Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally significant compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana . A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. We used transcriptomic analysis to identify glycosyltransferases that were upregulated in response to biosynthetic intermediates and produced plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produced a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases did not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.