Production kinetics of immunosuppressive fungal metabolites

Abstract

Artificial suppression of the immune system is vital to organ transplant therapy as well as treatment of auto-immune diseases. Unfortunately few non-toxic immunosuppressive drugs exist. In search for such pharmaceuticals with low toxicity, this work offers a preliminary investigation into characterization and optimization of production of compounds found recently to have immunosuppressive activity. One fungal strain from each of three genera (Emericella, Paecilomyces and Penicillium), produced bioactive metabolites in separate bioreactor fermentations. Preliminary screening showed that Penicillium produced citrinin, a known toxic compound. The two other strains produced an immunosuppressive metabolite. The unknown immunosuppressive metabolite was extracted from Emericella broth and characterized by HPLC and TLC. Results allowed comparison to known metabolites, suggesting vertisporin and especially vermiculin as likely candidates. More complete characterization by mass spectrometry requires pure samples. Several methods of purification were tried including HPLC, TLC, and a combination of membrane filtration and size-exclusion chromatography. Unfortunately, separation was impeded by depletion of the stock of bioreactor fermentation broth and deterioration of the acid extract. To circumvent these problems, fresh fermentation broth was produced on a regular basis in shake flasks. Thus, bioactive samples were available throughout the entire separation experiment. But broths from shake-flask fermentations did not contain the immunosuppressive metabolite although they did contain up to three metabolites with antibacterial activity, two of which were likewise produced in the bioreactor. One antibacterial metabolite produced in both types of fermentations was purified sufficiently to be characterized by mass spectrometry. Although not fully identified, its characteristics suggest that it may be a metabolic precursor of the immunosuppressive metabolite..