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Iridoids, Alkaloids, and Old Yellow Enzymes in Engineering of Secondary Metabolism

  • Author(s): Billingsley, John Martin;
  • Advisor(s): Tang, Yi;
  • et al.

While the definition of life remains controversial, metabolism is considered a staple characteristic through which organisms exist and evolve. Whereas primary metabolism provides critical life-sustaining nutrients through a relatively conserved set of chemical reactions, many organisms have developed secondary metabolic pathways which are not absolutely required for the survival of the organism. Instead, secondary metabolites, also known as natural products, confer a selective advantage for the producing organism. The secondary metabolite-mediated interactions between an organism and its ecological niche provide an avenue through which production of biologically active natural products may be evolutionarily advantageous. As a result, an enormous number of pharmaceuticals and agrochemicals used by humankind are natural products or natural product-derived. For this reason, tremendous interest exists in the discovery and application of natural product biosynthesis as a means for providing both existing and novel biologically useful molecules.

This thesis describes our approach in engineering natural product biosynthesis for the production and discovery of secondary metabolites. In the course of our studies, we have focused on two general classes of biologically relevant natural products, the iridoids and alkaloids. Investigation of natural product biosynthesis using the heterologous host Saccharomyces cerevisiae led to the discovery of the role played by old yellow enzymes in secondary metabolism. Using this information, we engineered S. cerevisiae for the production of the iridoid nepetalactol, a precursor to a class of plant natural products known as monoterpene indole alkaloids. Rational metabolic engineering permitted a 5.2-fold increase in the biocatalytic selectivity towards the desired iridoid product. We implemented this platform for the production of the industrially relevant nepetalactone, and through multivariate optimization showed an additional 5.8-fold increase in product titer. Furthermore, we developed a suite of synthetic biology tools which played a critical role in metabolic engineering, including a novel set of yeast vectors, a number of engineered yeast strains, as well as several CRISPR techniques for rapid genome editing in filamentous fungi and yeast. Finally, we utilized this technology to investigate an uncharacterized old yellow enzyme-containing gene cluster in filamentous fungi.

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