Metabolic Engineering for Microbial Taxol Biosynthesis

描述（由申请人提供）：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、膀胱癌和子宫颈癌、黑色素瘤和艾滋病相关的卡波西肉瘤都有疗效。尽管存在巨大的制造困难，但这一杰出的医疗记录帮助紫杉醇成为一种非常有吸引力的癌症治疗药物。首先从太平洋紫杉树的树皮中分离出来，目前的生产路线仍然依赖于分离植物衍生的紫杉醇中间体，通过化学合成大规模生产最终的活性成分。虽然这种半合成过程减轻了对自然资源的消耗，但它仍然是一个昂贵的过程，也阻碍了具有更大效力和更多样化药理谱的衍生物的合成。这些问题现在可以通过微生物细胞的工程来解决，在半合成生产路线中生产药物本身或其关键前体，这是本应用的主题。虽然微生物合成紫杉醇及其前体近年来一直在积极地进行，但代谢工程的最新进展为解决这一挑战提供了新的乐观。具体来说，我们在大肠杆菌中对类异戊二烯途径进行的工程设计使紫杉醇生物合成途径中第一个专用中间体紫杉二烯的产量增加了100多倍。此外，我们在大肠杆菌中表达了继杉二烯之后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、天然产物合成以及来自植物和其他来源的基因在细菌和酵母中的功能表达方面的专业知识，对紫杉醇生物合成至关重要，支持了拟建研究的总体目标，即微生物代谢工程，以有效合成紫杉醇及其前体。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，并与上游类异戊二烯途径一起优化其活性，以获得最大的生物合成速率；(b)确定紫杉醇途径中剩余的未知基因（约占总数的1/3），并在细菌和酵母中表达，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，使紫杉醇产量最大化。通过协调途径和生物反应器工程，我们的目标是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紫杉醇令人印象深刻的抗癌特性。总的来说，如果生物合成途径可以通过更简单的异源宿主重建，那么紫杉醇的生产将会得到帮助（因此，其治疗作用将会扩大），这种异源宿主在培养生长速度、可扩展性和可用于改变和优化生产的遗传操作技术方面提供了进步。此外，异源紫杉醇生物合成途径将极大地扩大生物合成各种紫杉醇衍生物的机会，这些衍生物具有更高的功效和更广泛的抗癌特性。

DESCRIPTION (provided by applicant): 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.