Metabolic Engineering for Microbial Taxol Biosynthesis

说明(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用特性，对卵巢癌、乳腺癌、肺癌、头颈部癌、膀胱癌和子宫颈癌、黑色素瘤和艾滋病相关的卡波西肉瘤具有明显的疗效。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，尽管存在巨大的制造困难。首先从太平洋红豆杉树皮中分离出来，目前的生产路线仍然依赖于分离从植物中提取的紫杉醇中间体，用于通过化学合成大规模生产最终有效成分。尽管这种半合成工艺减轻了对自然资源的消耗，但它仍然是一种昂贵的工艺，也阻碍了合成具有更大效力和更多样化药理光谱的衍生物。这些问题现在可以通过微生物细胞工程来解决，以生产药物本身或其在半合成生产路线中的关键前体，这是本申请的主题。虽然微生物合成紫杉醇及其前体近年来一直在积极进行，但代谢工程的最新进展使人们在应对这一挑战方面有了新的乐观。具体地说，我们在大肠杆菌中进行的类异戊二烯途径工程使紫杉醇生物合成途径中第一个专用中间体紫杉烯的产量增加了100多倍。此外，我们还在大肠杆菌中表达了紫杉醇途径中紫杉二烯之后的下一个基因。这些成就，加上研究团队在途径构建、优化以及天然产物合成和在细菌和酵母中表达对紫杉醇生物合成至关重要的植物和其他来源的基因的功能表达方面的专业知识，支持了拟议研究的总体目标，即通过微生物代谢工程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，并结合上游类异戊二烯途径优化它们的活性，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(约占总数的三分之一)，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，以最大限度地提高紫杉醇的产量。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/升范围的紫杉醇。与公共健康相关：更高效的生产方法将有助于充分利用紫杉醇令人印象深刻的抗癌特性。总体而言，如果生物合成途径可以通过更简单的异源宿主重组，这种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，预计紫杉醇的生产将得到帮助(因此，其治疗效果也会扩大)。此外，异源紫杉醇生物合成途径将极大地扩大生物合成多种紫杉醇衍生物的机会，使其具有更好的疗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.