Search Results (1,729)

Glutathione S-transferases (GSTs) are an important family of enzymes involved in plant detoxification, maintenance of redox homeostasis, and responses to abiotic stresses. However, the evolutionary characteristics and functional roles of the potato GST pan-gene family have not yet been systematically investigated at the pan-genome level. In this study, based on high-quality potato genomes constructed from 45 diploid accessions, GST gene family members were systematically identified, and their evolutionary features and expression patterns were analyzed. Phylogenetic analysis classified the GST family into six subgroups, among which the soft-core gene StGST7 and the near-core genes StGST8 and StGST16 were assigned to the Phi and Tau subgroups, respectively. Selection pressure analysis indicated that five StGST genes may have undergone positive selection, whereas most of the remaining genes were mainly subjected to purifying selection. Structural variation significantly affected the expression of StGST42 and the conserved domains of its encoded protein. Expression profiling revealed that GST family members exhibited clear tissue-specific expression patterns and responded differentially to drought, salt, high temperature, ABA, and IAA treatments. Co-expression network analysis revealed significant positive and negative correlations between multiple transcription factors and StGST gene expression, suggesting their potential involvement in the coordinated regulation of StGST genes. Further analyses demonstrated that StGST7 was significantly differentially expressed under multiple stress conditions, and its heterologous expression enhanced yeast tolerance to salt and drought stress. This study revealed the evolutionary characteristics and potential functions of the potato GST gene family and provides a theoretical basis for elucidating the molecular mechanisms underlying its regulation of environmental adaptation. Full article

The emerging threat of antibiotic-resistant pathogens and the limitations that conventional cancer chemotherapies display have created an urgent need for the development of innovative therapeutic strategies. Combining the pleiotropic biological roles of antimicrobial peptides (AMPs) and nanomaterials through their conjugation presents a promising possibility of targeting both microbial membranes and malignant cells. In the present study, we engineered a novel bioactive material by immobilizing the insect-derived AMP Papiliocin onto multi-walled—decorated with polyethylene–glycol—carbon nanotubes (PEG-MWCNTs) to prevent proteolytic degradation of the peptide and enhance its cellular delivery. Recombinant Papiliocin was cloned, heterologously expressed, purified and conjugated onto the PEG-MWCNT carrier. Successful expression and conjugation were validated via immunoblotting and Fourier transform infrared (FT-IR) spectroscopy, respectively. Further physicochemical characterization of the bionanocomposites was conducted using Dynamic Light Scattering (DLS) and Zeta potential measurements. Biologically, the biofunctionalized material exhibited potent, broad-spectrum antimicrobial activity both on Staphylococcus aureus and Escherichia coli, inhibiting almost 90% of the latter’s growth, highlighting the bioconjugate’s specific interactions with the Gram-negative pathogens’ membranes. Furthermore, it significantly reduced biofilm formation in Candida albicans, as indicated by the TCP assay. In parallel with its antimicrobial effects, CNTs-PEG–Papiliocin significantly reduced cancer cell viability and induced apoptosis via the extrinsic apoptosis pathway in HeLa cells, a response assisted by efficient intracellular delivery. Notably, cytotoxicity assays demonstrated lesser cytotoxic effect against non-tumorigenic HaCaT cells relative to the cancerous cell line. Collectively, these findings indicate the Papiliocin–biofunctionalized CNTs as a versatile, dual-action therapeutic agent with potential for antimicrobial activity and anticancer mode of action. Full article

With the acceleration of industrialization, water pollution caused by ammonia-nitrogen compounds has become a severe environmental challenge. In recent years, significant breakthroughs in biological nitrogen removal technology have been achieved alongside the discovery of novel ammonia-nitrogen-degrading bacterial strains. This study delves into the metabolic pathways and molecular mechanisms of ammonia-nitrogen degradation by Gordonia sp. TD-46. A comprehensive understanding of the strain’s nitrogen metabolic pathways and the functions of key genes was achieved by optimizing its ammonia-nitrogen degradation conditions, analyzing its whole-genome sequence, and conducting heterologous expression of crucial genes. The results demonstrated that when ammonium chloride served as the nitrogen source, sodium acetate as the carbon source, with a C/N ratio of 25, pH of 7, and an inoculum size of 15%, the strain achieved an ammonia-nitrogen degradation rate exceeding 80% under these conditions. Whole-genome sequencing analysis identified genes involved in nitrogen metabolism, including glnA, gdhA, narB, narGHI, nirBD, and nasBDE. These genes indicate that the nitrogen metabolism pathway of strain TD 46 follows the assimilatory nitrate reduction pathway (NO₃⁻ → NO₂⁻ → NH₄⁺) and the ammonia assimilation pathway (NH₄⁺ → Gln → Glu). Thus, strain TD-46 is capable of efficient nitrogen assimilation. Full article

