Lin Yuan

Lignocellulosic biorefineries are limited by the high cost of cellulolytic enzymes. Consolidated bioprocessing (CBP), which integrates saccharification and fermentation in one step, offers a solution to this challenge. In this study, a cellulase-hyperproducing mutant of Talaromyces pinophilus, Y117, was generated from the parental strain TP117 via sequential ultraviolet irradiation and NTG (N-methyl-N′-nitro-N-nitrosoguanidine) mutagenesis. Enzymatic secretion and lignocellulose degradation capacities were evaluated, focusing on agricultural residues, particularly corncob. Y117’s performance was compared with TP117 and Trichoderma reesei Rut-C30 (TR30) under high-solids fermentation. Furthermore, the lactate dehydrogenase A (ldhA) gene from Rhizopus oryzae was heterologously expressed in Y117 to direct hydrolyzed sugars toward lactic acid (LA). Y117 exhibited significantly enhanced enzymatic secretion, achieving FPase activity of 8.9 IU/mL and a substrate utilization rate of 72.2% at 125 g/L corncob solids. Y117 outperformed TP117 and TR30 in cellulase, xylanase, and CMCase activities, as well as growth under high-solids fermentation conditions. In the LA fermentation process, Y117 produced 14.20 g/L LA, a notable improvement compared to TP117 (5.33 g/L) and TR30 (2.71 g/L). While LA productivity and yield currently remain below bacterial benchmarks, the unique CBP capability of Y117 provides a foundation for further metabolic engineering toward industrial viability. The engineered T. pinophilus Y117 demonstrates promising potential as a CBP platform for efficient straw-to-LA conversion, providing a sustainable approach for third-generation biobased materials production.

Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis–degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary 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.

Endoglucanase plays a vital role in lignocellulose degradation, yet the functional diversity of glycosyl hydrolase 12 (GH12) endoglucanases remains underexplored. In this study, TpCel12a, a novel GH12 endoglucanase from Talaromyces pinophilus Y117 was identified and characterized. It exhibited optimal activity at pH 4.0 and 40 °C with a preference for xyloglucan. Size exclusion chromatography revealed a monomeric state of TpCel12a. Despite classification into the promiscuous subfamily I, TpCel12a showed strict specificity for xyloglucan and carboxymethyl cellulose, yielding XXXG-type oligosaccharides and glucose/cellobiose, respectively. A loop 3 insertion mutant, designated as TpCel12a(+TQA), enhanced substrate affinity (5-fold lower Km) but reduced activity, suggesting a trade-off between binding and catalysis. Surprisingly, TpCel12a inhibited the hydrolytic efficiency of native T. pinophilus crude cellulase on corn cob powder, coinciding with its undetectable secretion under microcrystalline cellulose induction. This study highlights TpCel12a’s unique biochemical properties and its unexpected antagonistic role in native cellulase systems, offering insights for engineering fungal enzyme cocktails.

Xyloglucan, a key component of plant cell wall polysaccharides, plays a crucial role in cell wall structural remodeling and biomass recalcitrance. This study reports the discovery and biochemical characterization of a novel glycoside hydrolase family 12 (GH12) xyloglucanase, TpXEG12a, from the biomass-degrading fungus Talaromyces pinophilus. Recombinant TpXEG12a exhibited exceptional catalytic efficiency toward xyloglucan, with a specific activity of 2375 U/mg, significantly higher than the typical range reported for GH12 xyloglucanases. The enzyme displayed optimal activity at pH 4.0 and 57 °C, with high stability in acidic conditions (pH 4–8) and moderate thermal stability. TpXEG12a demonstrated strict substrate specificity for xyloglucan, with no detectable activity against cellulose-related substrates, and primarily generated characteristic xyloglucan oligosaccharides (XXXG, XLXG/XXLG, XLLG) upon hydrolysis. Structural analysis revealed that TpXEG12a exists as a stable homodimer in solution, which likely contributes to its catalytic efficiency. Notably, TpXEG12a synergistically enhanced glucose release when combined with cellulase in lignocellulosic biomass degradation. These findings establish TpXEG12a as a promising candidate for industrial applications in biomass conversion, textile processing, and functional oligosaccharide production.

