Modular integration of nanopore sequencing, alphafold modeling, and statistical design boosts 1-hydroxyphenazine yield in Pseudomonas aeruginosa strain KAEH25

Résumé

Abstract 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 (R 2 = 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.

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