Transcriptome analysis of Stephania yunnanensis and functional validation of CYP80s involved in benzylisoquinoline alkaloids biosynthesis

doi:10.21203/rs.3.rs-5384973/v1

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Transcriptome analysis of Stephania yunnanensis and functional validation of CYP80s involved in benzylisoquinoline alkaloids biosynthesis

https://doi.org/10.21203/rs.3.rs-5384973/v1

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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.

Molecular Biology

Plant Molecular Biology and Genetics

aporphine alkaloids

biosynthetic gene clusters

biosynthesis

CYP80

Stephania yunnanensis

transcriptome

This study found that BIA biosynthetic genes in Stephania yunnanensis are not all highly expressed in roots and are clustered on chromosomes. Two CYP80s catalyze key steps in aporphine biosynthesis with strict conformational selectivity.

Stephania yunnanensis is one of the source plants of the traditional Chinese medicine ‘Shanwugui’, which is a plant from the genus Stephania in the family Menispermaceae of the order Ranales (Heming & Xianrui, 1980). Traditional Chinese medicine theory believes that S. yunnanensis has the effects of vomiting phlegm and foods, treating malaria, and detoxifying sores (Zhao et al., 2021). Previous chemical research has shown that the main active components of S. yunnanensis were four types of benzylisoquinoline alkaloids (BIAs), including 1-benzylisoquinolines (1-BIAs), aporphines, morphinans, and protoberberines (Dai et al., 2012; Hu et al., 2010; Wang et al., 2022), such as salutaridine, tetrahydropalmatine, roemerine, stephanine, and crebanine, etc. Crebanine, a kind of aporphine, was major active compound found in S. yunnanensis (Peng, 2014), recent studies have indicated that crebanine has neuroprotective (Yang et al., 2023), cardioprotective(Wang et al., 2016), anticancer (Tan et al., 2023; Yeh et al., 2024), anti-inflammatory and analgesic activities (Cui et al., 2022).

Since the extraction of BIAs from plants was an expensive process and the content was also affected by the growth conditions of the plants, this low yield and variability limited the commercial development of BIAs. The complexity of chemical synthesis further hindered the large-scale production of BIAs. The demand for BIAs ultimately promoted the development of BIAs metabolic engineering and synthetic biology. The biosynthetic pathways of BIAs were initiated with the enzymatic reaction of norcoclaurine synthase (NCS), which catalyzes the synthesis of norcoclaurine from dopamine and 4-hydroxyphenylacetic acid (4-HPAA) (Ghirga et al., 2017). Subsequent biochemical transformations, included hydroxylation, methylation, and isomerization processes involving an array of cytochrome P450s, methyltransferases, and reductases, such as norcoclaurine 6-O-methyltransferase (6OMT) (Menendez-Perdomo & Facchini, 2020), coclaurine N-methyltransferase (CNMT), (S)-N-methylcoclaurine 3’-hydroxylase (NMCH) (Liu et al., 2021; Pauli & Kutchan, 1998), and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) (Gurkok et al., 2016). These enzymes catalyzed the production of a series of crucial 1-BIA intermediates, including coclaurine (COC), N-methylcoclaurine (NMC), 3'-hydroxyl-N-methylcoclaurine (HNMC), and reticuline, which subsequently served as substrates for the biosynthesis of a diverse array of BIAs (Fig. 4).

Aporphine alkaloids like crebanine were the major compounds in S. yunnanensis, which had an unclear biosynthetic pathway. Recent studies have discovered that corytuberine synthase (CTS) like CjCYP80G2 from Coptis japonica (Ikezawa et al., 2008) and StCYP80G6 (Li et al., 2024) from Stephania tetrandra could catalytic C-C coupling of (S)-reticuline to the type Ⅰ aporphine (S)-corytuberine. Additionally, NnCYP80Q1 or StCYP80Q5 could catalytic C-C coupling of (R)-coclaurine and (R)-N-methylcoclaurine to the protoaporphines (R)-crotoflorine and (R)-glaziovine, two important precursors of type Ⅱ aporphines (Li et al., 2024; Pyne et al., 2023). Subsequently, the proaporphines crotoflorine and glaziovine underwent unknown oxidative rearrangements, followed by a series of hydroxylations, methylations, and the formation of methylenedioxy bridges, leading to the production of type II aporphines, including crebanine and nelumboferine (Li et al., 2024). These CYP80s were considered to be the starting points of the aporphines biosynthetic pathways, however, no CYP80s with similar catalytic C-C coupling functions had been found in S. yunnanensis.

