Intestinal inflammation associated with inflammatory bowel disease (IBD) is driven by complex interactions between host immunity and gut microbiota, necessitating alternative therapeutic strategies beyond conventional immunosuppressive drugs. In the present study, a dual-origin probiotic formulation comprising Lactobacillus plantarum AG40V (plant-derived) and Lactobacillus fermentum HO17HF (human-derived) was developed and evaluated for its safety and anti-inflammatory efficacy in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model in Sprague–Dawley rats. The formulation (1012 CFU/mL; 1:1 ratio) exhibited stability under refrigerated conditions and demonstrated significant in vitro functional antioxidant activities. Acute and sub-acute oral toxicity studies, conducted in accordance with OECD guidelines, confirmed the safety of the formulation, with no mortality or significant alterations in physiological, haematological, biochemical, or histopathological parameters. In the TNBS-induced colitis model, oral administration of the probiotic formulation resulted in marked attenuation of intestinal inflammation, as evidenced by reduced levels of nitric oxide, myeloperoxidase, and lipid peroxidation, along with enhanced catalase activity. Collectively, these findings demonstrate that the dual-origin probiotic formulation is safe and functionally effective in mitigating intestinal inflammation through modulation of oxidative stress and host inflammatory responses. This study highlights the translational potential of ecologically diverse probiotic consortia as a promising strategy for the management of inflammatory gastrointestinal disorders.
Replication of low-copy-number iteron plasmids is tightly controlled by initiator proteins that coordinate replication initiation with transcriptional autoregulation. In the F-plasmid system, RepE fulfills both functions through a balance between monomeric and dimeric forms. Here, random mutagenesis of the repE gene combined with high-stringency selection was used to identify variants that modulate plasmid copy number. Two point mutations, A78S and Q45L, were identified and shown by qPCR and ddPCR to increase the copy number of plasmid pFO1 relative to the parental repE(R118P) allele. The mutations exhibited growth-phase-dependent effects, with A78S having a stronger impact during exponential growth and Q45L during stationary phase. A double mutant showed a cumulative effect, particularly in exponential phase. RepE transcription increased in the presence of pFO1 and correlated with plasmid copy number, supporting a titration-based regulatory model. These findings demonstrate that engineering RepE variants enables tunable control of plasmid copy number, offering a useful strategy for optimizing gene dosage in synthetic biology and biotechnological applications. • RepE variants enable fi ne-tuning of plasmid copy number. • Gene dosage can be modulated without changing plasmid architecture. • RepE engineering off ers potential applications in biotechnology.
Cytochrome P450 reductases (CPRs) are microsomal diflavin oxidoreductases that transfer electrons from nicotinamide cofactors to class II cytochrome P450 monooxygenases (CYPs). CPRs can also reduce other proteins, including cytochrome b5, heme oxygenase, and components of the fatty acid elongation system, as well as nonphysiological acceptors such as cytochrome c, ferricyanide, and various drugs. Although structural and functional studies have provided valuable insights into CPRs, the available data remain insufficient to fully elucidate their electron transfer mechanism and interactions with diverse redox partners. Here, we report the structural and functional characterization of two fungal CPRs from Candida albicans (CalCPR) and Mycosarcoma maydis (formerly Ustilago maydis, UmaCPR) and compare their properties with previously characterized CPRs. N-terminal truncation enabled soluble expression in Escherichia coli, and kinetic parameters were determined using cytochrome c and ferricyanide. Although UmaCPR shares greater sequence similarity with ascomycete CPRs than with mammalian CPRs, its overall structure displays features more closely resembling mammalian CPRs, including its FAD-binding topology. We further investigated the ability of both CPRs to support C. albicans CYP52A21 activity and evaluated the effects of reaction environment and redox mediators on CYP–CPR reconstitution. Both truncated CPRs supported CYP activity, with CPR performance strongly influenced by the reaction environment. Addition of the redox mediator cytochrome b5 from M. maydis significantly enhanced product formation, including when a nonnative CPR was used. These findings highlight the structural diversity and functional plasticity of CPRs and demonstrate that the broader redox environment can strongly influence CYP–CPR efficiency, extending the complexity of electron transfer beyond the canonical CYP–CPR pairing. • CPRs from ascomycetes and basidiomycetes fungi exhibit distinct structural features. • The CPR from Mycosarcoma (Ustilago) maydis is structurally more similar to mammalian CPRs. • CYP-CPR activity is strongly influenced by reaction environment and cytochrome b5 and extends beyond the canonical protein pairing.
