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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085323-04
Application #
8248728
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
2009-04-10
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$587,698
Indirect Cost
$83,914
Name
Massachusetts Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Xu, Peng; Bhan, Namita; Koffas, Mattheos A G (2013) Engineering plant metabolism into microbes: from systems biology to synthetic biology. Curr Opin Biotechnol 24:291-9
Jiang, Ming; Zhang, Haoran; Pfeifer, Blaine A (2013) The logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin A produced through E. coli. J Vis Exp :e4346
Avalos, Jose L; Fink, Gerald R; Stephanopoulos, Gregory (2013) Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat Biotechnol 31:335-41
Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo et al. (2013) Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 97:5753-69
Jiang, Ming; Stephanopoulos, Gregory; Pfeifer, Blaine A (2012) Toward biosynthetic design and implementation of Escherichia coli-derived paclitaxel and other heterologous polyisoprene compounds. Appl Environ Microbiol 78:2497-504
Jiang, Ming; Stephanopoulos, Gregory; Pfeifer, Blaine A (2012) Downstream reactions and engineering in the microbially reconstituted pathway for Taxol. Appl Microbiol Biotechnol 94:841-9
Boghigian, Brett A; Salas, Daniel; Ajikumar, Parayil Kumaran et al. (2012) Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli. Appl Microbiol Biotechnol 93:1651-61
Boghigian, Brett A; Armando, John; Salas, Daniel et al. (2012) Computational identification of gene over-expression targets for metabolic engineering of taxadiene production. Appl Microbiol Biotechnol 93:2063-73
Boghigian, Brett A; Zhang, Haoran; Pfeifer, Blaine A (2011) Multi-factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions. Biotechnol Bioeng 108:1360-71
Santos, Christine Nicole S; Koffas, Mattheos; Stephanopoulos, Gregory (2011) Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab Eng 13:392-400

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