The combination of transcriptomics and metabolomics is currently the most common method for studying the biosynthesis and regulation of plants secondary metabolites (Lai et al., 2024; Li et al., 2022; Li et al., 2023; Tong et al., 2022). To date, de novo transcriptomes of multiple BIA-producing plants have been reported, focusing on the Papaveraceae, Ranunculaceae, and Menispermaceae families within the Ranunculales (Hagel et al., 2015). Papaveraceae plants have been a primary focus due to their unique accumulation of morphinan alkaloids (Park et al., 2024; Pei et al., 2021; Xu et al., 2022; Zhao et al., 2019). As the pharmacological effects of other types BIAs have been discovered, the transcriptomes of other BIA-producing plants have also been reported to enable more in-depth research into BIA biosynthesis. The genome of S. yunnanensis has been previously reported (Leng et al., 2024), providing valuable genomic data for investigating its BIA biosynthetic pathways. However, for a deeper understanding of the BIA biosynthesis and its regulation, it is also necessary to have information on gene expression levels and metabolite profiles. Unfortunately, there are currently no reported transcriptome data available for S. yunnanensis.

In this study, we determined the relative concentrations of four BIAs in different tissues of S. yunnanensis to investigate the differential accumulation patterns of these BIAs. We also generated a de novo transcriptome of S. yunnanensis and identified hundreds of genes potentially related to BIA biosynthesis. Subsequently, using differential expression analysis and phylogenetic analysis, we identified some candidate genes involved in BIA biosynthesis in S. yunnanensis and conducted genomic-level analysis on them. We then validated the functions of CYP80s genes of S. yunnanensis in vitro. Our research will contribute to further in-depth studies on BIA biosynthetic pathways and their evolution in Stephania species, aiming to elucidate the complete biosynthetic pathways of BIAs represented by crebanine.

2.1 Plant materials, chemicals, reagents and strains

The S. yunnanensis plant samples were collected from Yunnan University of Traditional Chinese Medicine in Kunming City, Yunnan Province, China.

Authentic standards of (S)-N-methylcoclaurine and (R)-N-methylcoclaurine were obtained through commercial chemical synthesis by WuXi LabNetwork. (S)-norcoclaurine, (R)-norcoclaurine, (S)-coclaurine and (R)-coclaurine were derived from the chiral separation of commercial racemic standard. The racemic norcoclaurine and coclaurine were purchased from Baoji Herbest Bio-Tech Co., Ltd. Other standards were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (R)-crotoflorine was prepared in previous work (Li et al., 2024).

YPD medium was composed of 20 g L^− 1 peptone (OXOID), 10 g L^− 1 yeast extract (OXOID), and 20 g L^− 1 glucose (Solarbio), serving as a standard medium for cultivating yeast and preparing competent cells. YPL medium, used to induce gene expression in yeast, uses galactose (Solarbio) instead of glucose compared to YPD medium. Synthetic dropout minus uracil medium (SD-Ura, FunGenome) with 20 g L^− 1 glucose was employed to select positive colonies transformed with the pESC-Ura vector. For plates preparation, agar (DING GUO) at a concentration of 20 g L^− 1 was added as necessary.

WAT11 yeast strain with endogenous cytochrome P450 reductase replaced by one from Arabidopsis thaliana was maintained by this laboratory (Urban et al., 1997). pESC-Ura was purchased from Agilent Technologies. Escherichia coli Trans1 T1 strain from TransGen Biotech was used for routine plasmid assembly.

2.2 Alkaloid extraction and composition analysis

50 mg root, stem, and leaf freeze-dried powders of S. yunnanensis were mixed to 2 mL methanol and then sonicated extracts for 0.5 h. After centrifugation, the supernatants were filtered with a nylon syringe filter (0.22 µm). Quantitative analysis was conducted on a UPLC-QTOF-MS system (Waters Technologies). The Acquity UPLC was carried out using a T3 column (Waters Technologies, 2.1 mm × 100 mm, 2.7 µm particle size) at 38 ℃. The mobile phases consisted of eluent acetonitrile (A) and 0.1% aqueous formic acid (B) with a flow rate of 0.1 mL min^− 1 with the linear gradient elution program: 5% − 12% A from 0.0 min to 1.0 min, 12% A from 1.0 min to 15.0 min, 12% − 17% A from 15.0 min to 16.0 min, 17% A from 16.0 min to 66.0 min, 17% ~ 90% A from 66.0 min to 67.0 min, 90% A from 67.0 min to 72.0 min, 90% − 5% A from 72.0 min to 72.5 min, and 5% A from 72.5 min to 76.0 min. The Acquity UPLC system was coupled to a Waters Xevo G2-S QTOF mass spectrometer equipped with electrospray ionization (ESI). The instrument was operated in positive ion mode to perform full scan monitoring in the range of m/z 50–800. The other operating parameters were set as follows: capillary voltage of 0.5 kV; sample cone voltage of 40 V; extraction cone voltage of 4 V; source temperature of 100 ℃; desolvation temperature 300 ℃; and desolvation gas flow of 800 L h^− 1. The trap collision energy of low energy function was set at 6 eV, while ramp trap collision energy of high energy function was set at 30–50 eV (Xiao et al., 2018). Data acquisition and processing were performed using MassLynx software.

