Improving Nicotiana Benthamiana As Bioproduction System for Proteins and Small Molecules

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. In particular, 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). Therefore, to improve N. benthamiana as a bioproduction platform, we are using the RNA-guided Cas9 nuclease to deactivate the native enzymes that likely make unwanted modifications to the target metabolite. As a proof-of-concept, we have demonstrated activity of SpCas9 nuclease to make targeted double-strand breaks (producing insertion/deletions) that inactivate the peroxidase NbPOX1. Next, we have used transcriptomic and phylogenetic analysis to select candidate genes for inactivation from among hundreds of possible enzymes. Finally, we have adapted cell-free protein synthesis (CFPS) to manufacture candidate glycosyltransferases to screen their activity towards pathway intermediates geraniol and cis-trans-nepetalactol.