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems. Full article

Anthocyanin biosynthesis in eggplant (Solanum melongena L.) is highly light-dependent, and insufficient light severely impairs fruit coloration, which restricts the development of the eggplant industry. SmMYB75 is a key positive regulator of anthocyanin biosynthesis, but its regulatory partners remain unclear. In this study, seven SmYABBY genes were identified from the eggplant genome, all containing conserved zinc finger and YABBY domains. Expression analysis showed that SmYABBY1 was predominantly expressed in fruit peel and significantly induced by light, with a peak at 4 h after light exposure. The yeast two-hybrid and bimolecular fluorescence complementation assays indicated that SmYABBY1 interacts with SmMYB75 and the light signaling regulator SmCOP1 in the nucleus. The heterologous overexpression of SmYABBY1 in Arabidopsis enhanced anthocyanin accumulation and upregulated the expression of anthocyanin structural genes. Transient co-expression in tobacco leaves further demonstrated that SmYABBY1 synergistically enhances SmMYB75-mediated anthocyanin biosynthesis. The yeast one-hybrid and Dual-LUC assays revealed that SmYABBY1 does not directly bind to the promoters of SmMYB75, SmDFR, and SmANS but indirectly promotes their transcriptional activity. Our results illustrate that SmYABBY1 acts as a transcriptional co-activator, interacting with SmMYB75 to promote anthocyanin accumulation, while SmCOP1 is involved in this regulatory process. This study provides a molecular basis for improving eggplant coloration under suboptimal light conditions. Full article

Nuclear Factor Y (NF-Y) transcription factor family plays essential roles in plant growth, development, and abiotic stress responses. However, the NF-YA subfamily in cultivated strawberry (Fragaria × ananassa) has not been systematically characterized from a genome-wide range. In this study, 27 FaNF-YA genes were identified from the octoploid strawberry genome and classified into four phylogenetic groups. With bioinformatic methods, it was found that all FaNF-YA proteins contain a highly conserved CCAAT-binding domain, while their exon–intron structures and motif compositions vary among groups. Promoter cis-acting element analysis revealed various stress- and hormone-responsive motifs, including ABRE, MYB, MYC, and MeJA-responsive elements. With molecular biology methods, organ-specific expression profiling was generated and showed that FaNF-YA genes exhibit distinct spatial expression patterns, with extremely low transcript abundance in fruit. Under salt stress, several FaNF-YA groups (e.g., FaNF-YA14/16/18/22) were dramatically induced, which indicated their potential involvement in salt tolerance. Heterologous expression of FaNF-YA7 and FaNF-YA9 in yeast enhanced salt tolerance, and these two proteins did not exhibit transcription-activating activity in the yeast GAL4 system. This study provides a reference for understanding the roles of NF-YA genes in responses to abiotic stresses and potential targets for molecular breeding of stress-tolerant strawberry cultivars. Full article

The flower color of Rhododendron is primarily determined by anthocyanin biosynthesis, with cytochrome P450 CYP75 family members, particularly flavonoid 3′,5′-hydroxylase (F3′5′H), playing a central role. However, the composition and functional characterization of CYP75 genes in Rhododendron remain insufficiently explored. This study performed genome-wide identification of the CYP75 gene family using the Rhododendron simsii reference genome and functionally characterized the corresponding F3′5′H homolog cloned from Rhododendron × hybridum petals (red cultivar and pink cultivar). Seven RsCYP75 genes were identified, categorized into two subfamilies: RsCYP75A (A1–A5) and RsCYP75B (B1–B2), with a prominent cluster on chromosome 13. All encoded proteins contained a conserved cytochrome P450 domain and typical heme-binding motifs. Among these, RhCYP75A2 showed the highest expression level in red petals at full blooming period and was designated as RhF3′5′H. RhF3′5′H encodes a basic membrane protein with the characteristic F3′5′H motif, with its transcript most abundant in flowers. Transient overexpression of RhF3′5′H in red R. × hybridum petals resulted in a 9.74-fold increase in its transcript levels and a 1.25-fold increase in anthocyanin content compared to that in the control accompanied by the up-regulation of CHS, F3H, DFR and ANS. Conversely, RhF3′5′H silencing reduced anthocyanin accumulation but increased CHS and F3H transcript levels, suggesting a compensatory transcriptional response in the upstream anthocyanin pathway. Moreover, RhF3′5′H was heterologously expressed in E. coli Rosetta as an MBP fusion protein, purified, and identified by LC-MS/MS and ELISA. The protein showed the ability to promote anthocyanin accumulation. Molecular docking analysis demonstrated that RhF3′5′H can bind to naringenin and dihydrokaempferol. These results confirm that RhF3′5′H is a functional F3′5′H-type CYP75A enzyme and a positive regulator of anthocyanin accumulation in Rhododendron petals. This work enriches the CYP75 gene catalog in Rhododendron and provides candidate genes for future studies on flower color regulation and molecular breeding. Full article

