Optimization of the production process for the anticancer lead compound illudin M: improving titers in shake-flasks

The fungal sesquiterpenes Illudin M and S are important base molecules for the development of new anticancer agents due to their strong activity against some resistant tumor cell lines. Due to nonspecific toxicity of the natural compounds, improvement of the pharmacophore is required. A semisynthetic derivative of illudin S (Irofulven) entered phase II clinical trials for the treatment of castration-resistant metastatic prostate cancer. Several semisynthetic illudin M derivatives showed increased in vitro selectivity and improved therapeutic index against certain tumor cell lines, encouraging further investigation. This requires a sustainable supply of the natural compound, which is produced by Basidiomycota of the genus Omphalotus. We aimed to develop a robust biotechnological process to deliver illudin M in quantities sufficient to support medicinal chemistry studies and future preclinical and clinical development. In this study, we report the initial steps towards this goal.

After establishing analytical workflows, different culture media and commercially available Omphalotus strains were screened for the production of illudin M.Omphalotus nidiformis cultivated in a medium containing corn steep solids reached ~ 38 mg L^-1 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L^-1; corn steep solids 7.0 g L^- 1; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L^-1). Based on a reproducible cultivation method, a feeding strategy was developed considering potential biosynthetic bottlenecks. Acetate and glucose were fed at 96 h (8.0 g L^-1) and 120 h (6.0 g L^-1) respectively, which resulted in final illudin M titer of ~ 940 mg L^-1 after eight days. This is a 25 fold increase compared to the initial titer.

After strict standardization of seed-preparation and cultivation parameters, a combination of experimental design, empirical trials and additional supply of limiting biosynthetic precursors, led to a highly reproducible process in shake flasks with high titers of illudin M. These findings are the base for further work towards a scalable biotechnological process for a stable illudin M supply.