2.3 Transcriptome analysis

S. yunnanensis RNA was extracted using the R6827 Plant RNA Kit (Omega Bio-tek) according to the manufacturer's protocol. The RNA was quality-checked using Nanodrop, Qubit, and gel electrophoresis. Once the RNA samples were verified to be of acceptable quality, they were randomly fragmented using a Covaris sonicator. The entire library preparation process then involved end repair, A-tailing, adapter ligation, purification, and PCR amplification. Specifically, fragmented mRNA was used as a template with random oligonucleotides as primers to synthesize the first strand of cDNA in an M-MuLV reverse transcriptase system. The RNA strand was degraded with RNaseH, and the second strand of cDNA was synthesized using DNA polymerase I with dNTPs as substrates. The double-stranded cDNA underwent end repair, A-tailing, and sequencing adapter ligation. AMPure XP beads were used to select cDNA fragments of approximately 250–300 bp. After PCR amplification, the library was purified again using AMPure XP beads. Quality control of the library involved quantification with a Qubit 2.0 Fluorometer, followed by size verification with an Agilent 2100 bioanalyzer. Once the insert size met expectations, the effective library concentration was accurately quantified using qRT-PCR (with the effective library concentration exceeding 2 nM). The library preparation kit used was the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®, and sequencing was performed on an Illumina Novaseq 6000 platform.

To obtain comprehensive functional information of the transcripts, functional annotation was performed using seven databases, including Nr (Yangyang et al., 2006), Pfam (Finn et al., 2014), Uniprot (Apweiler et al., 2004), KEGG (Kanehisa et al., 2004), GO (Ashburner et al., 2000), KOG/COG (Tatusov et al., 2003), and PATHWAY (Kanehisa et al., 2004). Functional annotation primarily employed two methods: sequence similarity search and motif similarity search. For sequence similarity search, the protein sequences encoded by the transcripts were compared with existing protein databases, such as Uniprot, Nr (Yangyang et al., 2006), and the metabolic pathway database KEGG (Kanehisa et al., 2004), using diamond BLASTp (version: 2.0.6.144; parameters: –evalue 1e^− 5) (Buchfink et al., 2015) to obtain functional information and potential metabolic pathway information. KEGG annotation was performed using KOBAS (version: 3.0) (Xie et al., 2011) to associate with KEGG ORTHOLOGY and PATHWAY. The Uniprot (Apweiler et al., 2004) database records the correspondence between each protein family and functional nodes in Gene Ontology (Ashburner et al., 2000), allowing prediction of the biological functions executed by the protein sequences encoded by the transcripts. Based on the associations between databases, KOG/COG (Tatusov et al., 2003) annotation results were obtained, followed by classification statistics and plotting for KOG/COG (Tatusov et al., 2003). For motif similarity search, proteins typically consist of one or more functional regions, commonly referred to as domains. The different combinations of these domains result in a variety of proteins; thus, identifying protein domains is crucial for analyzing protein functions. Domain prediction was performed using hmmscan (version: 3.3.2; parameters: e-value 0.01) (Finn et al., 2011) to obtain conserved sequences, motifs, and domains of the proteins. The Pfam (Finn et al., 2014) database is a large collection of protein families based on multiple sequence alignments and hidden Markov models.

The expression level of transcripts was assessed by calculating their FPKM value. If the samples all contain biological replicates, used DESeq2 for differential expression analysis; otherwise, used edgeR. Herein, the differentially expressed genes (DEGs) were screened with the thresholds were a significance level of corrected value of padj < 0.05 and |log2FoldChange| > 1. Perform GO enrichment and KEGG pathway enrichment analysis on DEGs, with the padj < 0.05, which was considered significantly enriched among DEGs, respectively.

2.4 Analysis of candidate genes in the BIA biosynthetic pathways

To identify the candidate genes associated with BIA biosynthetic pathways of S. yunnanensis, local BLASTp by BioEdit, version 7.1.3.0 (Hall, 1999) was performed against S. yunnanensis sequences using query sequences of BIA-producing plants obtained from GenBank databases. The resulting transcripts with high degrees of identity were selected as candidate genes. A heatmap was generated using Tbtools with row scaling. Based on the reported genome of S. yunnanensis (Leng et al., 2024), we studied the chromosomal localization of these candidate genes and conducted synteny analysis of three species using TbTools.

