PHYSIOLOGICAL AND GENETIC-FACTORS FOR PROCESS-DEVELOPMENT OF CYCLOSPORINE FERMENTATIONS

The new immunosuppressive agent cyclosporine (Cyclosporin A, Cy) is the most prominent member of a group of cyclic peptide fungal metabolites (cyclosporins) produced by Tolypocladium inflatum in submerged fermentations. In the present study, kinetics and physiology of mycelial growth and Cy production by T. inflatum were examined. A new semi-synthetic medium was formulated, consisting of a single carbon/energy source, Bacto-peptone, potassium phosphate and potassium chloride. A wide variety of carbon sources supported growth and Cy production. 3% (w/v) sorbose gave the highest final Cy titer (105.5 mg/l), based on 10-day fermentations. The best specific Cy production was observed with 2% sorbose (14.3 mg Cy/g biomass) followed by 5% myo-inositol (13.4 mg Cy/g biomass). A feeding strategy consisting of sequential addition of two carbon sources such as sorbose and maltose was developed in order to reach higher volumetric production. Genetic studies were also conducted, focussing on the development of mutants for increased Cy production and for the synthesis of novel cyclosporins. In the course of these studies, viable protoplasts of T. inflatum have been isolated and regenerated.