Microbial Prodigiosin shows broad-spectrum bioactivity confirmed by experimental and computational analysis

Muhammad Rafiq^1,2Noor ul Huda³Noor Hassan^3*Hazrat Ali³Abdul Tawab³Rizwan Bashir³Naveed Iqbal^2,4Zara Rafaque⁵Faisal Ahmad^6,7Wang Yanyan¹Waqar Rauf³Anam Saqib³Iqra Jawad³Yingqian Kang^1,8,9*

• ¹Key Laboratory of Environment Pollution Monitoring and Disease Control Ministry of Education, Guiyang, China
• ²Balochistan University of Information Technology Engineering and Management Sciences, Quetta, Pakistan
• ³National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
• ⁴The Aga Khan University Department of Paediatrics and Child Health, Karachi, Pakistan
• ⁵Hazara University Mansehra, Mansehra, Pakistan
• ⁶National Institute for Health,, Islamabad, 44000, Pakistan
• ⁷DAI Fleming Fund Country Grant, 44000 Pakistan, Islamabad, Pakistan
• ⁸Guizhou Medical University School of Basic Medicine, Guiyang, China
• ⁹Guizhou Medical University, Guiyang, China

The growing demand for natural bioactive compounds highlights the need for antimicrobial and antioxidative metabolites from microbial sources. Among them, prodigiosin, a red pigment from Serratia marcescens, displays potent antioxidant and antimicrobial properties. However, optimizing its production and understanding molecular interactions remain challenging. In this study, we identified an optimized process for enhanced yield through the peptone meat extract (PM) media and incubation temperature of 30 o°C, which notably outperformed the other tested conditions and media. The purified red pigment was further characterized through column and thin-layer chromatography, UV-visible spectrophotometry, Fourier Transform Infrared (FT-IR) spectroscopy and Mass Spectrometry (ESI-MS/MS). The pigment demonstrated an Rf value of 0.93 through column chromatography and TLC. The structural characteristics were established through UV-Vis (λmax 536 nm), FT-IR, and ESI-MS/MS (m/z 324.3 amu), consistent with the prodiginine family. The characterized and purified prodigiosin showed excellent antibacterial activity against Gram-negative bacteria Escherichia. coli (28.2 ± 0.57 mm) and Gram-positive bacteria Bacillus. subtilis (23.58 ± 0.6 mm), together with antifungal activity against Fusarium oxysporum and Aspergillus niger. The Antioxidant analysis showed a dose-dependent radical scavenging activity reaching up to 37.5% at 1000 µg/mL. To understand the mechanistic pathwaysmechanisms, molecular docking revealed high binding affinities of the produced metabolite with key target sites as. FKS1 (-7.2 kcal/mol) for antifungal inhibition, FabH (-7.3 kcal/mol) against antibacterial inhibition, and Keap1 (-8.3 kcal/mol) for antioxidant activity. Our findings not only feature the prodigiosin's broad-spectrum bioactivity but also offer its interaction with molecular targets, providing the base for developing this metabolite as a natural therapeutic agent in multiple industrial applications, including pharmaceuticals and agriculture.