Salmonella enterica serovar Typhimurium strain YB1 is a genetically engineered obligate anaerobe with strong tumor-targeting potential. In this study, we evaluated YB1 as a delivery platform for Laz, a lipidated azurin-derived protein, using three secretion strategies: a native lipoprotein signal peptide (SPase II; YDL), a Sec-dependent secretory signal peptide (SPase I; YDN), and a Type III secretion signal (YDP). Laz expression was controlled by the aTc-inducible Pxyl/tet promoter on a pET29a backbone. To eliminate the confounding effects of metabolic burden, we constructed a TetR-matched control plasmid (named YC*) to be used as a comparator. Compared with YC*, Laz-expressing YB1 strains significantly reduced the viability of MCF-7 cells: YDL (50.69 ± 5.51%, p < 0.001), YDN (52.05 ± 4.69%, p < 0.001), and YDP (51.03 ± 3.92%, p < 0.001). This effect was dependent on induction conditions, with stronger responses observed at higher aTc concentrations. In vivo evaluation using the Ehrlich carcinoma mouse model revealed increased tumor necrosis and reduced mitotic activity in bacterially-treated groups. Among the constructs, YDL exhibited higher Laz expression and export, which was associated with more pronounced effects in vitro and consistent trends in vivo. Collectively, these findings support the feasibility of YB1-mediated Laz delivery as a bacterial delivery platform and highlight the importance of matched regulatory architecture in the design of engineered bacterial therapeutics. This study provides a functional evaluation of Laz-associated cytotoxic effects in the YB1 system while indicating areas for further investigation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-026-02057-x.

Fourteen fungal species were isolated from sewage water and soil, of which ten strains showed positive extracellular L-asparaginase production by plate assay. Among them, Aspergillus nidulans AUMC17371, A. flavus AUMC17373, A. flavus AUMC17374, and A. terreus AUMC17372 exhibited the highest enzyme activities (1.35, 1.25, 0.97, and 0.79 U/mL, respectively). Optimization of culture conditions revealed that maximum production was achieved using fructose as carbon source, asparagine as nitrogen source, pH 6, and 30°C. SDS-PAGE analysis confirmed enzyme production, with protein bands ranging from 35 to 45kDa. Antimicrobial screening revealed strong activity against Gram-positive and Gram-negative bacteria; however, only limited antifungal effectiveness against Candida albicans was exhibited. MIC results showed that L-asparaginase from A. nidulans AUMC17371 showed the strongest antibacterial effect against Staphylococcus aureus, followed by A. flavus AUMC17373 against S. aureus and Pseudomonas aeruginosa. Antibiofilm assays indicated inhibition ranging from 22 to 69%, with A. flavus AUMC17373 showing the most potent effect. Cytotoxicity testing revealed that A. flavus AUMC17374 enzyme exhibited anticancer activity against MCF-7 and HepG2 cell lines with IC50 values of 184 and 450µg/mL, respectively. Computational analysis supported these findings, with molecular docking demonstrating stable binding of L-asparagine to A. nidulans AUMC17371 L-asparaginase (- 4.67kcal/mol), and molecular dynamics indicating a structurally stable enzyme-substrate complex. Collectively, these results suggest that fungal L-asparaginase may serve as a promising candidate with antimicrobial, antibiofilm, and cytotoxic activities.

Fish-processing generates substantial viscera waste that is often discarded or sold at negligible prices, representing both an environmental burden and a missed economic opportunity. This study demonstrates the sustainable valorization of Catla catla viscera through green solvent extraction and urea complexation to obtain polyunsaturated fatty acid (PUFA)-rich fractions. Among extraction methods, the autoclave-assisted approach gave the highest lipid yield (16 ± 0.7%), while the classical Bligh & Dyer method preserved the most balanced PUFA profile (20.16%, with EPA 9.47% and DHA 6.82%). Green-solvent modifications (2-MeTHF and ethanol) maintained yields (~ 9.9%) while enhancing ω-3 PUFA recovery to 19.80%. Subsequent urea complexation increased total PUFA from 28.44% to 56.03% and ω-3 fractions to 49.86% at a 6:1 molar ratio. The PUFA-enriched fraction demonstrated measurable in vitro bioactivities, including antioxidant capacity (IC₅₀ < 0.2 mg mL⁻¹), enhanced wound closure of 78.79 ± 3%, and significant immunomodulation (TNF-α reduced to 112.68 pg/mL; TGF-β1 increased to 571.52 pg/mL) at 50% concentration. A preliminary economic estimation suggests that valorization of viscera into PUFA-rich lipids may substantially enhance value compared with raw disposal, although detailed techno-economic analysis and pilot-scale validation are required to confirm feasibility. This dual demonstration of bioactivity and valorization potential highlights viscera-derived lipids as promising nutraceutical and biomedical resources.

