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：
8248728
负责人：
GREGORY STEPHANOPOULOS
金额：
$ 58.77万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-10 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8248728
关键词：
15 year old Achievement Acquired Immunodeficiency Syndrome Address Amino Acids Anabolism Artemisinins 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 Health 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 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物特性，对卵巢，乳腺癌，肺，头颈部，头颈，膀胱和子宫颈，黑色素瘤以及与艾滋病相关的Karposi的肉瘤的肉瘤具有疗效。尽管制造业难以实现，但这种出色的药用记录已帮助紫杉醇成为一种非常有吸引力的癌症治疗方法。首先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取决于隔离植物衍生的紫杉醇中间体，以通过化学合成对最终的活性成分进行大规模生产。尽管这种半合成过程减轻了自然资源的损失，但它仍然是一个昂贵的过程，也可以防止具有更大效力和更多样化的药理光谱的衍生物的合成。现在，可以通过微生物细胞的工程来解决这些问题，以在半合成生产途径中产生药物本身或其关键前体，这是本应用的主题。尽管近年来已经积极追求紫杉醇及其前体的微生物合成，但代谢工程的最新进展允许在应对这一挑战方面产生新的乐观情绪。具体而言，我们在大肠菌菌中的类吸引途径的工程导致紫杉醇生物合成途径中第一个专用中间体生产的100倍以上。此外，我们在大肠杆菌中的纳税二烯后表达了紫杉醇途径的下一个基因。这些成就，以及研究团队在途径建设，优化和天然产品合成方面的专业知识，以及来自植物和其他来源的基因和酵母中的功能表达，这对于紫杉醇生物合成至关重要，这对拟议研究的整体目标至关重要。我们将通过以下三个特定目的来追求这一目标：（a）在紫杉醇途径中获得所有已知基因的功能表达，并与上游类异丙途径相结合以最大的生物合成率来优化其活性；（b）确定紫杉醇途径中的其余未知基因（约为总计的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的运作，以最大程度地生产紫杉醇。通过协调的途径和生物反应器工程，我们的目标是开发可扩展的微生物发酵系统，该系统能够在克/升范围内生产紫杉醇。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物业。通常，如果可以通过更简单的异源宿主重构生物合成途径，则预计紫杉醇产量将得到帮助（因此，其治疗影响会扩大），该途径可以通过更简单的异源宿主进行重构，从而在培养速度，可扩展性和基因操纵技术方面提供了进步，可改变和优化生产。此外，异源性紫杉醇生物合成途径将大大扩大生物合成的机会，这些机会具有更大的功效和更广泛的抗癌特性。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Methods and options for the heterologous production of complex natural products.

DOI：
10.1039/c0np00037j
发表时间：
2011-01
期刊：
NATURAL PRODUCT REPORTS
影响因子：
11.9
作者：
Zhang, Haoran;Boghigian, Brett A.;Armando, John;Pfeifer, Blaine A.
通讯作者：
Pfeifer, Blaine A.

Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli.

DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
0
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G

Distributing a metabolic pathway among a microbial consortium enhances production of natural products.

DOI：
10.1038/nbt.3095
发表时间：
2015-04
期刊：
Nature biotechnology
影响因子：
46.9
作者：
Zhou K;Qiao K;Edgar S;Stephanopoulos G
通讯作者：
Stephanopoulos G

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol.

DOI：
10.1007/s00253-012-4016-1
发表时间：
2012-05
期刊：
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
影响因子：
5
作者：
Jiang, Ming;Stephanopoulos, Gregory;Pfeifer, Blaine A.
通讯作者：
Pfeifer, Blaine A.

The future of metabolic engineering and synthetic biology: towards a systematic practice.

DOI：
10.1016/j.ymben.2012.02.001
发表时间：
2012-05
期刊：
METABOLIC ENGINEERING
影响因子：
8.4
作者：
Yadav, Vikramaditya G.;De Mey, Marjan;Lim, Chin Giaw;Ajikumar, Parayil Kumaran;Stephanopoulos, Gregory
通讯作者：
Stephanopoulos, Gregory