Based on the reference genes of BIA-producing plants and the obtained candidate genes, phylogenetic analysis was conducted using MEGA11 (Tamura et al., 2021) through the maximum likelihood estimation.

2.5 Cloning of candidate genes and eukaryotic expression of recombinant plasmids

All candidate genes were amplified from S. yunnanensis root or leaf cDNA using the primers listed in the supplementary information (Table S4) and PrimeSTAR® Max DNA Polymerase (TaKaRa), according to the manufacturer's instructions. All CYP450 genes were purified and constructed into the pESC-Ura vector (digested with BamH Ⅰ) using the Gibson Assembly Kit (TransGen Biotech). The recombinant vectors were transformed into competent E. coli Trans1 T1 and identified by colony PCR (TransGen Biotech) and Sanger sequencing (Ruiotechnology). The identified positive recombinant plasmids were extracted from Trans1 T1 using the Plasmid Purification Kit (Magen Biotech).

The recombinant plasmids pESC-Ura carrying candidate CYP450 genes were each transformed into WAT11 using the Frozen-EZ Yeast Transformation II Kit (Zymo Research). WAT11 transformed with empty pESC-Ura was employed as a control. The positive transformants were screened on SD-Ura medium containing 20 g L^− 1 glucose. The positive clones were incubated with shaking at 30°C for 20–24 hours until the OD600 reached 2–3. Cells were centrifuged to remove the SD-Ura medium. The cells were then resuspended in YPL induction medium and grown overnight at 30°C to induce recombinant protein expression.

2.6 Microsomes extraction, enzymatic activity assay and LC-MS analysis

Microsomes of recombinant yeast were prepared as previously described (Pompon et al., 1996), which has proven to be successful for previous such characterization. In vitro activity assays were performed in a 250 µL reaction system that included 100 mM Tris-HCl (pH 7.5, sangong) and 500 µM NADPH (Solarbio), 0.1 g microsomal protein, and 20 µM of the substrate. The reactions were incubated at 30 ℃ for 2 hours with 180 rpm shaking, then 5 µL ammonium hydroxide of was added to the reaction system to make the pH to about 10, and then extracted with 250 µL of ethyl acetate. After the ethyl acetate solution of the reaction product was concentrated to dryness, 150 µL of methanol was added to redissolve and centrifuged at 20,000 g for 15 min before LC-MS analysis. All enzymatic reaction products were detected using UPLC-QTOF-MS system (Waters Technologies). The Acquity UPLC was carried out as previously described. The linear gradient elution program: 5% − 30% A from 0.0 min to 6.0 min, 30% − 60% A from 6.0 min to 8.0 min, 60% − 90% A from 8.0 min to 8.5 min, 90% A from 8.5 min to 9.5 min, 90% − 5% A from 9.5 min to 10.0 min, and 5% A from 10 min to 11 min. Data acquisition and processing were performed using MassLynx software.

3.1 Determination of BIAs in S. yunnanensis

S. yunnanensis is a medicinal plant with a long history of use, with BIAs as its main active components (Dai et al., 2012; Hu et al., 2010). Due to its strict environmental requirements and long growth cycle, it had not yet been cultivated on a large scale. Recent studies have reported the presence of various aporphine alkaloids in S. yunnanensis (Wang et al., 2022) (Fig. S1). We analyzed three tissues of S. yunnanensis using liquid chromatography-mass spectrometry (LC-MS) and researched the relative contents of salutaridine, roemerine, stephanine, and crebanine to investigate the accumulation patterns of these four representative BIAs in S. yunnanensis (Fig. 1, Fig. S2). Surprisingly, we detected four representative BIAs in the roots, each corresponding to the purchased reference standards, but in the stems and leaves, there were hardly any or only minimal amounts detected (Fig. S2). Furthermore, crebanine was the most abundant among the four compounds (Fig. 1), which is consistent with previous studies (Peng, 2014). Among these compounds, salutaridine was identified as a morphinan, another major compound reported in S. yunnanensis (Peng, 2014); Additionally, roemerine and stephanine were considered important intermediates, along with the representative product crebanine, all of which belong to type Ⅱ aporphines (Wang et al., 2022). We also attempted to detect additional aporphines that have been reported in other Stephania species (Peng, 2014; Wang et al., 2022). However, we did not detect magnoflorine, a representative type Ⅰ aporphine, suggesting that S. yunnanensis may contain more type Ⅱ aporphines, with type Ⅰ aporphines being less prevalent. This result might explain why S. yunnanensis was primarily used for its tuberous roots in medicine rather than its aerial parts. However, under the current extraction and detection conditions, we did not find any representative bisbenzylisoquinoline cepharanthine, which contradicts another study that reported the presence of cepharanthine and other bisbenzylisoquinolines in the metabolome of S. yunnanensis (Leng et al., 2024). In summary, the analysis of these compounds indicated that numerous structurally diverse BIAs are present in S. yunnanensis, specifically accumulating in the roots, with aporphines, represented by crebanine, being the most abundant.

