Improvement of Cyclosporin A Production in the fungus <i>Tolypocladium inflatum</i> PTCC 5253 by Random Mutagenesis

Background: Cyclosporine A (CyA) is an immunosuppressive medication widely used in various medical situations, including transplantations, autoimmune diseases, and rheumatological conditions. One of the primary sources of CyA production is its biosynthesis by the fungus Tolypocladium inflatum; however, low production yield remains a significant challenge. Objectives: This research aimed to study the impact of UV radiation-induced random mutations on CyA production and fermentation broth viscosity in the strain T. inflatum PTCC 5253. Methods: To determine the baseline CyA production in T. inflatum PTCC 5253, the supernatant of a 14-day fermented original strain in semi synthetic medium (SSM), as well as standard concentrations of CyA, were evaluated for bioactivity against Aspergillus niger. The strain was then subjected to 254 nm UV irradiation at various durations, including 140 s, 160 s, 180 s, 200 s, and 220 s. The resulting mutants were fermented in SSM for 14 days, and their ethyl acetate (EtOAc) extracts were screened by LC/MS analysis for CyA detection. Additionally, the rheological parameters of the fermented media, including shear stress and viscosity, were determined. Results: The CyA production in T. inflatum PTCC 5253 was measured at 34.04 mg/L based on bioactivity evaluation and 37.5 mg/L based on LC-MS analysis. The UV radiation resulted in 20 revived mutants, 10 of which showed significantly elevated CyA yield. Notably, the isolate M4 exhibited a remarkable 14-fold increase compared to the original strain, producing 540 mg CyA/L of medium. Furthermore, increased CyA production in the selected mutants correlated with an elevation in medium viscosity, and the media exhibited shear-thinning behavior. Conclusions: In conclusion, random mutation induced by UV radiation can significantly enhance CyA productivity in T. inflatum PTCC 5253. Additionally, our results suggest a crucial correlation between CyA production and fermentation media viscosity.