Metabolic Engineering for Microbial Taxol Biosynthesis

微生物紫杉醇生物合成的代谢工程

基本信息

批准号：
8072238
负责人：
GREGORY STEPHANOPOULOS
金额：
$ 28.21万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8072238
关键词：
15 year old Achievement Address Amino Acids Anabolism Artemisinins Arts Bacteria Behavior Biological Biological Factors Biopolymers Bioreactors Biotechnology Bladder Breast Carotenoids Cell physiology Cells Cervical Melanoma Chemicals Codon Nucleotides Development Distal Drug Industry Engineering Escherichia coli Ethanol Fermentation Foundations Future Gene Amplification Gene Deletion Gene Expression Genes Genetic Glycols Goals Growth HIV Protease Inhibitors Head and neck structure Healthcare Indans Ingredients and Chemicals Laboratories Lead Libraries Lung Metabolic Metabolism Methodology Methods Modification Natural Resources Nature Operon Ovarian Carcinoma Paclitaxel Pathway Analysis Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Plant Genes Plant Roots Plants Process Production Productivity Propane Property Proteins Protocols documentation Recombinants Regulator Genes Research Resources Route Source Speed Synthetic Genes System Systems Biology Taxus Taxus brevifolia Techniques Technology Terpenes Therapeutic Time Tracer Work Xylose Yeasts anti-cancer therapeutic artemisinine bioprocess cancer therapy cellular engineering chemical synthesis crixivan design gene cloning gene function genetic manipulation glucose production interest isopentenyl pyrophosphate isoprenoid lycopene mevalonate microbial microbial host microorganism operation optimism prevent promoter public health relevance reconstitution sarcoma success synthetic biology taxadiene tool

项目摘要

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.

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用特性，已证明对卵巢癌、乳腺癌、肺癌、头颈癌、膀胱癌和子宫颈癌、黑色素瘤以及艾滋病相关的卡波西肉瘤有效。尽管制造困难重重，但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。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植物源性紫杉醇中间体，通过化学合成大规模生产最终活性成分。尽管这种半合成工艺减轻了对自然资源的破坏，但它仍然是一个昂贵的工艺，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工程来解决，以在半合成生产路线中生产药物本身或其关键前体，这是本申请的主题。尽管近年来紫杉醇及其前体的微生物合成一直在积极研究，但代谢工程的最新进展让我们对应对这一挑战有了新的乐观态度。具体来说，我们对大肠杆菌中的类异戊二烯途径进行了改造，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 倍以上。此外，我们在大肠杆菌中表达了紫杉二烯之后紫杉醇途径中的下一个基因。这些成就，加上研究团队在途径构建、优化、天然产物合成以及细菌和酵母中对紫杉醇生物合成至关重要的植物和其他来源基因的功能表达方面所展示的专业知识，支持了拟议研究的总体目标，即高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表达，并结合上游类异戊二烯途径优化其活性，以获得最大的生物合成率； (b) 鉴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产量。我们的目标是通过协调途径和生物反应器工程，开发能够生产克/升范围内的紫杉醇的可扩展微生物发酵系统。公共健康相关性：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。一般来说，如果可以通过更简单的异源宿主重建生物合成途径，从而在培养物生长速度、可扩展性和基因操作技术方面取得进展，预计将有助于紫杉醇的生产（因此，其治疗影响会扩大）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高功效和更广泛抗癌特性的多种紫杉醇衍生物的机会。