Ergothioneine (EGT), a naturally occurring amino acid derivative with potent antioxidant and cytoprotective properties, is widely applied in the food, cosmetic, and medical industries. Traditional production methods are limited by high costs, low efficiency, and environmental concerns, so microbial fermentation serves as a sustainable alternative for EGT production. In this study, Escherichia coli BL21 (DE3) was employed as the chassis strain. First, a basic EGT-producing engineered strain was constructed by heterologously expressing the egtB gene from Methylobacterium pseudosasicola along with the egtD and egtE genes from Mycobacterium smegmatis. This initial strain achieved a yield of 84.84 ± 1.64 mg/L of EGT in shake-flask cultures. To enhance production, solubility-enhancing tags were introduced to improve the soluble expression of the key enzymes, and metabolic pathways were rationally engineered to strengthen the supply of essential precursor amino acids. These modifications led to the development of a high-yield EGT strain. After optimizing the fermentation process, the best results were achieved using a medium with glycerol as the carbon source, 0.5 g/L of histidine, 1.5 g/L of methionine, and 1.0 g/L of cysteine, along with induction at 25 °C using 0.2 mM IPTG for 120 h. Under these conditions, the final EGT yield reached 385.70 ± 4.86 mg/L. The engineered strain for EGT synthesis and optimized fermentation strategy developed in this study offer a useful basis for further process development. Full article

Soil salinization is one of the major abiotic stresses limiting agricultural production. As an economically important fruit tree worldwide, grapevine generally exhibits weak salt tolerance. Therefore, identifying key stress-tolerance genes is of great significance for improving stress resistance in grapevines. In this study, the transcription factor gene VvWRKY57, which is induced by salt stress, was cloned from the grape cultivar Vitis vinifera ‘Shine Muscat’. Its function under salt stress was systematically evaluated via heterologous overexpression in Arabidopsis thaliana. The full-length CDS of the VvWRKY57 gene is 915 bp, encoding a protein of 305 amino acids. The protein contains a typical WRKY conserved domain, belongs to group II of the WRKY family, and is localized in the nucleus and cytoplasm. Expression pattern analysis showed that VvWRKY57 was expressed in roots, stems, and leaves of grapevine. Based on this expression profile, transgenic Arabidopsis thaliana plants overexpressing VvWRKY57 were generated to further investigate its role in salt tolerance. Subsequent salt tolerance assays revealed that, compared with wild-type plants, the overexpression lines exhibited stronger resistance phenotypes under salt stress. This study demonstrates for the first time that grape-derived VvWRKY57 functions in enhancing salt tolerance in model plants, providing a novel genetic resource and theoretical basis for crop salt-tolerance molecular breeding using this gene. Full article

