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Article

De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus

by
Mengling Wen
1,2,
Junlan Zeng
1,
Fei Qiu
1,
Fangyuan Zhang
1 and
Zhihua Liao
1,*
1
Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China
2
Faculty of Agriculture, Forestry and Food Engineering, Yibin University, Yibin 644000, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(11), 2460; https://doi.org/10.3390/agronomy14112460
Submission received: 20 September 2024 / Revised: 11 October 2024 / Accepted: 15 October 2024 / Published: 22 October 2024
(This article belongs to the Special Issue Metabolomics-Centered Mining of Crop Metabolic Diversity and Function)
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Abstract

:
Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

1. Introduction

Anisodus luridus (which used to be called Scopolia lurida), a perennial herb belonging to the genus Anisodus of the Solanaceae family, originating from Tibet, Yunnan, Nepal, is an important Tibetan medicinal plant, which produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine [1,2]. Compared to other plant species that produce TAs, such as Atropa belladonna, A. luridus is one of the most effective producers of TAs due to its high biomass and hyoscyamine content [2]. Therefore, A. luridus has become a valuable plant source for commercially producing TAs. However, the enzymes involved in TAs biosynthesis have not all been identified, and the TAs biosynthetic pathway has not been systematically studied.
Due to their anticholinergic activity, TAs are widely used in the treatment of postoperative nausea and vomiting, motion sickness, organophosphate poisoning, and Parkinson’s symptoms [3,4]. After more than a century of tireless efforts by generations of scientists, the scopolamine biosynthetic pathway was fully identified in 2021 [5]. The complete biosynthetic pathway of TAs involves a total of 13 enzymes and can be divided into three parts [6,7]. One part is the synthesis of tropine, beginning with ornithine, and ornithine decarboxylase (ODC [8]), putrescine N-methyltransferase (PMT [9]), N-methylputrescine oxidase (MPO [6]), type III polyketide synthase (PYKS [10,11]), tropinone synthase (CYP82M3 [11]), and tropinone reductase I (TRI [6,12]) are involved. The other part is the synthesis of phenyllactylglucose, starting from phenylalanine, and aromatic amino acid aminotransferase 4 (AT4 [13]), phenylpyruvic acid reductase (PPAR [14]), and phenyllactate UDP-glycosyltransferase (UGT1 [15]) are involved. The last part is the condensation of tropine and phenyllactylglucose, with a total of four enzymes involved, which are littorine synthase (LS [15]), littorine mutase (CYP80F1 [16]), hyoscyamine dehydrogenase (HDH [17]), and hyoscyamine 6β-hydroxylase (H6H [18]).
In the past two years, the whole genomes of six TA-producing Solanaceae species, such as A. belladonna, Datura stramonium, Anisodus tanguticus, Brugmansia arborea, Mandragora caulescens, and Anisodus acutangulus, have been sequenced, assembled, and annotated [6,7,19,20]. At the same time, the biosynthetic pathway of TAs has been thoroughly and comprehensively analyzed in phylogenetic evolution. Research shows that the TAs biosynthetic pathway likely emerged in the ancestral lineage shared by all Solanaceae plants. However, the absence or pseudogenization of these genes in non-TAs-producing species have led to a constrained distribution of TAs among Solanaceae members [7]. Furthermore, the five species producing TAs (A. belladonna, D. stramonium, A. tanguticus, B. arborea, and M. caulescens) share a common biosynthetic pathway for scopolamine biosynthesis even though they are distantly related lineages within the Solanaceae family [6,7].
The species most intensively studied in relation to the biosynthetic pathway of TAs is A. belladonna in which all genes involved in the biosynthesis of TAs have been identified. Yang et al. employed TAs biosynthesis genes functionally tested in A. belladonna as a template to search for homologous genes in the genomes of 15 plant species [7]. The results show that homologous genes could be detected in TAs-producing plants, such as A. tanguticus, B. arborea, and M. caulescens, and with high sequence identity (>80%). However, homologous genes underwent pseudogenization or multiple losses in non-TAs-producing plants, indicating that complete and functional TAs genes are a necessary condition for TAs production. Thus, whether A. luridus possesses all homologous genes for TAs biosynthesis and whether TAs are synthesized by the same biosynthetic pathway as A. belladonna remain unclear. However, only PMT, TRI, CYP80F1, and H6H have been reported in A. luridus, among which TRI and H6H have been studied in more depth [1,2]. In 2017, The TRI cDNA sequence was cloned and performed a phylogenetic analysis, tissue profiling, an enzyme assay, and metabolic engineering [2]. In 2018, Lan et al. cloned the full-length H6H cDNA and performed metabolic engineering in hairy root cultures of A. luridus [1]. To date, the other nine genes related to scopolamine biosynthesis in A. luridus have not been reported.
Nicotiana benthamiana has been used in fundamental research for decades as a model plant. The infiltration of Agrobacterium strains carrying pathway genes into leaf tissues enables the conversion of endogenous metabolites, derived from the products of photosynthesis, to target compounds. The utilization of N. benthamiana has now broadened to encompass the elucidation and reconstitution of diverse biosynthetic pathways [21]. To date, several classes of secondary metabolites have been successfully produced in N. benthamiana, such as terpenes [22,23,24], phenolics [25,26,27], and alkaloids [28]. N. benthamiana has been employed as a plant chassis to clarify the biosynthetic pathways of a large number of alkaloids, including strychnine [29], the benzoxazinoid 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-b-d-glucopyranose [30], the colchicine precursor N-formyldemecolcine [31], cocaine [28], diosgenin [32], and piperine [33]. To date, the de novo synthesis of TAs in N. benthamiana has not been reported.
In this study, based on transcriptome data from different tissues of A. luridus, we identified 13 candidate TAs biosynthesis genes, which are highly consistent with the amino acid sequences of functionally tested TAs genes in A. belladonna. Finally, 13 candidate TAs biosynthesis genes were confirmed to be involved in the biosynthesis of TAs in A. luridus, and the de novo synthesis of TAs was performed for the first time in N. benthamiana.

