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、膀胱癌和宫颈癌、黑色素瘤和艾滋病相关的卡波西肉瘤具有有效性。这种杰出的医学记录帮助紫杉醇成为一种非常有吸引力的癌症治疗方法，尽管制造困难重重。紫杉醇最初是从太平洋紫杉树的树皮中分离出来的，目前的生产路线仍然依赖于从植物中分离出紫杉醇中间体，然后通过化学合成大规模生产最终的活性成分。虽然这种半合成方法减轻了对自然资源的消耗，但它仍然是一种昂贵的方法，也阻止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化来解决，以在半合成生产途径中生产药物本身或其关键前体，这是本申请的主题。近年来，紫杉醇及其前体的微生物合成一直在积极进行，代谢工程的最新进展使人们对解决这一挑战持新的乐观态度。具体来说，我们在大肠杆菌中对类异戊二烯途径的工程改造导致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倍以上。此外，我们已经在大肠杆菌中表达了紫杉醇途径中继紫杉二烯之后的下一个基因。杆菌这些成就，沿着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来源的对紫杉醇生物合成至关重要的基因在细菌和酵母中的功能表达方面的专业知识，支持了拟议研究的总体目标，即，工程化微生物代谢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表达，并优化它们与上游类异戊二烯途径结合的活性，以获得最大的生物合成速率;（B）鉴定紫杉醇途径中剩余的未知基因（约占总量的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，以最大限度地提高紫杉醇产量。我们的目标，通过协调的途径和生物反应器工程，是一个可扩展的微生物发酵系统能够生产紫杉醇在克/升范围内的发展。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，则预计紫杉醇生产将得到帮助（因此，其治疗效果扩大），该宿主提供了培养生长速度，可扩展性和遗传操作技术的进步，可用于改变和优化生产。此外，异源紫杉醇生物合成途径将极大地扩大生物合成具有更大功效和更广泛抗癌性质的多种多样的紫杉醇衍生物的机会。

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.