Integration of full-length transcriptomics and targeted metabolomics to identify benzylisoquinoline alkaloid biosynthetic genes in Corydalis yanhusuo

Abstract

Corydalis yanhusuo W.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients in Corydalis yanhusuo. However, few studies have investigated the BIA biosynthetic pathway in C. yanhusuo, and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs in C. yanhusuo were quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway in C. yanhusuo was constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway in C. yanhusuo. Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.

Chromosome-level genome assembly of Aristolochia contorta provides insights into the biosynthesis of benzylisoquinoline alkaloids and aristolochic acids

“…The important intermediate ( S )-reticuline that acts as the central branch point of several BIAs is then synthesized from ( S )-norcoclaurine through the catalysis of four enzymes: 6OMT, CNMT, NMCH, and 4′OMT [ 67 , 72 – 76 ]. Finally, magnoflorine (yellow), sanguinarine (blue), and berberine (green) are produced from ( S )-reticuline through the catalysis of multiple enzymes [ 15 , 64 , 77 ]. Genome-wide analysis predicted 91 nonredundant genes that were probably involved in the biosynthesis of BIAs in A. contorta ( Table S10 ).…”

Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast

“…In addition to fusion expression with hydrophilic chaperons, we can also focus on searching for orthologous BBE candidates (X. Liu et al, 2017;Xu et al, 2021) or improving BBE activity via protein engineering to optimize the sanguinarine biosynthetic pathway.…”

“…Zhao et al found that BBE was mainly expressed as inclusion bodies in Escherichia coli and the functional expression level could be significantly increased by fusion expression with maltose binding protein and glutathione S‐transferase (W. Zhao et al, 2021). In addition to fusion expression with hydrophilic chaperons, we can also focus on searching for orthologous BBE candidates (X. Liu et al, 2017; Xu et al, 2021) or improving BBE activity via protein engineering to optimize the sanguinarine biosynthetic pathway.…”

Transcriptome Analysis of Stephania tetrandra and Characterization of Norcoclaurine-6-O-Methyltransferase Involved in Benzylisoquinoline Alkaloid Biosynthesis

“…Gene cloning and functional characterization of these candidate CYP450s will be performed in our subsequent study. Compared with the BIAs production plants, such as Coptis chinensis ( He et al, 2018 ) and Corydalis yanhusuo ( Xu et al, 2021 ), tetrandrine was the unique compound of S. tetandra . However, the gene related to this biosynthesis pathway was unknown.…”

“…Recently, transcriptome analysis and metabolic profiling have been extensively used to explore and identify critical genes related to the biosynthesis and regulation of secondary metabolites in medicinal plants, such as Gloriosa superba ( Nett et al, 2020 ), Cinnamomum camphora ( Chen et al, 2018 ), and Atractylodes macrocephala ( Ruan et al, 2021 ). Using this integrative approach, several crucial genes ((S)-coclaurine N-methyltransferase, (S)-N-methylcoclaurine-3-hydroxylase) have been identified in Corydalis yanhusuo ( Xu et al, 2021 ), Coptis deltoidea ( Zhong et al, 2020 ), and Erythrina velutina Willd ( Chacon et al, 2021 ). These results shed light on the biosynthesis of BIAs in S. tetrandra .…”