Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum .

The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered Dendrobium catenatum as a chassis plant for the production of dendrobine through the screening and pyramiding of key biosynthesis genes. Initially, previously predicted upstream key genes in the methyl-D-erythritol 4-phosphate (MEP) pathway for dendrobine synthesis, including 4-(Cytidine 5'-Diphospho)-2-C-Methyl-d-Erythritol Kinase ( CMK ), 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase ( DXR ), 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase ( MCT ), and Strictosidine Synthase 1 ( STR1 ), and a few downstream post-modification genes, including Cytochrome P450 94C1 ( CYP94C1 ), Branched-Chain-Amino-Acid Aminotransferase 2 ( BCAT2 ), and Methyltransferase-like Protein 23 ( METTL23 ), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes ( SG ) were then stacked and transiently expressed in the leaves of D. catenatum , resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control ( EV ). Further, RNA-seq analysis identified Copper Methylamine Oxidase ( CMEAO ) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of CMEAO increased dendrobine content by two-fold. Additionally, co-expression analysis of the differentially expressed genes (DEGs) by weighted gene co-expression network analysis (WGCNA) retrieved one regulatory transcription factor gene MYB61 . Overexpression of MYB61 increased dendrobine levels by more than two-fold in D. catenatum . In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of D. catenatum to produce dendrobine, thereby improving its medicinal value.