抗肿瘤安丝菌素生物合成途径系统优化

Ansamitocin is a extremely high-potency cytotoxic agent and belongs to maytansinoid antibiotics. The antibody-conjugated drug Kadcyla displays significant inhibitory activity against the HER2-positive metastatic breast cancer and has been approved by the United States Food and Drug Administrtion in February 2013. Actually, the limited supply of ansamitocin results in not only inhibiting the development of antibody-conjugated drug, but also led to the high cost. In this project, we will illuminate the molecular mechanism for the high production of ansamitocin and discovery of the limited factors in ansamitocin biosynthesis by metabolic engineering, synthethtic biology and fermentation optimization strategies. In this investigation, the substrates and inermediates will be enriched by engineering of primary biosynthetic pathway or introcucing of heterologous gene circuite. The gene clusters which are competing with ansamitocin biosynthesis pathway for subtstrates and cofactors will be disrupted. Additonlly, the transfermation rate of the enzymes involved in ansamitocin post-PKS modification will be optimized by modulating the transcription level of corresponding gene. The regulatory genes involved in the specificity regulation of ansamitocin biosynthesis will be allocated to improve their transcriptional level. Finally, the high yield producer strain will be constructed by genetic manipulation. Moreover, the environmental response of the strain and the fermentation conditions will be measured and improved to significantly increase the production of ansamitocin. These research will not only facilitate the industrialization of ansamitocin to afford sufficient material for antibody-conjugated drug development, but also greatly reduce the expense for the patients.

安丝菌素为美登木素类抗生素，具有极强的抗肿瘤活性。该类化合物的抗体耦联药物在治疗乳腺癌中效果卓著，已经通过美国FDA的认证。目前，安丝菌素产生菌发酵产量太低的现状，不仅严重制约了抗体药物的大规模开发，而且导致了患者使用成本极高。 因此，本项目拟以安丝菌素产生菌为材料，以其生物合成途径中限制因子的系统解析与改造为切入点。通过代谢途径优化或引入异源高效合成系统，提升生物合成中所需前体物和底物的供给；进一步中断分流底物的基因簇；然后，使用底物喂养策略发掘后修饰中的限制因素，进行定向消除；在利用调控元件优化配置，提高基因簇中功能基因的转录水平的基础之上，全面解析安丝菌素高产的分子机理。最后，合理整合相关的高产因素，利用代谢工程和合成生物学方法定向优化安丝菌素生物合成途径，建立安丝菌素发酵过程中的环境应答评估体系，完善工程菌的发酵工艺，实现其发酵产量的大幅跃升，为安丝菌素抗体药物的开发提供保障。

安丝菌素为美登木素类抗生素，具有极强的抗肿瘤活性。该类化合物的抗体耦联药物在治疗乳腺癌中效果卓著，已经通过美国FDA的认证。目前，安丝菌素产生菌发酵产量太低的现状，不仅严重制约了抗体药物的大规模开发，而且导致了患者使用成本极高。. 因此，本项目以安丝菌素产生菌为材料，以其生物合成途径中限制因子的系统解析与改造为切入点。系统优化了安丝菌素的发酵条件，详细测定了A. pretiosum ATCC 31280 精细基因组序列，开展了该菌株的比较基因组学研究，构建了前体相关的中心碳代谢网络，进行了A. pretiosum ATCC31280-RNA-Seq 转录组测定及其强启动子的筛选。在系统构建安丝菌素生物合成中体检积累的相关突变株的基础上，优化了安丝菌素生物合成前体底物的获得策略，实现了目标产物的快速制备。建立了通过安丝菌素生物合成中间体的喂养体系，实现了快速发掘其生物合成限速步骤和安丝菌素产生菌快速遗传改造的方法 。经过系统的探索，发现了安丝菌素生物合成底物替代的规律，建立了各种生物合成单元所供给途径的定向优化的体系，并通过敲除A. pretiosum ATCC31280菌株中的竞争PKS 途径提高AP-3的产量，全面解析安丝菌素高产的分子机理。最后，合理整合相关的高产因素，利用代谢工程和合成生物学方法定向优化安丝菌素生物合成途径，完善工程菌的发酵工艺，实现其发酵产量的大幅跃升，为安丝菌素抗体药物的开发提供保障。