Abstract

Taxol is a natural compound that possesses impressive anticancer medicinal properties with demonstrated efficacy against carcinomas of the ovary, breast, lung, head and neck, bladder and cervix, melanomas, and AIDS-related Karposi's sarcoma. This outstanding medicinal track record has helped taxol become a very attractive cancer treatment despite formidable manufacturing difficulties. First isolated from the bark of the pacific yew tree, the current production route still depends on isolating a plant-derived taxol intermediate for large-scale manufacture of the final active ingredient by chemical synthesis. Although this semi-synthetic process has eased the toll taken on natural resources, it is still an expensive process that also prevents the synthesis of derivatives with greater potency and a more diverse pharmacological spectrum. These problems can now be addressed through the engineering of microbial cells to produce the drug itself or its key precursor in the semi-synthetic production route, which is the subject of the present application. While microbial synthesis of taxol and its precursors have been actively pursued in recent years, recent advances in metabolic engineering allow a new optimism in addressing this challenge. Specifically, our engineering of the isoprenoid pathway in the bacterium Escherichia coli has led to the increase by more than 100-fold of the production of the first dedicated intermediate in the taxol biosynthetic pathway, taxadiene. Additionally, we have expressed the next gene in the taxol pathway after taxadiene in E. coli. These accomplishments, along with demonstrated expertise of the research team in pathway construction, optimization, and natural product synthesis and functional expression in bacteria and yeasts of genes from plants and other sources that are critical for taxol biosynthesis, support the overall objective of the proposed research, namely, the engineering of microbial metabolism for the efficient synthesis of taxol and its precursors. We will pursue this objective through the following three specific aims: (a) Obtain functional expression of all known genes in the taxol pathway and optimize their activity in conjunction with the upstream isoprenoid pathway for maximum biosynthetic rate;(b) Identify the remaining unknown genes in the taxol pathway (approximately 1/3 of the total) and express them in bacteria and yeast in order to complete the full biosynthetic pathway;(c) Optimize culture conditions and bioreactor operation to maximize taxol production. Our goal, through coordinated pathway and bioreactor engineering, is the development of a scalable microbial fermentation system capable of producing taxol in the gram/liter range. PUBLIC HEALTH RELEVANCE: More efficient production methods would help capitalize on the impressive taxol anticancer properties. In general, it is expected that taxol production would be aided (and, hence, its therapeutic impact expanded) if the biosynthetic pathway could be reconstituted through a simpler heterologous host, one that offered advances in culture growth speed, scalability, and genetic manipulation techniques available to alter and optimize production. Additionally, a heterologous taxol biosynthetic pathway would drastically expand the opportunities of biosynthesizing a vast diversity of taxol derivatives with greater efficacy and broader anticancer properties.

紫杉醇是一种天然化合物，具有令人印象深刻的抗癌药物特性，对卵巢癌，乳腺癌，肺癌，头颈癌，膀胱癌和宫颈癌，黑色素瘤和艾滋病相关的卡波西氏肉瘤均有疗效。尽管制造困难，但这种出色的药用记录已经帮助紫杉醇成为一种非常有吸引力的癌症治疗方法。首先从太平洋紫杉树的树皮中分离出来，目前的生产途径仍然依赖于分离植物衍生的紫杉醇中间体，以通过化学合成大规模制造最终活性成分。虽然这种半合成方法减轻了对自然资源的影响，但它仍然是一种昂贵的过程，也阻止了具有更大效力和更多样化的药理谱的衍生物的合成。现在可以通过微生物细胞的工程化来解决这些问题，以产生药物本身或其在半合成生产途径中的关键前体，这是本申请的主题。虽然近年来人们一直在积极地进行紫杉醇及其前体的微生物合成，但代谢工程的最新进展使人们对应对这一挑战有了新的乐观态度。具体而言，我们对大肠杆菌中类异戊二烯途径的工程设计使紫杉醇生物合成途径中的第一个专用中间体紫杉二烯的产量增加了100倍以上。此外，我们在大肠杆菌中的紫杉二烯后表达了紫杉醇途径中的下一个基因。这些成就，以及研究团队在途径构建，优化和天然产物合成以及来自植物和其他对紫杉醇生物合成至关重要的基因的细菌和酵母中的功能性表达的专业知识，支持了拟议研究的总体目标。 ，即用于有效合成紫杉醇及其前体的微生物代谢工程。我们将通过以下三个具体目标来实现这一目标：（a）获得紫杉醇途径中所有已知基因的功能性表达，并结合上游类异戊二烯途径优化其活性以获得最大生物合成速率;（b）鉴定剩余的未知基因在紫杉醇途径中（约占总量的1/3）并在细菌和酵母中表达它们以完成完整的生物合成途径;（c）优化培养条件和生物反应器操作以使紫杉醇产量最大化。我们的目标是通过协调途径和生物反应器工程，开发一种可扩展的微生物发酵系统，能够生产克/升范围内的紫杉醇。公共卫生相关性：更有效的生产方法将有助于利用令人印象深刻的紫杉醇抗癌特性。一般而言，如果生物合成途径可以通过更简单的异源宿主重建，预期紫杉醇的产生将得到帮助（因此，其治疗效果得到扩展），这种宿主提供了培养物生长速度，可扩展性和遗传操作技术的进步。可用于改变和优化生产。另外，异源紫杉醇生物合成途径将极大地扩展生物合成大量多样的紫杉醇衍生物的机会，具有更高的功效和更广泛的抗癌特性。

----------------------------------------------------------------------

欲流之远者，必浚其源泉。
欢迎关注JUNYUAN，我们的理念是科技改变生活！

在这里，你可以检索美国国立卫生研究院经费数据库NIH，更重要的是，我们将同您一起探索生命科学带来的技术创新。