Abstract Isodon suzhouensis is a valuable medicinal plant found in the northern Anhui province of China. Its primary and most potent therapeutic ingredient is the diterpenoid glaucocalyxin A (Wangzaozin B). The biosynthesis pathway of glaucocalyxin A and the key genes involved are currently unknown. The objective of this study is to propose the potential biosynthesis pathway for glaucocalyxin A and identify key genes related to its production. To achieve this objective, we integrated transcriptome and metabolomics analyses of I. suzhouensis to mined differentially expressed genes (DEGs) involved in glaucocalyxin A biosynthesis. One DEG, IsCPS1, was identified and then cloned through RT-PCR amplification and subjected to bioinformatics analysis. The IsCPS1 protein was subsequently isolated and purified, and its in vitro enzyme activity was tested. Molecular docking of IsCPS1 and substrate GGPP was conducted to identify the key activity sites of IsCPS1. Furthermore, IsCPS1 was overexpressed in A. thaliana and the DEMs between wild type and transgenic lines were identified. Our study revealed a total of 6311 metabolites from the leaves and stems of I. suzhouensis. Among these, 418 metabolites, including glaucocalyxin A (wangzaozin B), were up-regulated in the leaves, while 354 metabolites were down-regulated. We also obtained 49.02 Gb of clean data and identified 47 149 unigenes, with 12768 DEGs found in the leaves compared to the stems. The enrichment analysis of these DEGs showed that pathways related to the formation of phenylpropanoid compounds, flavonoids, and terpenoid compounds were significantly enriched. We identified 30 important DEGs, including HMGR, CPS, KS, GA20ox, GA2ox, GA3ox, and CYP76AH1 (FRS). Several of these genes were up-regulated and are likely responsible for the tissue-specific accumulation of the diterpenoid glaucocalyxin A. Additionally, we successfully cloned the IsCPS1 gene, and found that IsCPS1 showed in vitro activity in transforming GGPP to ent-CPP. The molecular docking results indicated that D383 in the “DXDD” motif and K250 were key activity sites that bind with the alkyl carbon chain and diphosphate part of the substrate GGPP. Heterologous overexpression suggested that IsCPS1 may be involved in the production of diterpenes such as gibberellin and glaucocalyxin A. This study provides valuable basic metabolomic and genetic data resources for I. suzhouensis and sheds preliminary light on the biosynthesis mechanism of glaucocalyxin A. IsCPS1 could be a potential gene for molecular breeding to increase the content of glaucocalyxin A in I. suzhouensis.

Transcriptomic and Metabolomic Analysis of Isodon suzhouensis Reveals a Potentially IsCPS1 Gene for Glaucocalyxin A Biosynthesis

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发表日期：

2025年4月1日

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F. Liu

摘要

Abstract

Isodon suzhouensis is a valuable medicinal plant found in the northern Anhui province of China. Its primary and most potent therapeutic ingredient is the diterpenoid glaucocalyxin A (Wangzaozin B). The biosynthesis pathway of glaucocalyxin A and the key genes involved are currently unknown. The objective of this study is to propose the potential biosynthesis pathway for glaucocalyxin A and identify key genes related to its production. To achieve this objective, we integrated transcriptome and metabolomics analyses of I. suzhouensis to mined differentially expressed genes (DEGs) involved in glaucocalyxin A biosynthesis. One DEG, IsCPS1, was identified and then cloned through RT-PCR amplification and subjected to bioinformatics analysis. The IsCPS1 protein was subsequently isolated and purified, and its in vitro enzyme activity was tested. Molecular docking of IsCPS1 and substrate GGPP was conducted to identify the key activity sites of IsCPS1. Furthermore, IsCPS1 was overexpressed in A. thaliana and the DEMs between wild type and transgenic lines were identified. Our study revealed a total of 6311 metabolites from the leaves and stems of I. suzhouensis. Among these, 418 metabolites, including glaucocalyxin A (wangzaozin B), were up-regulated in the leaves, while 354 metabolites were down-regulated. We also obtained 49.02 Gb of clean data and identified 47 149 unigenes, with 12768 DEGs found in the leaves compared to the stems. The enrichment analysis of these DEGs showed that pathways related to the formation of phenylpropanoid compounds, flavonoids, and terpenoid compounds were significantly enriched. We identified 30 important DEGs, including HMGR, CPS, KS, GA20ox, GA2ox, GA3ox, and CYP76AH1 (FRS). Several of these genes were up-regulated and are likely responsible for the tissue-specific accumulation of the diterpenoid glaucocalyxin A. Additionally, we successfully cloned the IsCPS1 gene, and found that IsCPS1 showed in vitro activity in transforming GGPP to ent-CPP. The molecular docking results indicated that D383 in the “DXDD” motif and K250 were key activity sites that bind with the alkyl carbon chain and diphosphate part of the substrate GGPP. Heterologous overexpression suggested that IsCPS1 may be involved in the production of diterpenes such as gibberellin and glaucocalyxin A. This study provides valuable basic metabolomic and genetic data resources for I. suzhouensis and sheds preliminary light on the biosynthesis mechanism of glaucocalyxin A. IsCPS1 could be a potential gene for molecular breeding to increase the content of glaucocalyxin A in I. suzhouensis.