(509e) Genome Editing of Nicotiana Benthamiana As an Improved Plant-Based Bioproduction System for Secondary Metabolites (Faculty Candidate)

The wild tobacco relative Nicotiana benthamiana is a commonly used plant for manufacturing proteins and reconstituting metabolic pathways which produce complex metabolites such as fragrances or medicines. Importantly, the plant can transiently express heterologous multi-enzyme pathways in just a few days. However, small molecule compounds and their intermediate pathway metabolites produced in N. benthamiana are often over-glycosylated, oxidized/reduced, acylated or modified with glutathione by enzymes native to N. benthamiana. This unwanted activity has challenged efforts to reconstitute the 11-step pathway to strictosidine (an alkaloid precursor to the anti-cancer drug vinblastine) as well as many other pathways. Therefore, to improve N. benthamiana as a bioproduction platform, we are using Cas9-mediated genome engineering to deactivate the endogenous enzymes which are modifying the heterologous metabolites. We have used transcriptomic and phylogenetic analysis of an improved genome assembly to select candidate genes for inactivation from among thousands of possible enzymes and have generated several knockout lines. To further guide selection of knockout candidates, we leveraged cell-free protein synthesis and biofoundry-assisted DNA assembly to express candidate glycosyltransferases and have subsequently measured glycosylation activity towards strictosidine pathway intermediates geraniol and cis-trans-nepetalactol. We anticipate that edited N. benthamiana lines will reduce unwanted metabolite derivatization and enable increased production of target molecules.