Bacterial cellulose (BC)-based dressings exhibit favorable hydrogel characteristics, including high biocompatibility, moisture regulation, and mechanical adaptability, making them suitable candidates for biomedical applications. In this study, an integrated approach was employed to develop multifunctional, bioactive bionanocomposites. A cellulose-producing bacterial strain, Limosilactobacillus fermentum 6BC (accession number OM978241.1), was isolated from spoiled grapes and identified through 16 S rRNA gene sequencing. The structural and chemical characteristics of the synthesized BC were analyzed using Transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). To enhance the biological functionality of BC, zinc oxide (ZnO) nanoparticles were incorporated via a sono-coprecipitation method to produce ZnO-decorated BC (BCZO). The resulting BCZO was subsequently integrated into a hydroxypropyl ethyl cellulose (HPEC) matrix, forming a bionanoplatform designed for the immobilization of bioactive agents, specifically curcumin (Cc) and propolis extract (Pp). TEM was used to examine the nanoscale distribution of ZnO, while FTIR, XRD, SEM, and EDS were employed to characterize the composite structure and confirm drug incorporation. The fabricated scaffolds were further subjected to comprehensive antimicrobial, antioxidant, cytocompatibility, and in vitro wound healing assessments to evaluate their biological performance. The antimicrobial assays demonstrated effective inhibition of L. monocytogenes, S. aureus, E. coli, Salmonella sp., and the fungus C. albicans. The bionanoplatforms also exhibited concentration-dependent antioxidant activity in DPPH and ABTS assays (ascorbic acid as control). The cytocompatibility tests on human skin fibroblasts (HFB-4) showed excellent cell viability across all formulations. Among them, Cc/Pp50@BCZO/HPEC displayed the exciting antimicrobial and antioxidant performance, coupled with desirable cytocompatibility. Thus this study offers a systematic framework for engineering cellulose-based bioplatforms as bioactive materials for potential drug delivery and wound-healing applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02826-6.

BackgroundPeanut meal, a high-protein agricultural by-product, faces challenges as animal feed due to anti-nutritional factors, poor protein digestibility, aflatoxin contamination, and imbalanced amino acids. Microbial fermentation is one of the most effective methods to reduce anti-nutritional factors and enhance the nutritional value of peanut meal. Compared to single-strain fermentation, microbial-enzyme co-fermentation exhibits enhanced degradation efficiency, accelerates nutrient release, improves product safety by reducing mycotoxins and anti-nutritional factors, enhances sensory properties, and increases fermentation consistency and stability. However, research on microbial-enzyme co-fermentation of peanut meal remains limited, particularly regarding the co-fermentation of Weizmannia coagulans and Bacillus subtilis with enzyme preparations, which has yet to be systematically investigated. Therefore, this study aims to evaluate and optimize the use of Weizmannia coagulans for microbial-enzyme co-fermentation to enhance the nutritional quality and reduce anti-nutritional factors in peanut meal.ResultsPeanut meal fermentation with Weizmannia coagulans BC01, Bacillus subtilis BS27, and the synergistic enzyme system (acid protease and hemicellulose) was optimized through single factor experiments and response surface methodology. This process led to significant reductions in crude fiber (from 7.05 to 2.88%) and anti-nutritional factors, with trypsin inhibitors decreasing from 0.30 to 0.03% and phytic acid from 1.43 to 0.35%. Aflatoxin B1 was reduced from 43.87 µg/kg to 6.20 µg/kg. Nutritional quality improved markedly, with flavonoid content increasing from 1.2 to 3.01%, reducing sugars increasing from 0.7 to 7.02%, and total acids increasing from 1.65 to 5.92%. Protein composition and digestibility were markedly improved, with crude protein, acid-soluble protein, and small peptide contents increasing by 18.2%, 546%, and 447%, respectively. Additionally, the degree of protein hydrolysis and in vitro digestibility rose to 40.77% and 68.91%, respectively. Total amino acid content increased by 14.3%, contributing to a more balanced amino acid profile.ConclusionThe study indicates that microbial-enzyme co-fermentation involving W. coagulans effectively reduces anti-nutritional factors in peanut meal while enhancing its nutritional value. These findings provide a sustainable approach to transforming peanut meal into high-value animal feed, offering a practical solution to address the protein feed supply gap. Further validation via animal trials is necessary to evaluate its efficacy as a replacement for conventional protein feed sources.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02816-8.

