Complex engineering of <i>Solanum</i> alkaloids structural diversity in <i>Nicotiana benthamiana</i>

Transient expression in Nicotiana benthamiana offers a powerful and versatile platform for rapid production of complex specialized metabolites. Steroidal glycoalkaloids (SGAs) and steroidal saponins produced by members of the Solanaceae family are known for their diverse structures and activities including antimicrobial, anticancer, antiviral, and anti-inflammatory. Attempts to reconstitute their complete biosynthetic pathway have been unsuccessful to date. In this study, we identified a different tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM2 (SlGAME2-NEW) enzyme as a xylosyltransferase in the penultimate step of α-tomatine and dehydrotomatine biosynthesis. The discovery of SlGAME2-NEW facilitated the engineering of tomato SGAs and steroidal saponin biosynthesis in N. benthamiana. Expressing from 9 to 15 genes in combination we efficiently engineered a total of 20 steroidal 'end products' (both alkaloids and saponins) typically produced by tomato and eggplant and ones merely found in wild tomato species. Furthermore, we engineered the biosynthetic pathway of the steroidal saponins uttroside B, dehydrouttroside B, and their stereoisomers [25(S)-uttroside B and 25(S)-dehydrouttroside B]. Production of these metabolites in N. benthamiana opens broad possibilities for examining and exploring their therapeutic potential. This study therefore makes a significant contribution to the application of synthetic biology for producing high-value steroidal metabolites in heterologous plant hosts.