Understanding and Engineering Alkaloid Biosynthesis

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My group’s work encompasses the area of natural product biosynthesis, with an emphasis on the chemistry, enzymology and metabolic engineering of plant natural product pathways.

We are actively working to identify uncharacterized alkaloid biosynthetic enzymes in the medicianl plant Catharanthus roseus. Transcripts that encode putative biosynthetic enzymes displaying the expected sequence homology, and/or having an expression profile consistent with the metabolite production levels, will be cloned, subjected to heterologous expression and evaluated by in vitro biochemical assay. Alternatively, we will use Viral Induced Gene Silencing (VIGS) to silence the gene candidate in planta and then assess the resulting natural product profile in silenced C. roseus seedlings. We will subject and newly discovered enzymes to in depth mechanistic analysis. Furthermore, we also note that a number of alkaloid biosynthetic enzymes show homology to enzymes from primary metabolism; for example, the N-methyl transferase described in Section 1 shows sequence homology to a C-methyl transferase involved in tocopherol biosynthesis. We propose to compare the sequences of these enzymes and use site directed mutagenesis to explore how nature may have potentially evolved an enzymatic function from a preexisting one.

Finally, we have also developed strategies to enable C. roseus to produce “new-to-nature” products. By silencing tryptophan decarboxylase, which makes the alkaloid precursor tryptamine, we have generated a C. roseus culture that lacks all alkaloids. Thus, adding unnatural tryptamine analogs to the culture medium of the “silenced” system results in the formation alkaloids derived exclusively from the unnatural starting substrates. We are in the process of initiating screening efforts to determine the medicinal activity of these compounds.

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