Regulatory Network Dissection and Engineering of EPS Biosynthesis for Enhanced Biofilm and Functional Food Matrix Stability

The intricate regulatory landscape governing exopolysaccharide (EPS) biosynthesis in Enterococcus faecium probiotic strains was dissected employing multi-layered systems biology approaches integrating genomics, transcriptomics, and metabolomics. Whole genome sequencing with >۱۵۰x coverage revealed a complex EPS gene cluster architecture comprising glycosyltransferases, polysaccharide polymerases, and regulatory operons intricately modulated by multiple two-component systems (notably liaFSR and walRK) alongside alternative sigma factors (o^B). Transcriptomic profiling under industrially relevant osmotic (≥۰.۵ M NaCl) and thermal (۴۵°C) stresses demonstrated significant upregulation (۳-۵ fold, p < ۰.۰۱) of key EPS biosynthetic genes, highlighting adaptive transcriptional reshaping to enhance polymer output. Metabolomic LC-MS/MS analyses quantified elevated intracellular pools of nucleotide sugar precursors-UDP-glucose and GDP-mannose-registered at ۲.۸- and ۳.۲-fold increments respectively, corroborating enhanced substrate flux into EPS assembly pathways. CRISPR-Cas ۱۲a-mediated precision genome editing targeting transcriptional repressors within EPS operons yielded a ۴۷% increase in EPS yield (quantified at ۴.۶ ± ۰.۳ mg/mL) and a concurrent ۵۵% amplification in biofilm biomass, verified by crystal violet assays. Engineered strains exhibited superior physicochemical stability, maintaining matrix viscosity and cohesiveness under simulated industrial shear stress (up to ۵۰۰ s^-۱) and pH oscillations (۳.۵-۶.۵), critical for fermented food product integrity. Advanced metabolic flux modeling using constraint-based reconstruction and dynamic flux balance analysis integrated multi-omics datasets, achieving predictive accuracies exceeding ۹۰% for EPS production dynamics and biofilm formation rates. This modeling facilitated in silico optimization of pathway bottlenecks and guided rational strain engineering strategies. Collectively, the study provides a comprehensive quantitative framework linking regulatory networks, metabolic flux rerouting, and functional phenotype enhancement in E. faecium. The findings elucidate molecular mechanisms underpinning EPS-mediated biofilm robustness and functional food matrix stabilization, presenting a scalable blueprint for probiotic strain optimization aimed at industrial fermentation and food biotechnology applications.

کلیدواژه ها:

Exopolysaccharide biosynthesis ، Regulatory network ، Genome editing ، Biofilm ، Metabolic flux modeling ، Probiotic