Chinese hamster ovary (CHO) cells are the predominant host for recombinant protein production. While transient gene expression (TGE) systems provide a faster, more cost-effective alternative to stable gene expression (SGE), their application is often hampered by low protein yields. Here, we optimized TGE systems based on high-density suspension transfection using 40 kDa polyethylenimine (PEI) by engineering vector regulatory elements (Kozak, polyA, introns, leader) and optimizing transfection conditions. Vector optimization led to a substantial enhancement in recombinant protein, with EGFP and SEAP levels increased by 5.33-fold and 4.13-fold, respectively (p < 0.05). By applying the optimized vector in combination with identified optimal conditions, a DNA concentration of 4 µg/mL, a DNA: PEI mass ratio of 1:5, and a repeated transfection strategy, we achieved a transfection efficiency of 76.34% while maintaining cell viability above 85%. Moreover, the combined optimization of vectors and transfection conditions robustly enhance the production of therapeutically relevant proteins, increased the expression of recombinant type III human collagen and human serum albumin by 4.86-fold and 5.50-fold, respectively (p < 0.05). In summary, high-density suspension transfection markedly boosts transient recombinant protein expression in CHO cells by improving transfection efficiency, expanding the population of recombinant cells, and maintaining high cell viability. This approach presents a promising and effective strategy for high yield transient transgene expression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-025-01999-y.

The maize rhizobacterium Enterobacter cloacae Mz49 exhibits diverse plant growth-promoting and stress-adaptive traits, indicating strong potential for bioinoculant development. Mz49 was isolated from the Egyptian rhizosphere, and demonstrated in vitro production of indole-3-acetic acid (64.89 µg/mL), antioxidant activity (IC₅₀ = 11.71 µg/mL), and anti-inflammatory properties (IC₅₀ = 63.1 µg/mL). Whole-genome sequencing revealed a 5.34 Mb genome with 5,158 protein-coding genes linked to nutrient acquisition, abiotic stress tolerance, and secondary metabolite biosynthesis. Genes associated with nitrogen fixation (nirBD, ureABCDEFGJ, amtB), phosphate solubilization (pqqIFL, phoA, pstIPN), and phytohormone synthesis (iaaT, ysnE, ipdC, ppdC, amiE) were identified. Genome analysis also revealed phytase-related genes (agp, suhB), which contribute to the release of bioavailable phosphorus, an essential nutrient for maize growth. This genetic potential highlights the strain’s contribution to enhanced plant growth and nutrient-use efficiency, particularly under phosphorus-limited conditions. Additionally, stress-response genes (sodABC, katE, betB, proABCY, dnaJK, cspA) were identified, conferring resistance to oxidative, osmotic, thermal, and cold stresses. The presence of heavy metal resistance genes (arsenic, copper, zinc, nickel) suggests suitability for use in contaminated soils. AntiSMASH analysis identified biosynthetic gene clusters for siderophores, arylpolyenes, and non-ribosomal peptides. GC–MS profiling further detected bioactive compounds, including 2, 3-butanediol, D-pinitol, succinic acid, tyrosol, and azelaic acid, which are associated with plant growth promotion and defense responses. Collectively, these findings highlight Mz49’s potential for sustainable maize cultivation, particularly in phosphorus-limited or stress-prone soils. Future research should prioritize field trials to validate its efficacy and assess potential risks associated with its application.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-025-01981-8.