Hygrocybe species are a group of brightly colored mushrooms widely distributed across the world, and they represent the most species-rich genus within Hygrophoraceae. In this study, Hygrocybe nanfengmianensis, a new species in subg. Hygrocybe sect. Hygrocybe subsect. Macrosporae, from Jiangxi Nanfengmian National Nature Reserve is described. Hygrocybe nanfengmianensis is characterized by a yellow to orange pileus, pale yellow to yellow lamellae, clavate basidia (30–39 × 8–10 µm), 2 or 4 sterigmata, and possesses clamp connections at the base. Phylogenetic analysis of the species was performed using the ITS and the LSU sequence data. A full description, illustrations, and phylogenetic analysis results of the new species are provided.

Proteins perform biological functions through the specific recognition of their interaction partners. Evolution has been sculpting the interaction patterns of native proteins that conflict with the overall folding of minimal frustration but are specific for binding. However, the strategy for precisely pinpointing the specificity-determining sites remains lacking, and the regulatory mechanism through which these sites control the binding specificity is not fully elucidated. In this work, we proposed a novel strategy that integrates local energetic frustration analysis with deep learning-based sequence generation method ProteinMPNN to identify specificity-determining sites, using the two-component signaling (TCS) system as a model. In light of the precise identification of two specificity-determining sites, we found that the cognate specificity of TCS is marginal and can be modulated by individual mutations at specificity-determining sites. The order of mutations at these two specificity-determining sites can significantly impact the evolutionary intermediates, which in turn regulates cognate specificity through epistatic effects. Moreover, mutational degeneracy is pervasive in preserving cognate specificity and preventing crosstalk. The critical role of these two sites in tuning the specificity were extensively validated using AF3-predicted structural confidence and bacterial two-hybrid assays. This work establishes a generalizable strategy for identifying specificity-determining sites, and provides insights into how evolution shapes the binding specificity.

Talaromyces pinophilus is a filamentous fungus with notable lignocellulose-degrading capacity based on enzyme activities and protein secretion potential, making it a compelling candidate for industrial biotechnology applications. In this study, we present the genomic characterization of the highly cellulolytic strain Y117, a domesticated variant of T. pinophilus, based on whole-genome sequencing and comparative genomic analysis with eleven related strains. Comprehensive analysis of CAZymes, transcription factors, and secondary metabolite diversity in T. pinophilus strains revealed that the exceptional lignocellulose degradation capacity of Y117 is driven by its unique genomic architecture. Key genomic features that distinguish Y117 include (1) significant expansion of glycoside hydrolase (GH) and carbohydrate-binding module (CBM) families, (2) loss of fungal-RiPP-like clusters, and (3) absence of the developmental regulator BrlA. These genomic adaptations could indicate a metabolic trade-off favoring hydrolytic enzyme production over secondary metabolism and sporulation. Our findings provide fundamental insights into fungal lignocellulose degradation mechanisms while establishing Y117 as a promising chassis for metabolic engineering applications in industrial enzyme production and heterologous protein expression.

A Gram-stain-negative, aerobic, non-spore-forming, nonmotile, rod-shaped, and yellow-pigmented bacterium, designated strain JXAS1T, was isolated from a freshwater sample collected from Poyang Lake in China. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the isolate belonged to the genus Flavobacterium, being closest to Flavobacterium pectinovorum DSM 6368T (98.61 %). The genome size of strain JXAS1T was 4.66 Mb with DNA G+C content 35.7 mol%. The average nucleotide identity and in silico DNA–DNA hybridization values between strain JXAS1T and its closest relatives were below the threshold values of 95 and 70 %, respectively. The strain contained menaquinone 6 (MK-6) as the predominant menaquinone and the major polar lipids were phosphatidylethanolamine, one unidentified glycolipid, and one unidentified polar lipid. The major fatty acids (>5 %) were iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C15 : 0, iso-C17 : 0 3OH, iso-C15 : 0 3OH, and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). Based on phylogenetic, genotypic, and phenotypic evidence, the isolated strain represents a new species in the genus Flavobacterium, and the name Flavobacterium poyangense is proposed. The type strain is JXAS1T (=GDMCC 1.1378T=KCTC 62719T).