The dynamic changes of physicochemical properties, flavor and microbial communities in 1-, 3-, 5-, and 10-year aged Citri Reticulatae Pericarpium (CRP) was systematically analyzed, so as to reveal the quality formation mechanism of CRP. Results revealed that: (1) CRP color deepened progressively with time, total flavonoid content and antioxidant capacity of CRP significantly rose, total amino acids continuously decreased; (2)the main volatile compounds of CRP included alkenes, alcohols and esters, and it showed an initial accumulation followed by degradation trend, with 5-year aged CRP possessing the most complex flavor profile, where gamma-terpinene, benzaldehyde, and nonanol served as key age-specific flavor compounds; (3) microbial diversity peaked in 5-year aged CRP, with Proteobacteria as the dominant bacterial phylum and Ascomycota as the dominant fungal phylum, while specific genera (Vibrio, Xeromyces) were significantly associated with aging stages; (4) correlation analysis demonstrated that fungi, particularly Aspergillus and Wallemia, showed stronger associations with physicochemical properties, antioxidant capacity, and flavor development, and were more strongly linked to complex acid, aldehyde, and ketone flavors, whereas bacteria were mainly associated with basic flavor formation. The traditionally recognized attribute of "older is better" for CRP, in terms of the investigated physicochemical, antioxidant, volatile, and microbiological indicators, appears to manifest through the dynamic integration of flavonoid accumulation, enhanced antioxidant capacity, optimized flavor compounds, and microbial community succession. The 5-year CRP represents a key transition phase for flavor complexity and microbial diversity (within the sampled aging intervals), while long-term aging promotes the enrichment of functional metabolites. This study elucidates the multidimensional evolutionary patterns of CRP aging, providing systematic data support and theoretical foundations for its quality assessment, age authentication, and mechanistic research.

Flavonoid monomers from Citri Reticulatae Pericarpium, particularly nobiletin, can enhance the aroma quality of cigar tobacco leaves (CTLs) during fermentation. However, the nobiletin (herein abbreviated as CCPS)-mediated changes in chemical components and microorganisms of cigar tobacco leaves at different fermentation days remain unclear.CCPS was added to Dexue No. 1 CTLs at 0.3% (w/w), and the CTLs was fermented at 35 °C and 75% relative humidity for 30 days. To elucidate the changes of volatile and non-volatile constituents and microorganisms in CCPS-treated samples at different fermentation days, this study integrated multi-omics technologies consisting of metagenomics, HS-GC-IMS, HS-SPME-GC-MS and untargeted metabolomics for investigation. Results indicated that the total sugar, reducing sugar, protein, and pectin content of CTLs significantly decreased by 22.36–60.99% during fermentation. CCPS significantly altered leaf physicochemical indices and enzyme activities. The amino acid content presented a significant tendency of initial increase followed by decrease. The total amount of volatile organic compounds (VOCs) showed increasing trend. Alcohols, ketones, and aldehydes were the main small molecule flavor compounds, and heterocyclic aroma-producing substances were significantly enriched. Core genera (Alternaria, Aspergillus, Staphylococcus) strongly correlated with flavor metabolites. Metabolomics combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that amino acid metabolism may serve as the core regulatory pathway during CTL fermentation. This study provides theoretical evidence for elucidating the changes in chemical components and microbial communities of CTLs across different fermentation days mediated by CCPS.

