Optimization of Heterologous Glucoraphanin Production In Planta

:glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target forproduction in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphanebefore and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin inheterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there maybe missing auxiliary genes that play a role in improving productionin planta.Inaneffort to identify auxiliary genes required for highglucoraphanin production, we leveraged transient expression inNicotiana benthamianato screen a combination of previouslyuncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategyalleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosyntheticpathway provides a pathway amenable for high glucoraphanin production.