Molecular tools for advancing microbial biosynthesis of plant natural products

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Isis Trenchard, Christina D. Smolke

Stanford University, United States

The success of the artemisinin project from Amyris has demonstrated that microbial biosynthesis of natural product pharmaceuticals is a viable production platform to address global drug needs. Plant secondary metabolites, like artemisinin, are a rich source of established and potential drug candidates. However, our capacity to extend artemisinin’s success to other plant pathways is limited by our ability reliably engineer complex natural product pathways in microbial hosts. Plant pathways are particularly challenging due to the number and types of associated enzymes and in particular, the prevalence of cytochrome P450s. We have focused on engineering Saccharomyces cerevisiae as a microbial production platform for the benzylisoquinoline alkaloids (BIAs), a large class of plant secondary metabolites that exhibit a wide range of pharmacological activities, including anti-HIV, anticancer, and antimicrobial activities. Through engineering BIA biosynthetic pathways in yeast we have sought to (1) create a reliable and scalable source of valuable drugs and drug candidates and (2) gain an understanding of the effects of complex pathway expression on the host system and develop generalizable optimization strategies that will broadly advance the development of microbial production platforms for complex plant natural products. We have engineered strains of Saccharomyces cerevisiae capable of producing various protoberberine and benzophenanthridine alkaloids. In particular, strains that produce the key branch point intermediates reticuline and scoulerine have been developed and used to produce the compounds cheilanthifoline and stylopine through the addition of two sequential cytochrome P450 transformations. The number and types of chemical transformation steps achieved in this work represents one of the most complex examples in the field of metabolic engineering. We have developed a number of pathway optimization strategies for improving pathway flux and functional microbial expression of plant cytochrome P450s including spatial control over pathway enzymes and transport control over intermediate metabolites.