Enhancement of Dendrobine Production by CRISPR/Act3.0-Mediated Transcriptional Activation of Multiple Endogenous Genes in Dendrobium Plants.

Dendrobine, a significant medicinal compound, typically accumulates at low concentrations within several Dendrobium species, including Dendrobium nobile, Dendrobium catenatum, and Dendrobium moniliforme. As D. nobile and D. catenatum are established ingredients in traditional Chinese medicine and have been cultivated extensively, they present ideal plant chassis for upscaling dendrobine production for industrial and research applications. This study employed two approaches: the ectopic overexpression of seven genes through multigene stacking and the activation of multiple key endogenous genes in the dendrobine synthesis pathway using CRISPR/Act3.0 in either D. nobile or D. catenatum. These methods enhanced dendrobine production in transiently infiltrated leaves by 30.1% and transgenic plants by 35.6%. The study is the first to apply CRISPR/Act3.0 to Dendrobium orchids, enhancing dendrobine production, and thus laying a solid foundation for further improvements. CRISPR/Act3.0 is a recently developed technique that demonstrates high efficiency in model plant species, including rice, maize, and Arabidopsis. By combining CRISPR with transcriptional regulatory modules, activation of multiple endogenous genes in the metabolic pathway can be achieved. The successful application in orchid molecular breeding reveals promising potential for further exploration.