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

Abstract

Background 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. Results 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. Conclusion 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.

Optimization of the production process for the anticancer lead compound illudin M: process development in stirred tank bioreactors

“…To identify critical process parameters, the optimized shake flask process for the production of illudin M [ 8 ] was performed using the online Respiration Activity Monitoring System RAMOS® [ 26 ]. By setting the aeration rate to 4.7 mL min −1 in the RAMOS flasks it was possible to resemble the hydrodynamics and gas composition of the conventional 500 mL Erlenmeyer flasks with membrane caps.…”

“…We hypothesized that by achieving a similar process profile in stirred tank bioreactors, comparable titers to the shake flask process would be achieved. We recently reported that the feed of acetate and/or the associated increase in pH, reduced the growth of the producer organism and concluded that an optimal quantity of active biomass during the production phase was crucial for improved product titers [ 8 ].…”

“…S1), and high concentrations of undissociated acetate affect the growth of the organism and thus the production pattern. We have previously reported an inhibitory effect on O. nidiformis by feeding acetate and maintaining cultures at pH 4.2 in shake flask experiments [ 8 ] and hence, we considered the potential toxicity of acetate if pH changes are used for growth control in the bioreactor. For the cultivation in stirred tank bioreactors, we investigated the lowest pH value of the culture, which could cause growth inhibition when applying identical feeding conditions as in the optimized shake flask process.…”

“…We therefore aimed to develop a biotechnological process, enabling a stable illudin M supply using the natural producer Omphalotus nidiformis . The first step was the improvement of product titers by developing an optimized fed-batch process in shake flasks, which could be suitable for subsequent process transfer into a scalable system such as stirred tank bioreactors [ 8 ].…”

“…The optimized parameter setup was transferred to a 10 L process in a stainless steel bioreactor that delivered ~ 500 mg L −1 of illudin M; this concentration is significantly higher than any previously reported using stirred tank bioreactors. This is the second of a series of three reports addressing improvements of the biotechnological production of illudin M [ 8 , 25 ]. The aim of this work was the transfer of the optimized shake flask process into stirred tank bioreactors and set the base for further scale-up development ensuring a stable supply of illudin M for further pre-clinical and clinical studies.…”