Department of Microbial Drugs, Helmholtz Centre for Infection Research, Brunswick, Germany.
German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Brunswick, Germany.
Microb Cell Fact. 2022 May 28;21(1):98. doi: 10.1186/s12934-022-01827-z.
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 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L; corn steep solids 7.0 g L; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L). 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) and 120 h (6.0 g L) respectively, which resulted in final illudin M titer of ~ 940 mg L 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.
真菌倍半萜类化合物 Illudin M 和 S 因其对某些耐药肿瘤细胞系的强烈活性，成为开发新型抗癌药物的重要基础分子。由于天然化合物的非特异性毒性，需要改善药效团。伊鲁福仑（Irofulven）是一种伊鲁定 S 的半合成衍生物，已进入治疗去势抵抗性转移性前列腺癌的 II 期临床试验。几种半合成伊鲁定 M 衍生物显示出对某些肿瘤细胞系的体外选择性和治疗指数的提高，这鼓励了进一步的研究。这需要稳定的天然化合物供应，而天然化合物是由蘑菇属的担子菌产生的。我们旨在开发一种稳健的生物技术工艺，以提供足够数量的伊鲁定 M，以支持药物化学研究以及未来的临床前和临床开发。在这项研究中，我们报告了朝着这一目标迈出的最初步骤。
在建立分析工作流程后，筛选了不同的培养基和市售的蘑菇菌株，以生产伊鲁定 M。在含有玉米浸膏的培养基中培养的蘑菇 nidiformis 达到了约 38mg/L，为优化设定了起点。改进的种子制备与简化的培养基（葡萄糖 13.5g/L；玉米浸膏 7.0g/L；改良的 Dox 肉汤 35mL）相结合，大大缩短了培养时间并提高了产量（约 400mg/L）。基于可重复的培养方法，开发了一种考虑潜在生物合成瓶颈的补料策略。在 96 小时（8.0g/L）和 120 小时（6.0g/L）时分别补加乙酸盐和葡萄糖，经过八天的培养后，最终的伊鲁定 M 产量达到了约 940mg/L。与初始产量相比，这是 25 倍的增长。
在严格规范种子制备和培养参数后，通过实验设计、经验试验和补充限制生物合成前体的组合，在摇瓶中实现了高度可重复的过程，并且伊鲁定 M 的产量很高。这些发现是进一步开发稳定的伊鲁定 M 供应的可扩展生物技术工艺的基础。