Silver nanoparticles (AgNPs) have attracted considerable interest for their distinctive physicochemical properties and wide-ranging applications in nanomedicine, environmental catalysis, and antimicrobial applications. However, sustainable and robust biosynthesis methods remain a challenge. In this study, we report the biosynthesis of thermostable AgNPs using the secretome of Geobacillus stearothermophilus GF16, a thermophilic and metal-resistant bacterium isolated from the hydrothermal volcanic area of Pisciarelli, Italy. The synthesis was performed without specialized growth media, relying solely on the cell-free bacterial supernatant, and was systematically optimized by varying precursor concentration, temperature, pH, and reaction time. The nanoparticles were characterized by UV-Vis spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, scanning (SEM) and transmission (TEM) electron microscopy. Morphological analysis showed predominantly subspherical nanoparticles with average diameters of 17 ± 5nm (SEM) and 16 ± 5-7nm (TEM), depending on precursor concentration. Thermogravimetric analysis demonstrated excellent thermal stability with retention of structural integrity up to 120°C, an exceptional feature among biogenic AgNPs. The obtained AgNPs exhibited remarkable radical scavenging activity, reaching up to 79% in DPPH and 75% in ABTS assays at 100µg/mL, highlighting a level of antioxidant performance rarely observed in AgNPs of bacterial origin. In addition to their redox properties, the nanoparticles demonstrated efficient catalytic activity as evidenced by the complete degradation of Congo Red in 20min and 4-nitrophenol in 35min. Time-kill assays and minimum inhibitory concentration (MIC) also showed a broad-spectrum antimicrobial potential with complete inhibition of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella Typhimurium at 100µg/mL. Interestingly, MIC values were significantly lower than those reported for comparable AgNPs. Notably, the nanoparticles also displayed hemocompatibility, validated by hemolysis assays performed on both healthy and β-thalassemic erythrocytes, with hemolysis rates consistently below the 2% safety threshold. Overall, this study presents the first comprehensive characterization of AgNPs biosynthesized by a thermophilic bacterium, highlighting their multifunctional potential. The use of a thermophilic bacterium as a robust and flexible microbial nanofactory offers a novel eco-friendly and scalable strategy for AgNP production. The resulting nanoparticles exhibit unique thermal stability, broad-spectrum bioactivity, and clinically relevant hemocompatibility, underscoring their promising applicability in nanomedicine, green catalysis, and environmental remediation.

BackgroundGluten proteins from wheat, rye, and barley play a substantial role in human nutrition. At the same time, they can trigger several different immune reactions. This, together with their influence on the quality of grain products and their emerging role as biomaterials, makes them an interesting target for further study. The proteins’ propensity for aggregation challenges heterologous eukaryotic production systems. The yeast Komagataella phaffii has demonstrated excellent qualities as a production host for heterologous proteins and was therefore investigated as a platform strain.ResultsA gene coding for the rye (Secale cereale) prolamin 75k γ-secalin was cloned and inserted into K. phaffii; protein expression was verified via mass spectrometry and immunoblotting and quantified via ELISA. Different parameters were investigated regarding their effect on target protein production and endoplasmic reticulum (ER) homeostasis, including the induction temperature and co- and post-translational import into the ER. At 28°C, the cells produced 1.69-fold more 75k γ-secalin than at 20°C. The introduction of the MATα-pro-region, in conjunction with either the MATα-pre- or OST1-pre-signal, led to significantly lower 75k γ-secalin accumulation, 0.20- and 0.18-fold, respectively. No mutant showed significant changes in the unfolded protein response compared to a non-producing strain.ConclusionsK. phaffii is a suitable host for prolamin production. The absence of a significant unfolded protein response during 75k γ-secalin expression indicates little challenge of ER-homeostasis by the aggregation-prone protein. It underscores K. phaffii’s imminent role in protein production. The significantly decreased protein yield through the common protein secretion leader component MATα-pro demonstrates the need for further investigation into the role of secretion signals in optimizing K. phaffii as a production platform for repetitive, aggregation-prone proteins.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02809-7.