Citri reticulatae pericarpium (CRP), a traditional Chinese medicine and food homologous material, is rich in bioactive flavonoids with multiple pharmacological effects. However, traditional organic solvent extraction methods for flavonoids suffer from low efficiency, high cost, and environmental pollution. In this study, ultrasound-assisted deep eutectic solvent extraction (UAE-DES) was developed as an efficient and green alternative for flavonoid extraction from CRP. The 41 DESs were screened, and choline chloride-acetamide (mole ratio 1:3) with 30% water content was identified as the optimal solvent. The extraction performance was improved by 92% compared to 70% acidified methanol. Response surface methodology (RSM) based on BoxBehnken Design (BBD) was further used to optimize the extraction process, with the optimal conditions determined as follows: solid-liquid ratio 1:28 (g/mL), extraction temperature 30 degrees C, extraction time 61 min, and ultrasonic power 210 W. Under these conditions, the maximum hesperidin content (the most abundant flavonoid in CRP) reached 27.46 +/- 0.33 mg/g. Fourier transform-infrared spectrometry (FTIR) verified the formation of strong hydrogen bonds between choline chloride and acetamide, which enhanced flavonoid solubility. Scanning electron microscopy (SEM) observations revealed that UAE-DES treatment caused more severe disruption of CRP cell structures compared to water and 70% methanol extraction, facilitating flavonoid release. Molecular dynamics (MD) simulations further elucidated the extraction mechanism at the molecular level: The multicomponent (chloride ion, acetamide, choline cation) in Des synergizes to keep flavonoids monodisperse, increasing solvent accessibility and mass transfer efficiency. In conclusion, UAE-DES is a promising green extraction technology for CRP flavonoids, and the insights into the molecular interaction mechanism provide a theoretical basis for the design and application of DES in plant active ingredient extraction.

Introduction:It is reported that addition of tangerine peel extract (flavonoids) can significantly improve the quality and flavor of tobacco leaves during fermentation. Nobiletin, tangeretin and hesperidin are the predominant flavonoids present in tangerine peel. However, their effects on the fermentation process of cigar tobacco leaves (CTL) have not yet been elucidated. Methods:Three major tangerine peel flavonoids, hesperidin (CPG), nobiletin (CCPS) and tangeretin (JPS), were applied during the fermentation of CTL) to evaluate their effects on fermentation outcomes. Gas chromatography-ion mobility spectrometry (GC-IMS) and microbiome analysis based on 16S rRNA and internal transcribed spacer sequencing were employed to characterize volatile flavor compounds and microbial community dynamics during the 30 days of fermentation. Results:A total of 157 volatile compounds were identified in CTL in this study. All tangerine peel flavonoid treatments improved the flavor quality of tobacco leaves by modulating the microbial community. The CPG group was enriched in esters and acids (e.g., n-amyl formate and propyl acetate), contributing to fruity aroma, with high relative abundances of Firmicutes and Staphylococcus, and a slight increase in Bacillus. The CCPS group accumulated abundant ketones (e.g., 3-methyl-2-cyclopenten-1-one, 3-pentanone, and 2-hydroxy-2-methyl-4-pentanone), providing caramel-like sweetness and enhancing the smoke body, while the enrichment of Sphingomonas, Rhizobium and other genera elevated bacterial diversity. The JPS group formed characteristic volatile compounds showed the highest fungal richness among all treatments. The mixed flavonoid (HHHT) group exhibited the highest abundance of Firmicutes and Staphylococcus, the lowest proportion of Proteobacteria, nearly undetectable harmful microorganisms, and the lowest microbial diversity. All treatment groups shared certain microbial trends, but distinct microbial profiles were observed among the CPG, CCPS, JPS, and HHHT groups, with significant correlations between key microorganisms and volatile flavor compounds. Discussion:In this study, the influence of tangerine peel flavonoids on CTL flavor quality and microbial community was systematically analyzed. These findings demonstrated that tangerine peel flavonoids enhanced flavor of CTL by modulating microbial communities during fermentation, and this provides new insights into CTL processing.

High pressure processing (HPP) and ultrasound-assisted extraction (UAE) can effectively shorten extraction time and increase extraction efficiency of the cold brew (CB) process. However, their application in CB citri reticulatae pericarpium (CRP) and the underlying mechanisms of flavor modulation remain poorly understood. In this study, CB-CRP beverage was prepared with HPP-assisted, UAE-assisted, and HPP+UAE-assisted extraction from 1, 3, 5, and 10 years CRP. Results revealed that the total soluble solids (TSS), total sugars, flavonoids, polyphenols, and volatile organic compounds (VOCs) and antioxidant activity of CB-CRP increased after assisted extraction. The combined application of HPP and HPP+UAE-assisted extraction exhibited the most pronounced effects. The kinds and total content of VOCs of CB beverages prepared from 10-year-aged CRP increased from 45 to 81, and from 2.44 to 5.98 μg/mL, respectively. Moreover, the combined HPP+UAE extraction promoted the enrichment of fatty and woody aroma-related compounds, which drove a shift in the flavor profile from fresh to a richer woody type. And this endowed the CB-CRP water with a more complex and multidimensional aroma profile.