Background/Objectives: The wall-associated kinases (WAKs) and WAK-like proteins (WAKLs) comprise a unique receptor-like kinase subfamily in plants, which have been shown to regulate plant development and defense responses by sensing cell wall-derived components, such as pectin or pectin fragments. In this study, we aimed to characterize the function of WAKL10 in flg22-triggered immunity in Arabidopsis. Methods: Through functional analyses of WAKL genes in Arabidopsis, we identified WAKL10 as the most pronouncedly induced WAKL member in response to flg22 treatment. Gain- and loss-of-function genetic analyses were performed to assess its role in flg22-triggered immune responses, including mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) burst, and defense gene induction. Transgenic Arabidopsis plants expressing a kinase domain-deleted mutant (WAKL10-ΔK) were generated. Co-immunoprecipitation assays were conducted to examine interactions with FLAGELLIN-SENSITIVE 2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Heterologous overexpression of WAKL10 in tomato was also tested for bacterial disease resistance. Results: WAKL10 positively regulates flg22-triggered immune responses. Interestingly, WAKL10-ΔK retains the capacity to potentiate these responses. Co-immunoprecipitation assays showed that both wild-type WAKL10 and WAKL10-ΔK constitutively associate with FLS2 and BAK1. Overexpression of WAKL10 in tomato confers enhanced bacterial disease resistance. Conclusions: The extracellular domain of WAKL10 promotes FLS2-BAK1 complex formation, thereby contributing to flg22 signaling. This study reveals a new function of WAKLs, distinguished from their proposed role in sensing cell wall components. The functional conservation of WAKL10 suggests its potential application in engineering disease resistance in crop plants. Full article

Carbonic anhydrases (CAs) efficiently catalyze CO₂ reversible hydration, critical for carbon capture and sequestration (CCS), but naturally low yield limits industrial use. Yarrowia lipolytica, an unconventional yeast, is an ideal heterologous expression host with robust adaptability, post-translational modification capacity, and versatile genetic tools. In this study, 10 α-, β-, and γ-class CAs were successfully expressed in Y. lipolytica, and two top-performing candidates were identified: Methanosarcina mazei γ-CA (MmaCA) and Sulfurihydrogenibium azorense α-CA (SazCA). Their production was further optimized via promoter and gene dosage adjustment, cultural condition optimization and auxiliary protein co-expression. The optimized intracellular MmaCA activity reached 960 U/mL (64.42-fold improvement), and the extracellular SazCA activity peaked at 925 U/mL (70.08-fold enhancement). CO₂ mineralization experiments confirmed both recombinant CAs significantly accelerated CaCO₃ precipitation, demonstrating a promising CCS application potential. To our knowledge, this is the first systematic investigation of CA heterologously expressed in Y. lipolytica, providing a novel strategy for the highly efficient production of CAs to enable their application in industry. Full article

The surface spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key target for the development of Coronavirus Disease 2019 (COVID-19) vaccines. Nevertheless, the mutations in the S protein, particularly in its receptor-binding domain region, have resulted in a reduced or complete loss of immunogenicity and/or protective efficacy in early vaccines against the Omicron variant and subvariants. Accordingly, continuous efforts are required to develop effective vaccines against multiple Omicron subvariants to reduce current and future threats. In this study, we designed an mRNA vaccine targeting the S protein of a recent Omicron-XEC subvariant (XEC-S-mRNA) and assessed its immunogenicity, including its broad neutralizing activity, and its protective efficacy against multiple Omicron subvariants. Our results demonstrated that the lipid nanoparticle-formulated mRNA vaccine formed an appropriate particle size with strong stability and successful antigen expression. It elicited durable cellular immune responses and broad neutralizing antibodies against multiple early and recent Omicron subvariants, thereby cross-protecting transgenic mice from challenge with a heterologous Omicron strain (KP.3). Moreover, the vaccine-induced neutralizing antibodies alone were sufficient to prevent Omicron-KP.3 infection. Overall, this study shows promise for further development of the candidate vaccine against current and future Omicron infections. Full article

dTDP-L-rhamnose (Deoxythymidine diphospho-L-rhamnose) is a crucial active sugar nucleotide that serves as the key glycosyl donor for the synthesis of rhamnose-containing polysaccharides in bacteria, holding broad application potential in pathogen-associated molecular mimicry and vaccine development. In this study, the rhamnose synthase gene cluster (Pa-RmlABCD) was successfully cloned for the first time from the marine bacterium Pseudoalteromonas agarivorans Hao 2018. Four key enzymes—Glc-1-P thymidylyltransferase (Pa-RmlA), dTDP-glucose-4,6-dehydratase (Pa-RmlB), dTDP-4-keto-6-deoxyglucose 3,5-epimerase (Pa-RmlC), and dTDP-4-keto-rhamnose reductase (Pa-RmlD)—were heterologously expressed in Escherichia coli. A one-pot four-enzyme synthesis system was constructed, and the successful synthesis of dTDP-L-rhamnose was verified by Q Exactive Focus. After correction for recovery (92% ± 2%), the actual yield reached 3.47 mg/L with a conversion rate of 53.4% ± 1.1%. Combined with bioinformatics analysis, tertiary structure modeling, and molecular docking simulations, the sequence characteristics, substrate binding modes, and catalytic mechanisms of Pa-RmlABCD were systematically elucidated. By characterizing the marine-derived Pa-RmlABCD system and achieving efficient one-pot synthesis, this work opens up a new avenue for the sustainable production of dTDP-L-rhamnose, with the potential to alleviate the current industrial supply constraints. Full article

