Reconstruction of plant natural product pathways in genetically well-characterized microbial organisms such as Saccharomyces cerevisiae is a sustainable and scalable method of producing high value pharmaceutical compounds. Strictosidine is the universal precursor to thousands of monoterpene indole alkaloids (MIAs) such as vinblastine and camptothecin. MIAs are indispensable pharmaceutical ingredients, but are also expensive due to difficulties in production and isolation from plant producers. In this proposal, we will use strictosidine biosynthesis as a model system to explore the use of newly developed yeast-based technologies at UCLA and Stanford Genome Technology Center (SGTC) for high-titer production of strictosidine in yeast. Our labs and others have shown that critical parts of this biosynthetic pathway are subject to considerable crosstalk with the endogenous yeast redox active enzymes, resulting in significant loss of flux toward irrecoverable shunt products. Our preliminary efforts have led to increase in product titer of the intermediate nepetalactol, and suggest a more global approach aimed at the different intermediates in the pathway will lead to significant improvements. This collaborative proposal will leverage the Tang labs expertise in natural product biosynthesis with the new synthetic biological tools developed for yeast by SGTC. This will pave the way for complete reconstitution of important MIAs in yeast, as well as elucidation of hitherto unknown MIA biosynthetic pathways involving strictosidine. Together we will address four aims: 1) Use high-throughput pathway construction to achieve improved baseline production of strictosidine; 2) establish metabolite-responsive growth screenings for strictosidine and other key biosynthetic pathway intermediates; 3) employ new genome-engineered tools to rapidly create, screen and genotype yeast strains that can achieve high level of strictosidine production starting from the improved baseline strain; and 4) heterologous production and downstream pathway exploration of complex MIAs, such as vinblastine and camptothecin, starting from strictosidine.

Public Health Relevance

Plant natural products and plant natural product-derived drugs have been indispensible towards the treatment of different diseases and the prophylactic maintenance of healthy lifestyle. In this proposal, we aim to use synthetic biology strategies for yeast-based, high-titer production of strictosidine, a central plant monoterpene indole alkaloid that is the precursor to many therapeutically important plant natural products.

Agency
National Institute of Health (NIH)
Institute
National Center for Complementary & Alternative Medicine (NCCAM)
Type
Research Project (R01)
Project #
3R01AT010001-03S1
Application #
10120163
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Hopp, Craig
Project Start
2018-07-01
Project End
2023-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095