Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai, 200438, People's Republic of China.
Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People's Republic of China.
ACS Synth Biol. 2020 Aug 21;9(8):2009-2022. doi: 10.1021/acssynbio.0c00100. Epub 2020 Jul 15.
Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered . Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.
埃坡霉素作为一类新型微管稳定抗癌药物，对紫杉烷类耐药细胞具有很强的生物活性，并显示出治疗晚期乳腺癌的临床活性。此外，它们在中枢神经系统损伤和阿尔茨海默病方面也具有很大的潜力。然而，由于原始生产商的发酵周期长，以及非核糖体肽/聚酮（NRP/PK）巨合酶基因遗传工程的挑战，埃坡霉素的应用受到严重限制。在这里，我们通过重新组装一个新型的 56kb 埃坡霉素生物合成基因簇来解决这些问题，根据 RNA-seq 分析优化每个基因的启动子，并在工程菌中完成前体合成途径。此外，我们通过优化培养条件来消除细胞自溶的瓶颈。最终，在摇瓶中，经过六天的发酵，埃坡霉素的产量提高到 82mg/L。总的来说，我们不仅构建了埃坡霉素高产菌，以进一步进行药物开发，还为 NRP/PK 化合物的高产提供了合理的策略。
