Two CYP72 enzymes function as Ent‐labdane hydroxylases in the biosynthesis of andrographolide in Andrographis paniculata

Andrographis paniculata (Burm.f.) Wall. Ex Nees in Wallich (A. paniculata), an annual medicinal herb of the Acanthaceae family, is widely cultivated for its various medicinal utilities in Southeast and South Asia. Its total extract and monomeric components have a broad range of pharmacological effects including anti-inflammatory, anti-microbial, hepatoprotective and anticancer (Subramanian et al., 2012). Numerous bioactive secondary metabolites have been isolated from the leaves and roots of A. paniculata, andrographolide, an ent-labdane diterpenoid, is considered the main bioactive compound (Subramanian et al., 2012). For example, Xiyanping®, a traditional Chinese medicine injection made of andrographolide sulfonate, is widely used to treat upper respiratory tract infection, viral pneumonia and bronchitis in China. Due to their medicinal properties, andrographolide biosynthesis has been intensively investigated, genomic data and terpene synthase functions have been reported (Sun et al., 2019). However, the enzymes responsible for structural modification that form the key pharmacologically active groups in its biosynthetic pathway remain unknown. The modification steps in andrographolide biosynthesis include hydroxylations at C3, C14, C18 and lactone ring formation at C15–C16. This series of oxidation processes were supposed to be mediated by cytochrome P450 enzymes (CYP450s). In order to accurately screen the CYP450s in andrographolide biosynthesis pathway, we constructed the differential bio-accumulation samples of andrographolide seedlings (Figure S1). After 100 μM MeJA treatment, the production of andrographolide demonstrated significant enhancement at 24 h post-inoculation (hpi) and reached 37.8 mg/g DW at 72 hpi in the leaves, which is approximately 10 times greater than that in the control (Figure 1a). We then constructed the expression atlas and investigated the time-series expression changes of A. paniculata. The expression profiles of samples at 12 hpi, 24 hpi and 48 hpi exhibited significantly different patterns compared to the samples collected at 0 hpi (Figure S2). By applying a cutoff of a four-folds difference in FPKM and a false discovery rate of less than 0.05, we identified that the expression levels of 4463 genes were up-regulated at 12 hpi, 24 hpi or 48 hpi in comparison to the control samples (Figure 1b). As an upstream pathway for terpene synthesis, the expression of all genes in mevalonate (MVA) and methylerythritol phosphate (MEP) pathways were strongly induced by MeJA treatment. Most of them reached the highest level of genes expression in 12 hpi or 48 hpi. As expected, the expression level of GPP synthase (GPPS), FPP synthase (FPPS) and GGDP synthase (GGPPS) increased dramatically, and maintained a relatively high level, the expression of ApCPS2 significantly increased up to about 30-folds compared with control samples (Figure 1c). The transcriptome data revealed that 154 CYP450 genes were significantly increased or decreased in at least one period during induction and 96 ones are derived from CYP71 clan (Figure S3a,b), many of the currently discovered diterpenoid biosynthetic P450s belong to this clan (Zheng et al., 2019). Among 154 CYP450 genes, the expressions of 54 genes were increased in all induced time points and 32 of them belong to CYP71 clan (Figure S3c). Ten of the CYP71 clan genes, four of CYP72 clan genes and one CYP85 gene were selected as candidates according to their progressive increase expression after induction (Figure 1d). The candidate P450 genes identified through coexpression screening were expressed in yeast and subsequently extracted their microsomes (Figure 1d). The catalytic function of CYP450 enzymes were validated via enzymatic reactions with six andrographolide compounds, which were predicted to be intermediates in the biosynthetic pathway of andrographolide (Figure S4). By analysing the enzymatic reaction products and comparing with the standards, TR79615 was found to catalyse the formation of andrographolide (1) using 14-deoxyandrographolide (2) as substrate (Figure 1e). TR79615 has been designated CYP72F1 according to systematic nomenclature. The product structure demonstrated that CYP72F1 can facilitate the C14 hydroxylation of 14-deoxyandrographolide while rearranging the double bond to positions C12 and C13 (Figures 1h and S5a). And two byproducts with the same molecular weight of andrographolide were produced (Figures 1e and S5b,c), which were speculated to be andrographolide isomers with different double bond positions. Plant CYP450 enzymes are capable of catalysing multiple substrates that share the same skeletal structure (Ma et al., 2021). CYP72F1 microsome was also found to react with andrograpanin, considering the specificity of substrate oxidation sites targeted by plant CYP450 enzymes, along with the polarity difference between the substrate and product, we hypothesize that CYP72F1 can catalyse the C14 hydroxylation and C12 and C13 double bond rearrangement of andrograpanin to form 3-deoxyandrographolide (Figure S6). With the continuous advancement of genomic data, an increasing number of the key genes involved in the biosynthesis pathway of plant diterpenes have been demonstrated to cluster within genomes (Ma et al., 2021). Based on reported genomic data of A. paniculata (Sun et al., 2019), we conducted genomic localization studies on CYP72F1 and other candidate genes in Figure 1d. The results indicated that seven CYP450 genes belonging to the CYP72 clan were clustered alongside CYP72F1 on chromosome 2, with minimal distance separating these genes (Figure 1f). Their expression trend of these CYP450s after MeJA induction was investigated (Figure S7), the expression of TR81244 was significantly up-regulated and then down-regulated after 24 h induction, which was similar to the expression pattern of MVA pathway genes and GGPPS (Figure 1c). These four CYP72 genes were then cloned and expressed in yeast for enzymatic reactions using andrographolide compounds as substrates. The results showed that TR81244 could catalyse the generation of new product peaks from andrograpanin (3) and the product was identified as 14-deoxyandrographolide (2) by comparing with the standard compound (Figures 1g and S8a). Consequently, TR81244 was named CYP72A399 and confirmed the catalytic activity of facilitating the C3 hydroxylation of andrograpanin (Figure 1h). CYP72A399 could also catalyse ent-cppalol and 16,19-dihydroxy-ent-copalol as substrates, according to the specificity of CYP450 catalytic position, we speculate that it also catalyses their C3 site to generate hydroxylation products (Figure S8b–f). Chromosome localization and collinearity analysis were performed on these CYP72 genes from chromosome 2, along with species rich in diterpenoids such as Salvia miltiorrhiza, Scutellaria baicalensis and Leonurus japonicus. These collinear genes are also clustered on the same chromosome or scaffold in other species (Figure 1i), which provides reference for further exploration of the clustering of terpenoid biosynthesis pathway genes. Since ent-labdane terpenoids are abundant in L. japonicus (Wang et al., 2022), we expressed the three L. japonicus CYP450s obtained by collinearity analysis in yeast to verify whether they have similar functions. Lej2023 was found to have the same catalytic function as CYP72A399, catalysing andrograpanin C3 hydroxylation to form 14-deoxyandrographolide (Figure S9). In plants, the CYP72 clan represents one of the largest groups of CYP450s involved in secondary metabolism, yet limited biochemical information of CYP72 clan genes were screened. The currently identified proteins in CYP72 clan facilitate complex biocatalytic processes such as the oxidations in the pathway of gibberellins (He et al., 2019), triterpenoids (Biazzi et al., 2015) and secologanic acid (Yang et al., 2019). The two CYP72 proteins identified in this study extend our understanding of the novel catalytic functions associated with the CYP72 family (Figure 1j). These CYP72 proteins are categorized into two subfamilies and are found clustered on the chromosomes of A. paniculata, they continuously catalyse the final steps in biosynthesis of andrographolide. The catalytic processes involving C14 hydroxylation and C3 oxidation are crucial for the formation of andrographolide derivatives, which are important for the enhancement of anti-tumour activities of andrographolide (Zhang et al., 2021). To sum up, this study reported two CYP450 genes of CYP72 clan in A. paniculata through terpenoid pathway coexpression and gene cluster analysis, these two CYP72 CYP450s catalysed the C3 and C14 hydroxylation and the C12–C13 double bond rearrangement, which are the key steps in andrographolide biosynthetic pathway. This work was supported by the National Key R&D Program of China (2020YFA0908000), Scientific and technological innovation project of CACMS (CI2023E002, CI2023D002 CI2024C009YNL), the National Natural Science Foundation of China (82204573), Key project at central government level: The ability to establish sustainable use of valuable Chinese medicine resources (2060302), the Fundamental Research Funds for the Central public welfare research institutes (ZZ16-YQ-039, ZZ14-YQ-044). Thanks to QINGFENG Pharma Group for providing materials and data support. The project was designed by HLQ, GJ and MY. MY and WJ participate in eukaryotic expression of candidate genes. WJ, LHX and TJH prepared plant materials and RNA samples for transcriptome sequencing and bioinformatics analyses. WJ and TJH carried out the metabolites analysis and the qPCR analysis; MY and WJ participated in the paper preparation. All authors read and approved the final manuscript. The authors declare no competing financial interests. High-throughput sequencing data corresponding to time-serialized gene expression profiles have been deposited in the NCBI Gene Expression Omnibus (GEO) (No. GSE83775). The Chromosome information of S. miltiorrhiza, S. baicalensis and L. japonicus in the IMP database (http://www.bic.ac.cn/IMP/#/). Appendix S1 Plant materials and growth conditions. Figure S1-S9 Supplementary Figures. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.