3.2 Transcriptome sequencing, assembly, and analysis of S. yunnanensis.

To explore the biosynthesis of aporphine alkaloids in S. yunnanensis, we prepared nine samples from the roots, stems, and leaves for transcriptome sequencing. RNA was extracted and cDNA libraries were constructed using Illumina and PacBio Sequel II for sequencing, resulting in 41,640,198 raw reads. De novo assembly yielded 50,119 transcripts, with an N50 length of 2,041 bp and an average length of 1,681 bp (Table 1).

Table 1

Assembly results of *de novo* transcriptome of *S. yunnanensis*
Item	Number
Seq Num	50,119
Seq Base (bp)	91,278,165
N50 (bp)	2,041
Max Length (bp)	7,046
Min Length (bp)	108
Average Length (bp)	1,821.23
Mean Length (bp)	1,681.00

Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment (Manni et al., 2021) indicated that the S. yunnanensis transcriptome contained 87.3% complete BUSCOs (Fig. S3a, Table S1). Annotation was performed for all 50,119 (100%) transcripts across various databases, with an annotated transcript N50 length of 1,518 bp (Table S2). Gene Ontology (GO) classification (Ashburner et al., 2000) was conducted to describe gene functions in three categories: cellular component, molecular function, and biological process (Fig. S3b).

To investigate the distribution of assembled transcripts in each tissue, their relative expression levels were determined by calculating the fragment per kilobase of transcript per million fragments mapped (FPKM) values of assembled transcripts. To compare the differentially expressed genes (DEGs) between roots and other tissues, we conducted a comparative analysis of the expression levels of assembled transcripts (Fig. 2). The results indicated that the number of DEGs between roots and leaves was the highest compared to other groups, with 1,780 up-regulated and 932 down-regulated (Fig. 2a & 2b). To further analyze the DEGs between roots and leaves, GO and KEGG clustering analyses were performed (Figs. 2c & 2d, Fig. S4). GO clustering revealed that most DEGs between roots and leaves were classified into “monooxygenase activity-related” categories, except for photosynthesis-related categories (Fig. 2c). On the other hand, KEGG clustering showed that a total of 685 DEGs were considered to be involved in secondary metabolism, with only a small number (11 DEGs, with 4 down-regulated and 7 up-regulated) clustered into the “isoquinoline alkaloid biosynthesis” category (Fig. 2d).

3.3 Analysis of BIA biosynthesis genes

Apart from the two reported SyNCS4, SyNCS5 (Leng et al., 2024), the biosynthesis of BIAs in S. yunnanensis has not yet been elucidated. The upstream pathways, from dopamine and 4-HPAA to reticuline, were largely conserved among BIA-producing plants (Lee & Facchini, 2010). Analysis of the annotation results revealed a total of 141 OMT, 117 NMT, 45 NCSs, and 505 P450s (Tables S5-S8). In recent studies, CYP80s involved in BIA biosynthesis have gained increasing attention for their roles in the synthesis of 1-BIAs, as well as bisbenzylisoquinoline alkaloids and aporphine alkaloids (Hao et al., 2024; Li et al., 2024). Notably, among the P450s, 25 were further annotated into the CYP80 family, which are likely involved in BIA biosynthesis. To investigate the BIA biosynthesis in S. yunnanensis, we identified candidate genes with high similarity to previously reported BIA biosynthesis genes from S. tetrandra (Li et al., 2022; Li et al., 2020; Li et al., 2023). We selected sequences with homology greater than 55% and coverage greater than 90% as candidate genes, and a total of 26 candidate transcripts for the BIA biosynthesis were identified from the S. yunnanensis transcriptome. Phylogenetic analysis and domain analysis indicated that these candidate transcripts have structural similarities to the reference genes (Fig. 3, Table S3). The sequence alignment revealed that these candidate genes share similar sequences with the reference genes (Fig. S5-S8). To analyze the expression patterns of these candidate transcripts in different tissues of S. yunnanensis, a heatmap was generated using the FPKM of the candidate genes (Fig. S9). The heatmap showed that, except for NCS, 4'OMT, and CYP80G6, most candidate transcripts did not exhibit distinct differential expression patterns and maintained high expression levels across multiple tissue parts. This may explain why only a few DEGs were clustered into the "isoquinoline alkaloid biosynthesis" category. The disparity between gene expression and compound accumulation suggests that BIA compounds transport might occur in S. yunnanensis. On the other hand, this disparity also suggests that the downstream genes of the BIA biosynthesis might not be highly expressed only in the roots, an important consideration for gene screening.

