Physiological and genetic factors for process development of cyclosporine fermentations

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Abstract
The new immunosuppressive agent cyclosporine (Cyclosporin A, Cy) is the most prominent member of a group of cyclic peptide fungal metabolites (cyclosporins) produced byTolypocladium inflatum in submerged fermentations. In the present study, kinetics and physiology of mycelial growth and Cy production byT. 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 ofT. inflatum have been isolated and regenerated.