Yeast surface display is a powerful strategy for enzyme immobilization and whole-cell biocatalysis; however, the intracellular processing of heterologous enzymes during secretion and anchoring remains poorly understood. In this study, a GH5 endoglucanase gene (eglS, 1.4 kb) from Bacillus subtilis, originally isolated from a paper mill effluent, was cloned into the pYD1 vector and expressed in Saccharomyces cerevisiae EBY100 using the Aga1–Aga2 surface display system. The recombinant strain produced clear degradation halos on carboxymethyl cellulose (CMC) plates, confirming cellulolytic activity at the whole-cell level. Zymographic analysis revealed multiple active enzyme forms depending on the cellular fraction analyzed. Intracellular extracts displayed active bands ranging from 70 to 57 kDa, consistent with immature or partially processed Aga2 fusion proteins, whereas cell wall-associated fractions showed active bands between 55 and 35 kDa, suggesting proteolytic processing during secretion and surface anchoring. The apparent specific activity of the cytoplasmic fraction was 5.33 ± 0.31 U mg⁻¹, while the cell wall-associated fraction exhibited a higher apparent specific activity (58.4 ± 10.1 U mg⁻¹). Although these values were obtained from non-purified fractions and therefore do not represent intrinsic enzymatic constants, they indicate a relative enrichment of catalytically active enzyme in the cell wall-associated fraction, consistent with functional surface display. The presence of multiple active enzyme forms and the enhanced catalytic efficiency observed in the cell wall-associated fraction suggest that the engineered yeast strain may serve as a promising whole-cell biocatalyst, with potential applications in consolidated bioprocessing (CBP) strategies for lignocellulosic biomass conversion. Full article

Petrochemical-based plastics are widely used due to their convenience and low cost, with polyethylene (PE) being the most produced globally. However, the lack of efficient and sustainable treatment methods for conventional plastic wastes has led to severe environmental pollution. A new fungus strain capable of degrading PE was isolated from soil samples collected at a waste disposal site in Henan province and identified as Aspergillus sydowii W144. After 30 days of incubation under solid-state culture conditions, the strain demonstrated significant oxidative depolymerization of low-density polyethylene (LDPE). FTIR results revealed a substantial increase in the carbonyl index of the LDPE film, while differential scanning calorimetry (DSC) analysis detected an enhanced crystallinity in the LDPE film. Notably, distinct pitting and erosion marks were observed on the surface of LDPE film using scanning electron microscopy (SEM). Quantitative analysis showed a weight loss rate of 6.39% and a reduction in Weight-Average Molecular Weight (Mw) by 50.93%. Among currently identified PE-degrading strains polyethylene, A. sydowii W144 exhibits particularly outstanding depolymerization efficiency, especially on untreated PE. Based on the whole-genome data of A. sydowii W144, a preliminary model of the putative polyethylene degradation pathway in A. sydowii W144 was constructed through homology-based sequence analysis and by referencing previously reported polyethylene degradation pathways. Laccase/multicopper oxidase plays a key role in the initial oxidation of PE. Heterologous expression of the candidate gene laccase4 in Pichia pastoris yielded an active enzyme (~56 kDa) with a laccase activity of 460 U/L, confirming its functionality. This study provides a novel microbial resource and potential enzymatic tools for PE biodegradation. The strain exhibits a promising application in complex ecosystems for PE pollution. IMPORTANCE: The polyethylene-degrading strain A. sydowii W144 isolated in this study exhibits highly efficient depolymerization capabilities, particularly under solid-state culture conditions. Genomic sequencing analysis enabled the construction of a potential polyethylene (PE) degradation pathway and facilitated the identification of key laccase and multicopper oxidase genes involved in this process. The isolation of this novel strain enriches the microbial resources available for PE waste treatment and offers new insights into the mechanisms of plastic biodegradation. Full article