Engineering Yeast towards High Titer Production of Monoterpene Indole Alkaloid Natural Products

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ABSTRACT 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.