Improving Nicotiana Benthamiana As Bioproduction System for Proteins and Small Molecules
International Conference on Plant Synthetic Biology and Bioengineering
2019
3rd International Conference on Plant Synthetic Biology, Bioengineering, and Biotechnology
Poster Session
Poster Session
Friday, October 4, 2019 - 5:00pm to 6:00pm
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.