High-fat diet (HFD) consumption poses a significant public health threat due to its strong association with obesity, hyperlipidemia, cardiovascular diseases, and metabolic syndrome. While Akkermansia muciniphila has emerged as a promising probiotic for ameliorating metabolic disorders, the underlying mechanisms-specifically its interplay with intestinal tryptophan metabolism-remain poorly understood. Here, we demonstrate that A. muciniphila supplementation mitigates HFD-induced metabolic dysfunction in zebrafish by orchestrating a gut microbiota-tryptophan metabolite-AhR signaling axis. Firstly, A. muciniphila treatment effectively reduced the final weight of zebrafish, partially mitigated weight gain caused by HFD, and improved survival rate. In addition, A. muciniphila treatment reduced zebrafish adiposity, hepatic steatosis, and endotoxemia while enhancing the antioxidant capacity of the liver. We further found that A. muciniphila exerted anti-apoptotic effects, improved chronic low-grade inflammatory responses, enhanced antioxidant capacity, and repaired intestinal barriers induced by HFD. These effects were partially abolished by antibiotic pretreatment, confirming microbiota dependency. Subsequent 16S rRNA sequencing analysis revealed that A. muciniphila supplementation reshaped gut microbiota, enriching Staphylococcus and Vibrionaceae, while depleting Acinetobacter, Perlucidica, and Massilia. In addition to reducing lipid synthesis, metabolomic profiled the substantial changes in microbiota-regulated tryptophan metabolism, including increased levels of indole-3-lactic acid, indole-3-acetaldehyde, and 5-hydroxyindole acetic acid in Amuc group. These metabolites activated aryl hydrocarbon receptor (AhR), upregulating downstream IL-22 and improving intestinal integrity and chronic inflammatory response. Pathway analysis and enzyme gene expression detection indicated that A. muciniphila may inhibit the KP pathway while promoting the microbiota-dependent tryptophan metabolic pathway. This study establishes a novel link by which A. muciniphila exerts protective effects against HFD-induced metabolic syndrome through microbiota-mediated enhancement of tryptophan metabolism.

Aflatoxin contamination of milk is a serious health concern. When animals ingested food contaminated with aflatoxin B1 (AFB1), it would be converted into aflatoxin M1 (AFM1) and secreted in the milk. This carcinogenic and hepatotoxic toxin could be overcome by biological methods. Therefore, this study aimed to assess the anti-aflatoxigenic effect of the probiotic Lactobacillus rhamnosus (L. rhamnosus) ATCC 7469, as well as its synbiotic combination with chitosan ZnO nanocomposite using ELISA. This is carried out by measuring AFM1 concentration in 90 milk samples, including 73 raw and 27 powdered milk samples. The average AFM1 concentration was 11.22 ± 11.31 and 10.62 ± 8.08 µg/kg, which exceeded the international regulatory limits for raw and powdered milk, respectively. All milk samples were treated with L. rhamnosus ATCC 7469 and a synbiotic combination of chitosan/ZnO nanocomposite and L. rhamnosus ATCC 7469. The results showed that the probiotic L. rhamnosus ATCC 7469, and the synbiotic combination significantly reduced the AFM1 concentration in milk (p value ≤ 0.05). The used probiotic bacteria showed binding to AFM1 from 10 to 83.8%, while the binding range increased to 34–92% after treating milk with the synbiotic combination. In conclusion, the biological treatment of milk using the probiotic, L. rhamnosus ATCC 7469, alone or in combination with chitosan/ZnO nanocomposite, is an efficient method for reducing the AFM1 concentration in milk. This study highlights the use of both metal nanoparticles (as a prebiotic) and probiotics, forming a synbiotic approach to control milk contamination with aflatoxins in the laboratory.

Heterologous overexpression of certain recombinant proteins in E. coli often triggers the formation of inclusion bodies (IBs). Traditionally viewed as inactive aggregates, IBs are now known to sometimes retain native-like folding and catalytic activity, termed catalytically active inclusion bodies (CatIBs). In this work, we investigated the CatIBs formed by recombinant LipAMS8, a cold-adapted lipase from an Antarctic Pseudomonas sp., expressed in E. coli iBL21(DE3)/pET32b. Overexpression yielded abundant insoluble aggregates which were gently isolated using 50 mM Tris–HCl, 50 mM NaCl, 1% Triton X-100 (pH 8.0), and subsequently solubilized in Tris–HCl (pH 8.0) without denaturants. Scanning electron microscopy revealed rod-like protein particles ranging from ~ 100 nm up to 1 μm. Biochemical characterization demonstrated that these LipAMS8 aggregates indeed function as CatIBs, exhibiting enzymatic activity with a defined optimum temperature, broad pH stability, specific metal ion responses, and remarkable tolerance to organic solvents. The LipAMS8 CatIBs retained significant residual activity (≥ 50%) across a wide pH range and various temperatures and showed only modest activity loss after prolonged storage (over 13 weeks at 4 °C and 8 weeks at 25 °C, maintaining > 50% activity). Notably, these aggregates displayed higher stability in extreme conditions (pH and organic media) compared to typical soluble enzymes. This study is the first to characterize a naturally formed lipase CatIB, highlighting that LipAMS8 CatIBs are produced in vivo without any artificial tags. The LipAMS8 CatIBs combine high catalytic activity with exceptional stability and solvent tolerance, underscoring an alternative strategy to obtain cold-active lipases in immobilized form. Such robust CatIB biocatalysts are highly sought after in industries for diverse biotransformations in challenging conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-025-01937-y.