Plasmid DNA (pDNA) is a cost-driving reagent for the production of gene therapies and DNA vaccines. Improving pDNA production in the most common production host (E. coli) has faced obstacles arising from the complex network of genes responsible for pDNA synthesis, with the specific enzyme(s) limiting pDNA yield remaining unidentified. To address this challenge, we employed an inducible genome-wide mutagenesis strategy, combined with fluorescent screening, to isolate E. coli NEB 5α strains with enhanced pDNA production. Following selection, we successfully isolated an E. coli strain (M3) with elevated plasmid copy numbers (PCNs) across multiple origin types. Specifically, we observed a 5.93-fold increase in PCN for the GFP reporter plasmid, a 1.93-fold increase for the gWiz DNA vaccine plasmid, and an 8.7-fold increase for the pAAV-CAGG-eGFP plasmid, all of which contain pUC origins. In addition, plasmids with p15A and pSC101 origins showed 1.44-fold and 1.68-fold increases in PCN, respectively. Whole-genome sequencing of the adapted strain M3 identified 85 mutations, including one in recG, which encodes an ATP-dependent DNA helicase. Replacement of the mutant recG with its wild-type counterpart in the mutant strain resulted in a 63% reduction in PCN, but the recG mutation alone was insufficient to increase PCN in the wild-type strain. These findings suggest that the recG mutation plays a synergistic role with other genomic mutations to drive PCN increases. Taken together, this study presents the development of a pDNA hyperaccumulating E. coli strain with promising applications in industrial and therapeutic pDNA production, while also offering important insights into key genes involved in pDNA production.

BackgroundRecombinant phytase production has recently gained increased recognition in phosphate recycling from phytate contained in plant-based side and waste streams. Until now, new phytase variants are evaluated at the end of the expression by standard offline screening procedures, where promising candidates with high activities and protein titers are identified. However, for large mutant libraries, this implies extensive laboratory work for a first screening of hundreds of clones. In this study, for the first time, two synergistic concepts for the primary screening of phytases were investigated.ResultsThe aim was to predict high recombinant protein producer strains as well as high volumetric activity phytase variants, based on the development of the respiratory activity over time of the host cell, in this case, Komagataella phaffii (Pichia pastoris). In a first step, the metabolic burden was investigated by cultivating a clone library in YPD medium in a µTOM device. It was found that strains expressing medium or high protein concentrations show clear characteristics of an elevated level of metabolic burden during constitutive expression. However, a high protein concentration does not imply a high enzymatic activity. Therefore, in a second approach, the screening was adapted to screen for phytase variants with high volumetric activity. To do so, a modified Syn6 MES medium was developed, where phytic acid was used as the only phosphate source. Thereby, only clones secreting active phytase and generating free phosphate were able to grow, which was monitored via the oxygen transfer rate. A correlation between the offline measured volumetric phytase activity and µmax was found. The clones were then ranked according to their online and offline performance and the results matched in 83% of the cases.ConclusionOnline monitoring of the oxygen transfer rates in 96-well plates allowed for the evaluation of the total protein concentration and the volumetric phytase activity already during the expression. Using these results, also the specific activity can be calculated. In the future, primary screening experiments of large enzyme mutant libraries can be conducted without offline activity assays, to identify promising candidates.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02806-w.