Furthermore, based on the reported S. yunnanensis genome (Leng et al., 2024), we examined the distribution and location of these candidate transcripts on the chromosomes (Fig. 5a). Sequence alignment revealed that 26 candidate transcripts corresponded to 18 genes in genome, they were arranged in descending order of homology and named according to their respective functional genes before being mapped onto the chromosomes. Notably, among these candidate genes, one SyNMCH, two SyCNMT and two Sy6OMT were located on pseudochromosome 3 in close proximity, suggesting that this region might form a BIA biosynthetic gene cluster (BGC). These were the genes involved in the three consecutive steps following NCS in the upstream BIA biosynthetic pathways. Similarly, another candidate gene SyCYP80G6 co-localized with two SyCNMT on pseudochromosome 11. Additionally, the candidate genes for CYP80Q5 were found on three different pseudochromosomes. Specifically, four were located on pseudochromosome 1, one was located on pseudochromosome 9, and another was co-localized with Sy4'OMT and SyNMCH2 on pseudochromosome 13. Among them, SyCYP80Q5-1 and SyCYP80Q5-2 shared over 98% identity, indicating they are tandem repeats, whereas they had 90% identity with SyCYP80Q5-3 (Fig. S10). The other three genes, SyCYP80Q5-4, SyCYP80Q5-5, and SyCYP80Q5-6, had low identity with other SyCYP80Q5 genes, approximately 59–65%. These chromosomal locations preliminarily suggested that the BIA biosynthetic genes in S. yunnanensis may exhibit a certain degree of clustered distribution. To investigate the distribution and association of these candidate genes in Stephania species, we conducted a synteny analysis using the reported genomes of two other plants, S. japonica and S. cepharantha (Leng et al., 2024) (Fig. 5b). Interestingly, the two tandemly duplicated genes of SyCYP80Q5 on chromosome 1 of S. yunnanensis, SyCYP80Q5-1 and SyCYP80Q5-2 had only one ortholog in S. cepharantha and none in S. japonica, but SyCYP80Q5-3 was conserved across the three species. Notably, the clusters observed in S. yunnanensis, were present on different chromosomes in the three Stephania species. This indicated that the clustering patterns of BIA biosynthetic genes, were conserved across the three species, suggesting that such clustering is likely conserved within Stephania as well. In summary, chromosome localization and synteny analysis have provided insights into the genomic-level associations of BIA biosynthetic genes in S. yunnanensis.

3.4 Functional verification and phylogenetic analysis of CYP80s

To elucidate the biosynthesis of the BIAs of S. yunnanensis, we conducted in vitro functional validation of the two key CYP80s involved in aporphine skeleton formation: CYP80G6 (CTS) and CYP80Q5. These enzymes specifically catalyzed the (S) or (R)- types of 1-BIA substrates to produce the corresponding type Ⅰ aporphines or type Ⅱ aporphines (protoaporphines). The further rearrangement and modification of these intermediates ultimately led to the formation of aporphines such as crebanine. Since the high identity between SyCYP80Q5-1 and SyCYP80Q5-2, we cloned only SyCYP80Q5-1 and another four, SyCYP80Q5-3, SyCYP80Q5-4, SyCYP80Q5-5, SyCYP80Q5-6, as well as SyCYP80G6, and validated their functions of them in vitro. Recombinant plasmids containing these candidate genes were constructed and expressed in Saccharomyces cerevisiae (WAT11), in which the endogenous cytochrome P450 reductase was replaced by one from Arabidopsis thaliana. Microsomes extracted from the recombinant yeast were used for in vitro enzyme assays and were detected by LC-MS. We found that only SyCYP80Q5-1, SyCYP80Q5-3 and SyCYP80G6 exhibited catalytic functions (Fig. 6, Figs. S11 & S12). Specifically, SyCYP80G6 catalyzes the conversion of (S)-reticuline and (S)-NMC into (S)-corytuberine and (S)-glaziovine (Figs. 6a-6d, Figs. S11a & S11c). Both SyCYP80Q5-1 and SyCYP80Q5-3 were capable of catalyzing the conversion of (R)-COC and (R)-NMC into the aporphine alkaloids (R)-crotoflorine and (R)-glaziovine (Figs. 6e-6h, Figs. S11b & S12). Furthermore, the catalytic efficiency of SyCYP80Q5-1 was slightly higher than that of SyCYP80Q5-3 when using either (R)-COC or (R)-NMC as substrates (Figs. 6f & 6h).

