Exploring Plant Alkaloid Biosynthetic Pathways in Yeast Using Untargeted Metabolomics and Proteomics

Plants produce some of the most potent human therapeutics and other industrially relevant molecules. Despite the wealth of plant natural products with therapeutic effects, very few are commercially available because of low production levels in native plants, generally slow growth, as well as limited genetic tractability of plants that makes it difficult to engineer plants.

Reconstitution of biosynthetic pathways in a heterologous host is a proven strategy for rapid and inexpensive production of complex molecules. Saccharomyces cerevisiae is one of the most common choices for a heterologous microbial host because of the abundance of available genetic tools for these organisms. However, a deep understanding of both the heterologous natural product biosynthetic pathway and its connectedness with the native yeast metabolism is essential for developing cell factories with high catalytic flux towards natural product biosynthesis.

Monoterpenoid indole alkaloids (MIAs) are secondary plant metabolites that exhibit a remarkable structural diversity, with >2,000 MIAs derived from a common precursor. Many of these alkaloids exhibit pharmaceutically valuable biological activities, which makes large-scale production using recombinant microbial cell factories very attractive.

In this study, untargeted metabolomics and proteomics were used to investigate and compare the cellular metabolism and proteome of wild-type yeast and yeast engineered to produce MIAs. This has provided valuable insights into the changes associated with genetically modified biochemical reactions and metabolic pathways. Here we will present the learnings from our integrated omics approach using compound annotation to enable automatic selection and generation of target lists for LC-MS/MS analysis. We will also present the integration of LC-MS and LC-MS/MS data, and rapid compound annotation using a variety of different compound databases, as well as illustrate experimental designs and omics outputs leveraged for guiding novel MIA biosynthetic pathway designs and optimize baker’s yeast as a chassis for fermentation-based manufacturing of plant natural products.