Modular integration of nanopore sequencing, alphafold modeling, and statistical design boosts 1-hydroxyphenazine...

This study establishes an integrated genome-to-structure-to-process framework that significantly enhances 1-hydroxyphenazine (1-OH-PHZ) biosynthesis in Pseudomonas aeruginosa KAEH25. Nanopore long-read sequencing completely resolved the 10,358 bp phenazine biosynthetic locus, confirming intact organization of core (phzB-phzG) and tailoring (phzH, phzS) genes. AlphaFold modeling and InterProScan analysis validated conserved catalytic domains across all pathway enzymes, confirming functional coherence. Plackett-Burman screening identified temperature, pH, glucose, peptone, inoculum size, and incubation time as significant production determinants, while response surface methodology optimized these factors to achieve a maximum 1-OH-PHZ titre of 24.85 µg mL⁻¹ under refined conditions (37 °C, pH 6.5-7, 1% glucose, 2 g L⁻¹ peptone, 10 mL inoculum, 48 h). The statistical model demonstrated exceptional explanatory power (R² = 0.9605; Adeq Precision = 19.11). Preliminary antimicrobial assays demonstrated selective activity of 1-hydroxyphenazine (1-OH-PHZ), with inhibition zones of 20 ± 1 mm against Escherichia coli, 13 ± 0.5 mm against Salmonella enterica serovar Typhimurium, 13 ± 0.7 mm against Klebsiella pneumoniae, and 8 ± 0.3 mm against Staphylococcus aureus, while no inhibition was observed against Enterococcus faecalis or Listeria monocytogenes. Collectively, this systems-guided approach provides a transferable platform for optimizing microbial secondary metabolites and validates 1-OH-PHZ as a promising lead for antimicrobial development.