IntroductionCigar fermentation is crucial for developing its characteristic aroma, exogenous microorganisms can be used to enhance fermentation. It is reported that the citrus reticulata ‘Chachi’ (Chenpi, a traditional fermented ingredient) extract can improve the flavor of cigarette. However, there is no report on the influence of Chenpi-derived microorganisms on the fermentation process and flavor quality of cigar tobacco leaves (CTLs) till now.MethodsA fermentation strain (Enterobacter hoffmannii, G5Z-2) was isolated from Chenpi, and it was applied as a bioaugmentation agent in CTLs fermentation. A multi-omics approach, including metagenomics and metabolomics, was employed to investigate its impact.ResultsInoculation with G5Z-2 significantly altered the microbial community structure, suppressing native Pseudomonas and reducing overall alpha diversity while enriching beneficial genera like Aspergillus and Staphylococcus. Metabolomic analysis revealed substantial restructuring of metabolic pathways, particularly the enrichment of amino acid metabolism (such as arginine biosynthesis and phenylalanine metabolism) and nicotinate/nicotinamide metabolism. This led to accelerated degradation of proteins and amino acids, providing precursors for Maillard reaction, and a marked increase (57.5%) in total volatile flavour compounds, including key aroma constituents from carotenoid and cembranoid degradation.ConclusionThe Chenpi-derived E. hoffmannii G5Z-2 optimises the fermentation process by modulating the microbial consortium and driving metabolic shifts towards favourable flavour development, demonstrating significant potential for improving the quality of Chinese-style cigars.

Although Citri Reticulatae Pericarpium (CRP) extracts can enhance cigar tobacco leaf (CTL) quality, the specific contributions of CRP-associated microbiota to CTL quality remain unexplored. This study investigated the potential of Bacillus cereus C3X-5, a strain isolated from CRP, as a bio-enhancer for improving CTL fermentation quality. The physicochemical analysis, metagenomics, and untargeted metabolomics were employed to systematically evaluate the strain’s impact. Results demonstrated that C3X-5 inoculation significantly elevated amylase and protease activities of CTL, and the sugar content and free amino acids content of CTL significantly reduced. Metagenomic analysis revealed that C3X-5 directed microbial community succession, reducing overall diversity while enriching beneficial functional genera such as Aspergillus. 66 key differential volatile organic compounds (VOCs) were identified with GC-IMS, which showed that C3X-5 treatment significantly increased desirable aroma compounds, including pyrazines (nutty, roasted notes) and ketones (fruity, sweet notes), while effectively suppressing green off-notes associated with 1-hexanal. Metabolomic and correlation network demonstrated that C3X-5 optimizes the CTL metabolic profile through a dual strategy: promoting key aroma-forming microbes and their metabolic pathways while competitively inhibiting fungi responsible for undesirable fatty acid derivatives. The CRP-derived strain C3X-5 enhanced CTL quality and provides a scientific basis for developing novel bio-starters for the cigar industry.

Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.

Microbial infections in deep wounds pose a major threat to human health. The development of hydrogel with both antimicrobial and injectable properties can effectively fill wounds while inhibiting microbial infections. In this study, we reported a copper ion cross-linked TEMPO-oxidized bacterial cellulose (OBC) nanofiber hydrogel. The dynamic nature of -COOH-Cu2+coordination imparted physicochemical versatility to Cu/OBC hydrogels, including injectability and optimal shear-thinning rheology. Duo to the Cu2+- mediated Fenton-like effect and its synergistic effect with glutathione (GSH), the obtained hydrogels could generate hydroxyl radical (·OH) and exhibited broad-spectrum antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Notably, the Cu/OBC/GHS hydrogel with low content of Cu2+ had good both hemolytic activity and cytocompatibility. Furthermore, the 2Cu/OBC/GSH10 hydrogel supported widespread cell attachment, improved cellular morphology, and enhanced viability, highlighting their potential as a biomaterial for sustained cell culture applications. This study provides a simple strategy to develop the injectable, antimicrobial, and biocompatible hydrogel.