BackgroundCis,cis-muconic acid (ccMA), an important platform chemical, can be produced from lignin related molecules (LRM) via a specific two-branch catabolic route known as the β-ketoadipate pathway, which is present in certain soil bacteria. This pathway enables high production yields because ccMA is a native intermediate in one of its branches. However, commonly obtained LRM, such as p-coumaric and ferulic acid, are typically metabolized through the branch that lacks the ccMA intermediate. To redirect these LRM toward ccMA production, the two branches must be functionally integrated. This is usually achieved by introducing a non-native enzymatic activity, specifically protocatechuate decarboxylase (PCADC), which catalyzes the conversion of protocatechuate to catechol. Nevertheless, this conversion often represents the rate-limiting step in the production process.ResultsHere, we established a growth-coupled selection system for screening PCADCs using the soil bacterium Acinetobacter baylyi ADP1 as the host. In this system, cell growth depends on the in vivo performance of PCADC, thereby enabling the selection of the optimal candidate for further ccMA production. In total, five PCADC candidates were screened. AGDC1, a gallic acid decarboxylase from the yeast Blastobotrys adeninivorans, was selected for production studies. In fed-batch cultivations, the engineered strain expressing AGDC1 achieved an 83% molar yield of ccMA from ferulate and p-coumarate, that were found in lignin hydrolysate derived from straw.ConclusionIn this study, we established a growth-based selection system for PCADCs. The outcome of the selection system was further validated in production cultivations using an engineered A. baylyi ADP1 strain. This study not only confirms the feasibility of AGDC1 in ccMA production in bacterial systems but also provides a practical screening system for future improvements.

Biosynthesis of CdSe quantum dots is the process of converting metal ions into semiconductor nanomaterials. Studies have shown that CdSe quantum dots synthesized by Saccharomyces cerevisiae are rich in ribosomal proteins, but the role of ribosomal proteins in the synthesis of CdSe quantum dots remains unclear. In this paper, ribosomal proteins enriched during the synthesis of CdSe quantum dots by S. cerevisiae were screened, and their effects on the synthesis of quantum dots were detected by gene knockout and over-expression. The results reveal that ribosomal protein RPL14B is involved in the synthesis of quantum dots. RPL14B binds cadmium ions during the nucleation of CdSe quantum dots and acts as a template, ultimately regulating the particle size of CdSe quantum dots by change the incubation time of CdCl2. In summary, this study elucidates the mechanism of ribosomal protein RPL14B regulation of CdSe quantum dot biosynthesis, laying a foundation for the precise regulation of CdSe quantum dot synthesis.

BackgroundThe global rise of antimicrobial resistance has accelerated the search for sustainable and eco-friendly therapeutic alternatives. This study evaluates fish gut waste (FGW) as a low-cost, renewable substrate for producing antifungal peptides through the proteolytic activity of Serratia liquefaciens NRC1.ResultOptimization of protease production using statistical designs resulted in a fourfold increase in enzyme yield. The protease demonstrated stability at neutral pH and moderate temperatures (40–50 °C), and efficiently hydrolyzed complex proteins such as collagen and keratin. Peptides generated from FGW hydrolysis exhibited significant antifungal activity against Candida auris (C. auris), with a MIC₅₀ of 5.1 ± 0.08 mg/ml. Unlike fluconazole, repeated peptide exposure did not induce resistance, nor did it alter the expression levels of key resistance genes (CDR1, ERG11), as confirmed by qRT-PCR. Peptide profiling using MALDI-TOF/MS, coupled with in silico analysis via AMPfun, identified multiple candidates with predicted antifungal properties.ConclusionThis research highlights the potential of fish gut waste-derived peptides as a sustainable and effective antifungal strategy against C. auris, offering an alternative to conventional antifungal drugs.