The medicinal plant Stephania yunnanensis is rich in aporphine alkaloids, a type of benzylisoquinoline alkaloid (BIA), with aporphine being the representative and most abundant compound, but our understanding of the biosynthesis of BIAs in this plant has been relatively limited. Previous research reported the genome of S. yunnanensis and preliminarily identified the norcoclaurine synthase (NCS), which is involved in the early stages of the BIA biosynthetic pathways. However, the key genes promoting the formation of the aporphine skeleton have not yet been reported. In this study, based on the differences in the content of crebanine and several other BIAs in different tissues, we conducted transcriptome sequencing of roots, stems, and leaves. We then identified candidate genes through functional annotation and sequence alignment and further analyzed them in combination with the genome. Based on this analysis, we identified three CYP80 enzymes (SyCYP80Q5-1, SyCYP80Q5-3, and SyCYP80G6), which exhibited different activities toward (S)- and (R)-configured substrates in S. yunnanensis and demonstrated strict stereoselectivity enroute to aporphine. This study provides metabolomic and transcriptomic information on the biosynthesis of BIAs in S. yunnanensis, offers valuable insights into the elucidation of BIA biosynthesis, and lays the foundation for the complete analysis of pathways for more aporphine alkaloids.

Background/Objectives: Stephania epigaea is a plant from the Menispermaceae family. Its root is an important traditional folk medicine, which is called Diburong in China. Diburong is rich in benzylisoquinoline alkaloids (BIAs), including cepharanthine, which has been demonstrated to exhibit significant anti-inflammatory, antiviral, antineoplastic, and anti-SARS-CoV-2 activities, as well as raising leukocytes. Cepharanthine is composed of (R)- and (S)-1-benzylisoquinoline alkaloid (1-BIA). (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting enzyme in BIA biosynthesis. However, its role in the cepharanthine biosynthetic pathway, particularly with the (R) stereoisomer substrate, remains largely unexplored. This study aimed to identify Se6OMTs involved in the cepharanthine biosynthetic pathway and elucidate the O-methyltransferases (OMTs) responsible for the production of (R)- and (S)-stereoisomer BIAs. Methods: In this study, three OMTs were cloned from S. epigaea and functionally characterized using nine 1-BIAs of (R)- and (S)-configurations as substrates. Results: Two O-methyltransferases, Se6OMT1 and Se6OMT3, showed efficient catalytic activity at the C6 position of both (R)- and (S)-norcoclaurine. Furthermore, Se6OMT3 demonstrated high catalytic activity at the C7 and C4′ positions of other (R)- and (S)-configuration 1-BIAs, which resulted in the generation of multiple products. Conclusions: This study focused on 6OMT enzymes in S. epigaea, identifying Se6OMTs involved in the cepharanthine biosynthetic pathway, determining the OMTs involved in the production of (R)- and (S)-stereoisomer BIAs. This research provides valuable insights into the substrate promiscuity of Se6OMTs on (R)- and (S)-configured 1-BIAs in S. epigaea and highlights the genetic components necessary for the metabolic engineering and synthetic biology approaches to cepharanthine production.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) serves as a key rate-limiting enzyme in the mevalonate pathway and plays a central regulatory role in the biosynthesis of tanshinones. To date, four HMGR family members (SmHMGR1–4) have been identified in Salvia miltiorrhiza. Here, we cloned and identified a novel member, SmHMGR5, by integrating multiple genomic datasets. Genomically, SmHMGR5 formed an inverted repeat with SmHMGR3 (98.04% homology) and phylogenetically clustered with SmHMGR2. Based on the expression patterns of the five HMGR genes, we further generated SmHMGR2 and SmHMGR5 knockout mutants using CRISPR/Cas9 technology and compared their effects on the accumulation of 12 tanshinones and 4 phenolic acids via UPLC-MS-based metabolomic analysis. Knockout of SmHMGR2 significantly suppressed the accumulation of seven tanshinones, whereas SmHMGR5 knockout downregulated only three tanshinones, and neither mutation affected phenolic acids. Notably, the major compound tanshinone IIA remained stable across different mutants, but tanshinone IIB was markedly reduced upon SmHMGR2 knockout, suggesting complex regulatory mechanisms in tanshinone biosynthesis. These findings provide new insights into the biosynthetic network of tanshinones and establish a theoretical foundation for metabolic engineering strategies aimed at enhancing the production of bioactive constituents in S. miltiorrhiza.

The medicinal plant Stephania yunnanensis is rich in aporphine alkaloids, a type of benzylisoquinoline alkaloids (BIAs), with aporphine being the representative and most abundant compound, but our understanding on the biosynthesis of BIA alkaloids in this plant have been relatively limited. Previous research has reported the genome of S. yunnanensis and preliminarily identified the upstream gene norcoclaurine synthase (NCS) in the BIA biosynthetic pathways. However, the key genes promoting the formation of the aporphine skeleton have not yet been reported. In this study, based on the differences in the content of crebanine and several other BIAs in different tissues, we conducted transcriptome sequencing of roots, stems, and leaves. We then identified candidate genes through functional annotation and sequence alignment, followed by transcriptomic and genomic analyses. Based on this analysis, we identified three CYP80 enzymes (SyCYP80Q5-1, SyCYP80Q5-3, and SyCYP80G6), which exhibited different activities towards (S)- and (R)-configured substrates in S. yunnanensisand demonstrated strict stereoselectivity enroute to aporphine. This study provides metabolomic and transcriptomic information on the biosynthesis of BIAs in S. yunnanensis and offers valuable insights into the elucidation of BIA biosynthesis, and lays the foundation for the complete analysis of pathways for more aporphine alkaloids.