The physicochemical properties, structural characteristics, antioxidant, radioprotective and lipid-lowering activities, as well as the underlying structure-activity relationships of polysaccharides extracted from Nostoc flagelliforme grown under normal (WL-EPS-1), salt stress (NaCl-EPS-1) and mixotrophic culture conditions (Glu-EPS-1) were studied. The results demonstrated that WL-EPS-1, NaCl-EPS-1 and Glu-EPS-1 were heteropolysaccharides comprising different proportions of monosaccharides and uronic acid, with different average molecular weights of 0.99 x 10(3), 1.09 x 10(3) and 1.18 x 10(3) kDa, respectively. Their intrinsic viscosity were significantly different, at 24.72, 29.98, and 41.06 dL/g, respectively. The functional groups of polysaccharides were not greatly affected, but the chemical composition, triple-helix structure, chain length and surface morphology were significantly influenced by culture conditions. In vitro bioactivity assays showed that the antioxidant activity generally increased in the order of WL-EPS-1 < NaCl-EPS-1 < Glu-EPS-1, while NaCl-EPS-1 had the best radioprotective effect, and Glu-EPS-1 had the best lipid-lowering effect. In addition, the structure-activity relationship of the polysaccharides was analyzed by partial least squares, which revealed that the most important factors affecting the antioxidant, radioprotective and lipid-lowering activities of polysaccharides were viscosity and molecular weight. This study provides a strategy for obtaining high-bioactivity polysaccharides by appropriate regulation of culture conditions, as well as opening new directions for the molecular modification of polysaccharides.

Grifola frodosa polysaccharides, especially β-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase ( GLS ), UTP-glucose-1-phosphate uridylyltransferase ( UGP ), and UDP-xylose-4-epimerase ( UXE ) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa , providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. Key points •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

Introductionβ-nicotinamide mononucleotide (β-NMN) is an essential precursor of nicotinamide adenine dinucleotide (NAD+) and plays a key role in supplying NAD+ and maintaining its levels. Existing methods for NMN production have some limitations, including low substrate availability, complex synthetic routes, and low synthetic efficiency, which result in low titers and high costs.MethodsWe constructed high-titer, genetically engineered strains that produce NMN through a new pathway. Bacillus subtilis WB600 was used as a safe chassis strain. Multiple strains overexpressing NadE, PncB, and PnuC in various combinations were constructed, and NMN titers of different strains were compared via shake-flask culture.ResultsThe results revealed that the strain B. subtilis PncB1-PnuC exhibited the highest total and extracellular NMN titers. Subsequently, the engineered strains were cultured in a 5-L fermenter using batch and fed-batch fermentation. B. subtilis PncB1-PnuC achieved an NMN titer of 3,398 mg/L via fed-batch fermentation and glucose supplementation, which was 30.72% higher than that achieved via batch fermentation.DiscussionThis study provides a safe and economical approach for producing NMN on an industrial scale.

