Integrated metabolite profiling and transcriptome analysis reveal candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids in Corydalis solida.

Abstract

Structurally diverse benzylisoquinoline alkaloids (BIAs) are found in specific plant families, some of which are desirable for their efficient production because of their strong biological activities. Corydalis plants (e.g., Corydalis yanhusuo) of the family Papaveraceae also produce various BIAs; thus, they have been used in traditional Chinese medicine. Because metabolic engineering and synthetic biology using microorganisms are promising technologies for the effective production of useful metabolites, elucidation of the biosynthetic pathway of each BIA is indispensable. Although several enzyme genes involved in the biosynthesis of Corydalis BIAs have recently been isolated, many remain unknown, such as the protoberberine alkaloid C-methyltransferase involved in the biosynthesis of corydaline, one of the main BIAs found in the tubers of Corydalis plants. In this study, we performed transcriptome analysis combined with metabolite profiling of different tissues of Corydalis solida. Based on the high accumulation of several BIAs, including protopine, allocryptopine, and corydaline, genes encoding putative biosynthetic enzymes, including cytochrome P450, methyltransferase, and oxidase proteins, that were highly expressed in the tubers were screened. Two OMT genes, CsOMT1 and CsOMT2, were highly expressed in the tuber, and further characterization using crude enzyme preparations demonstrated that CsOMT1 showed 7-O-methylation activity against reticuline, whereas CsOMT2 catalyzed 9-O-methylation of scoulerine, followed by 2-O-methylation of tetrahydrocolumbamine. Our findings provide valuable information for the isolation of novel biosynthetic enzyme genes in Corydalis species.