, and Streptomyces tendae SSPJ48. S. griseoincarnatus SSPJ4 emerged as the most promising isolate, demonstrating significantly higher enzyme activities. S. griseoincarnatus SSPJ4 was subjected to whole-genome sequencing using the GALAXY tool for assembly and annotation, with an assembled genome of 7.3 Mb, 72.38% G+C content, and 6678 genes. Biosynthetic gene clusters (23) were identified using antiSMASH, and 226 proteins were annotated with CAZy domains, of which 121 belonged to the glycoside hydrolase (GH) family. Supplementation of Celluclast® with the crude enzyme preparation from S. griseoincarnatus SSPJ4 enhanced the saccharification of alkali-pretreated paddy straw, resulting in a 1.55-fold higher sugar yield compared with Celluclast® alone. Conclusion: The findings demonstrate the considerable potential of S. griseoincarnatus SSPJ4 as a source of accessory lignocellulolytic enzymes for improving biomass saccharification. Its enzyme repertoire could be exploited to develop cost-effective and efficient enzymatic formulations for second-generation bioethanol production.
Prakriti Jhilta, Vikram Poria, Arjun Singh et al.· Iranian Journal of Microbiol...· 0 citations