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

In this work a-amylase was immobilized on magnetic Fe3O4 nanoparticles with polyethylenimine (PEI)/ polydopamine (PDA) coating or 3-aminopropyl triethoxysilane (APTES) for the first time via adsorption-precipitation-cross-linking. Compared with the free a-amylase, the resultant magnetic cross-linked a-amylase aggregates (PEI/PDA-M-CLEAs and N-M-CLEAs) exhibited excellent thermal and storage stability as well as pH stability. After storage at 25 C-degrees for 60 days, free a-amylase only retained 60% of its initial activity, while PEI/PDA-M-CLEAs and N-M-CLEAs retained 80% and 78% of their initial activities, respectively. Furthermore, N-M-CLEAs and PEI/PDA-M-CLEAs showed good reusability. After 6 repeated uses, PEI/PDA-M-CLEAs and N-M-CLEAs still maintained 65% and 62% of their initial activities, respec-tively. Especially, PEI/PDA-M-CLEAs and N-M-CLEAs exhibited higher starch hydrolysis efficiency than free a-amylase. The maximum dextrose equivalent (DE) values of starch hydrolysis by PEI/PDA-M-CLEAs and N-M-CLEAs reached 29.24% and 28.79% within 90 min, respectively. However, the maximum DE values of starch hydrolysis by the free a-amylase was only 27.89% even in 150 min. The magnetic cross-linked a-amylase aggregates could be introduced as effective biocatalyst for industrial applications in production of maltose syrups. (c) 2023 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Each year, economic losses in the food industry due to spoilage of grain, aquatic products and fruit are huge. People now express more concern about food safety and nutrition, therefore, the need for green preservatives is also growing. Epsilon-poly-L-lysine (ε-PL), a cationic polyamino acid with 25–35 L-lysine residues, possesses broad-spectrum antimicrobial activity, biodegradable properties, resistance to high temperature, and non-toxicity and can dissolve in water. So, it has been extensively applied in the field of preservatives for foodstuffs, agriculture and biomedicine. Thus, the chapter mainly focuses on the recent research on microbial synthesis, production enhancement, and antimicrobial mechanism, as well as improving food safety, its utilization in food packaging materials and agriculture of εPL .

Pure gelatin film often exhibits high hydrophilicity and a lack of antibacterial activity, hindering its practical application in the field of food preservation. To address these issues, we incorporated 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized bacterial cellulose (TOBC) nanofibers stabilized cinnamon essential oil (CEO) Pickering emulsions into the gelatin matrix to develop active food packaging films. The study revealed that the good distribution of emulsion droplets in the film matrix. While with increasing Pickering emulsion proportion, the microstructures of composite films were more heterogeneous, showing some pores or cavities. In addition, the insertion of TOBC-stabilized CEO emulsions could improve the elongation at break (EAB), water-resistance, UV blocking ability, and antibacterial activity of film, but reduced its tensile strength (TS) and water vapor barrier properties (WVP). Notably, the film prepared with 4 % TOBC-stabilized CEO Pickering emulsion demonstrated enhanced preservation of strawberries. Overall, the as-prepared gelatin-based active composite films have considerable potential for food packaging.

Skin wounds are repaired by a complex series of events and overlapping phases in which bacterial infection and insufficient angiogenesis at the wound site delay the healing process. Thus, functional wound dressings with enhanced antibacterial activity and angiogenic capacity have attracted attention. Herein, bacterial cellulose (BC)-based dressings were successfully fabricated by functionalization with a polydopamine (PDA) coating and copper sulfide nanoparticles (CuS NPs). Under 808 nm laser illumination, the BC/PDA/CuS composite membranes exhibited outstanding adjustable photothermal and photodynamic activities as well as controlled Cu2+ release, endowing the composite membranes with synergetic antibacterial activity. Specially, a bactericidal efficiency of 99.7 % and 88.0 % for Staphylococcus aureus and Escherichia coli was achieved after treatment with BC/PDA/CuS5 sample under NIR irradiation (0.8 W/cm2, 10 min), respectively. Moreover, the BC/PDA/CuS5 composite membrane could enhance the angiogenesis due to the released Cu2+. In vivo experiments revealed that the BC/PDA/CuS5 composite membrane dressing could accelerate the wound closure process of the full-thickness skin defects with S. aureus by synergistically reducing inflammation, enhancing collagen deposition, and promoting vascularization under NIR irradiation. Additionally, the BC/PDA/CuS5 composite membrane exhibited high biocompatibility and biosafety. This work offers a new strategy to prepare multifunctional BC-based dressing for clinical wound healing.

Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.