Additionally, we discussed the activity of SyCYP80Q5-1 and SyCYP80G6 on a broader range of BIA substrates and other configurations (Fig. S13 & S14). As expected, SyCYP80Q5 and SyCYP80G6 exhibited strict substrate specificity and stereoselectivity, which was consistent with the results of other CYP80s (Li et al., 2024). We performed a phylogenetic analysis of the CYP80 family, and the results showed that the three SyCYP80s clustered within the CYP80 family (Fig. 7), and they were distributed across various clades of the CYP80 family. The functions of these enzymes matched the previously reported enzymes (Li et al., 2024), indicating that these three enzymes play similar roles in the BIA biosynthesis in S. yunnanensis.

The biosynthetic pathways of BIAs have not been fully elucidated, mainly due to the complex transformations from 1-BIAs to various types of BIAs scaffolds and the subsequent downstream modifications, including C-C coupling, C-O coupling, hydroxylation, and methylation, etc. (Li et al., 2024; Meng et al., 2024). This study reported the de novo transcriptome of S. yunnanensis, and through the combined analysis of the transcriptome, metabolome, and genome, identified 18 candidate genes involved in BIA biosynthesis in S. yunnanensis. The functions of three CYP80s were validated in vitro, they specifically catalyzed the C-C coupling of (S)- or (R)-configured 1-BIA substrates, resulting in the formation of aporphines or protoaporphines. This is an important step in the biosynthetic pathways of aporphines (Li et al., 2024).

In medicinal plants where roots or rhizomes are used, the active components are often highly concentrated in the underground parts, with corresponding functional genes and regulatory factors showing significantly higher expression levels compared to the aboveground parts (He et al., 2018; Liu et al., 2017; Liu et al., 2023; Tong et al., 2022; Wang et al., 2015; Zhan et al., 2019). In this study, we analyzed the representative BIAs of S. yunnanensis and found that the major active component, crebanine and three other BIAs were highly accumulated in the roots, similar to other root-based medicinal plants. Notably, no upstream 1-BIAs were detected in the metabolomes of any tissues, possibly due to their complete consumption or concentrations below the detection limit. Furthermore, under the extraction and detection conditions of this study, the representative bisbenzylisoquinoline alkaloid, cepharanthine, was not detected.

Then we sequenced the transcriptomes of S. yunnanensis using next-generation sequencing, followed by de novo assembly and functional annotation. Previous studies have shown that P450s are involved in almost the entire biosynthetic pathways of BIAs, specifically CYP80, which participates in hydroxylation and downstream C-O and C-C coupling reactions (An et al., 2024; Hao et al., 2024; Li et al., 2024; Meng et al., 2024). CYP719 catalyzed the formation of the methylenedioxy bridge (Hori et al., 2018; Ikezawa et al., 2003; Li et al., 2024; Menendez-Perdomo & Facchini, 2023), a characteristic group of many active BIA components, including crebanine. We identified 25 transcripts annotated as “CYP80” and 11 were annotated as “CYP719” in the de novo transcriptome of S. yunnanensis. Through annotation and sequence alignment, we identified 26 candidate transcripts involved in 10 steps of the BIA biosynthetic pathways. The heatmap showed that, except for a few genes, the majority were not specifically expressed in the roots but were highly expressed across multiple tissues. This pattern of metabolite accumulation and differential gene expression levels, while uncommon, is not unprecedented in BIA-producing plants. Similar mechanisms involving transporter proteins facilitating the movement of compounds within the plant have been reported previously in C. japonica (Sakai et al., 2002; Shitan et al., 2003)d somniferum (Dastmalchi et al., 2019). We speculate that similar mechanisms may also exist in S. yunnanensis, facilitating the internal transfer of compounds through processes such as endocytosis and exocytosis. In summary, the transcriptome data obtained in this study are valuable for elucidating the biosynthetic pathways of BIAs, including crebanine.

