Unlocking the Chemical Diversity of Plant Natural Product Pathways: Dehydrogenases in Alkaloid Biosynthesis

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Plant-derived natural products exhibit a variety of biological activities that have ramifications for both the medicinal and agricultural industry arenas. Unfortunately, these compounds are often produced in low amounts, or are found in plants that are slow-growing, or difficult to obtain. Understanding the biosynthetic pathways that generate these compounds will allow exploitation of these compounds for a wider variety of applications. Historically, plant pathway elucidation has been a challenge. However, recent advances in next-generation sequencing technology and bioinformatic approaches have made the rich metabolism of plants more accessible.

Our group has focussed on the pathways of the monoterpene indole alkaloids of the medicinal plant Catharanthus roseus. This class of ~3000 natural products, all derived from a common biosynthetic intermediate, displays extraordinary chemical diversity. However, the steps of this pathway in which the extraordinary chemical diversity emerges are still not understood. We recently reported the first gene that acts at the crucial branch point where the structures of the alkaloids begin to diverge, an alcohol dehydrogenase name tetrahydroalstonine synthase (THAS). Here we describe how we will capitalise on this discovery, as well as our recent development of a production platform for de novo production of these alkaloids in yeast, to rapidly generate complex plant-derived alkaloids. We plan to identify and characterise additional biosynthetic enzymes in the alcohol dehydrogenase class that act at this crucial branch point of the pathway to generate stereoisomers of tetrahydroalstonine. We further propose to use structural biology to understand the mechanism of these enzymes and to manipulate the substrate and product specificities by mutagenesis. Finally, all discovered enzymes and enzyme mutants will be transformed into our yeast production platform to demonstrate that we can generate complex alkaloids de novo.

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