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).

Proteins perform their biological functions through specific recognition of their interaction partners, but approaches for pinpointing specificity-determining sites and elucidating how they govern binding specificity remain limited. Here, using two-component signaling systems (TCSs) as a model, we combine local energetic frustration analysis with ProteinMPNN-based sequence generation to identify two specificity-determining sites. We find that cognate specificity in TCSs is intrinsically marginal and can be reconfigured by single mutations at these sites. The order in which mutations arise shapes evolutionary intermediates and tunes specificity through epistatic effects. We further show that mutational degeneracy is widespread and contributes to maintaining cognate specificity while limiting crosstalk. The critical roles of these two sites in tuning specificity are supported by AF3-predicted structural confidence and bacterial two-hybrid assays. This work establishes a generalizable strategy for identifying specificity-determining sites and provides mechanistic insight into how evolution shapes protein binding specificity.

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.

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.

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.