BGCs, composed of tightly arranged genes that collectively participate in the biosynthesis of specific metabolites, allow organisms to coordinate and efficiently regulate gene expression and synergistic metabolic reactions (Li et al., 2017). With the reported high-quality genome of S. yunnanensis (Leng et al., 2024), this study also examined the chromosomal localization of the 18 candidate genes and found a tendency for several candidate genes to cluster. Further collinearity analysis showed that this clustering phenomenon is conserved among different Stephania species, indicating important functions in their evolutionary processes. Notably, the tandem duplication of SyCYP80Q5-1 and SyCYP80Q5-2 on chromosome 1 of S. yunnanensis was not conserved among different species, whereas another SyCYP80Q5-3 gene on chromosome 13 was widely conserved. This might explain why all Stephania species can produce aporphines and protoaporphines, but differ in the types and amounts they contain. Furthermore, the number of CYP80Q5 copies in S. yunnanensis was significantly higher than CYP80G6, which was consistent with the finding that there are more type II aporphines present in S. yunnanensis. In conclusion, the differences and conservation of the BIA biosynthetic pathways in Stephania suggest that there are still variations within the genus. This may explain why there are significant differences in the types and contents of BIAs among different Stephania species.

As an important gene family in the BIA biosynthetic pathways, CYP80, this study further validated the functions of three CYP80s from S. yunnanensis in vitro, showing that they all had the ability to catalyze substrate C-C coupling with configurational selectivity. Specifically, SyCYP80G6 specifically catalyzed the production of corresponding aporphines or protoaporphines from (S)-type substrates; SyCYP80Q5-1 and SyCYP80Q5-3 specifically catalyzed the production of corresponding protoaporphines, the precursor of type Ⅱ aporphines, from (R)-type substrates. The functions of CYP80 are complex and diverse, and there might be some evolutionary correlation. It is noteworthy that, although SyCYP80G6 can also catalyze (S)-type substrates to produce aporphines and protoaporphines, similar to other CYP80Gs, we did not detect any representative reported (S)-type aporphines, such as magnoflorine or mecambroline, in S. yunnanensis. However, the expression level of SyCYP80G6 is not low. This discrepancy between metabolites and gene expression suggests that there may be mechanisms in S. yunnanensis's aporphine biosynthesis that we have yet to understand. It is possible that the metabolic flux is diverted towards (R)-reticuline, leading to the biosynthesis of (R)-type aporphines, morphinans, and protoberberines.

In conclusion, the combined approach of metabolite analysis and transcriptome sequencing identified 26 candidate transcripts responsible for the biosynthesis of BIAs, including crebanine, in S. yunnanensis, these genes might be responsible for BIAs biosynthesis in S. yunnanensis. Furthermore, genome analysis of the 18 candidate genes showed clustering on chromosomes, suggesting the presence of related BGCs, and collinearity analysis indicated these BGCs are conserved among different Stephania species. Finally, we identified three CYP80s from the S. yunnanensis transcriptome data as involved in BIAs biosynthesis using in vitro enzymatic reaction. Overall, our work provides valuable genetic information on S. yunnanensis and reveals the biosynthesis of BIAs in this medicinal plant.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the National Key R&D Program of China (2020YFA0908000), the National Natural Science Foundation of China (82011530137, 31961133007), Scientific and technological innovation project of CACMS (CI2023D002, CI2023E002) and Key project at central government level: The ability to establish sustainable use of valuable Chinese medicine resources (2060302).

Author contributions

LH and JGuo conceived and designed the entire research plans; WS and QL performed most of the experiments; LL, XL, JGan, YM, TC, and PS participated in some of the experiments; WS analyzed results of all the experiments; WS, JGuo, JW, YZ, XM, and LH wrote the manuscript; QL and YM revised the manuscript. All authors read and approved the manuscript.

Data availability

The raw data from the transcriptome sequencing have been uploaded to public database under the accession number XXXXXXXX. All other subsequent data are provided in the supplementary materials.

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The authors declare no competing interests.

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Transcriptome analysis of Stephania yunnanensis and functional validation of CYP80s involved in benzylisoquinoline alkaloids biosynthesis

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Abstract

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Key Message

1. Introduction

2. Materials and methods

2.1 Plant materials, chemicals, reagents and strains

2.2 Alkaloid extraction and composition analysis

2.3 Transcriptome analysis

2.4 Analysis of candidate genes in the BIA biosynthetic pathways

2.5 Cloning of candidate genes and eukaryotic expression of recombinant plasmids

2.6 Microsomes extraction, enzymatic activity assay and LC-MS analysis

3. Results

3.1 Determination of BIAs in S. yunnanensis

3.2 Transcriptome sequencing, assembly, and analysis of S. yunnanensis.

3.3 Analysis of BIA biosynthesis genes

3.4 Functional verification and phylogenetic analysis of CYP80s

4. Discussion

Declarations

Competing Interests

Funding

Author contributions

Data availability

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