Sophorolipids (SLs) are microbial biosurfactants with broad industrial potential, but their large-scale application relies on robust high-producing strains with desirable functional properties. In this study, Starmerella bombicola CGMCC 1576 was subjected to nitrogen (N+) ion beam mutagenesis to improve SL production and product performance. A mutant library containing 124 isolates was constructed, and two stable high-producing mutants, 2–21 and 3–5, were obtained through high-throughput screening and shake-flask validation. Mutant 2–21 preferentially accumulated acidic sophorolipids (ASL) and showed the highest total sophorolipid (TSL) production, whereas mutant 3–5 produced more lactonic sophorolipids (LSL). HPLC profiling showed that the mutants retained the major SL components of the parental strain but differed markedly in component abundance and product distribution. Transcriptional analysis revealed upregulation of genes associated with fatty acid utilization, SL biosynthesis and metabolite transport, particularly in mutant 2–21 at 96 h, suggesting enhanced precursor conversion and product accumulation. Functional assays further showed composition-dependent differences in surface activity and mildness. LSL-rich fractions exhibited stronger surface activity, while ASL-rich fractions showed lower zein solubilization and hemolytic activity. In 100-L bioreactor fermentation, mutant 2–21 produced 224.31 g/L TSL, confirming its scalability and production robustness. These results demonstrate that N+ ion beam mutagenesis is an effective strategy for developing high-producing S. bombicola strains and tailoring SL composition for functional applications.
The rumen microbiome plays an important role in converting dietary carbohydrates into metabolites associated with host physiology, yet the relationships between microbial carbohydrate metabolism and host lipid metabolism remain incompletely understood. Here, we compared Japanese Wagyu and Nanyang cattle to investigate microbiota–metabolite–host associations in relation to breed-associated differences in intramuscular fat (IMF) deposition. Metagenomic analysis revealed differences in rumen microbial community composition and functional profiles between breeds, including differential abundances of carbohydrate-active enzyme (CAZyme) families associated with polysaccharide degradation. Wagyu cattle exhibited higher abundances of glycoside hydrolase (GH) families GH5 (cellulose degradation) and GH92 (alpha-mannan degradation), whereas GH13 (starch degradation) and GH43 (arabinoxylan and arabinan degradation) were enriched in Nanyang cattle, together with differences in carbohydrate-degrading genera, including Prevotella, Fibrobacter, and Butyrivibrio. These microbial functional differences were accompanied by distinct fermentation profiles, with higher ruminal acetate and butyrate concentrations in Wagyu cattle and higher propionate concentrations in Nanyang cattle. Proteomic analysis of the longissimus dorsi muscle identified breed-associated differences in proteins involved in lipid synthesis, lipid storage, and energy metabolism, including FABP4, FASN, PLIN1, ACSS2, and IDH1, which showed higher abundances in Wagyu cattle. Correlation analyses identified associations among microbial taxa, CAZyme families, fermentation metabolites, and host proteins related to lipid metabolism and IMF deposition. Overall, these findings reveal coordinated breed-associated differences in rumen microbial functions, fermentation characteristics, and host lipid metabolic profiles, providing insights into microbiota–host metabolic associations related to IMF deposition in cattle. • Rumen carbohydrate-active enzyme (CAZyme) profiles differ between Wagyu and Nanyang cattle. • Wagyu cattle show higher acetate and butyrate levels. • Microbiota–volatile fatty acids (VFAs)–proteome axis is linked to intramuscular fat (IMF) deposition