2. Materials and Methods

2.1. Plant Materials

A. luridus plants were cultivated in the medicinal plant garden of the Xizang Agricultural and Animal Husbandry College (Nyingchi, Tibet, China). N. benthamiana was grown in a greenhouse under a photoperiod of 16 h/8 h day/night at 25 °C and used for functional verification in vivo.

2.2. RNA Sequencing and Expression Profiling Analyses

Total RNA was extracted from different tissues of the secondary root (SR), primary root (PR), stem, leaf, flower, and fruit derived from 3 independent A. luridus plants using the RNAsimple Total RNA Kit (TIANGEN) according to the manufacturer’s instructions. And total RNA was subjected to RNA-seq analysis using an Illumina Genome Analyzer at Biomarker Technologies (Beijing, China). Based on FPKM values, the expression patterns of the candidate TAs biosynthesis genes in different tissues were analyzed, and a heat map was generated using TBtools-II [34].

2.3. Real-Time PCR Analysis

Real-time PCR (RT-PCR) was performed using a CFX Connect™ Real Time PCR System (BIO-RAD) with SYBR-Green (TIANGEN, Beijing, China). The primer sequences used are shown in Supplementary Table S1. Each reaction was carried out in a 10 μL volume consisting of 5 μL of SYBR, 4 μL of diluted template (1 μL of the generated first strand cDNA diluted by 19 μL of RNase-Free ddH2O), and 0.5 μL of each of the two gene-specific primers. Two steps were used for RT-PCR: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. With PGK as internal reference, relative gene expression values were calculated using the 2−∆∆Ct method.

2.4. Homologous Gene Retrieval

To search for homologs in the A. luridus transcriptome database, we utilized the functionally tested TAs biosynthesis genes of A. belladonna as a reference [6,17,35], employing BLAST search and the Hidden Markov Model (HMM). The obtained gene sequences from both methods were integrated, and we manually corrected the errors in the automatic annotations.

2.5. Bioinformatics Analysis

Candidate TAs biosynthesis genes of A. luridus were analyzed using Expasy-ProtParam for their physical and chemical properties and WoLF PSORT [36] for prediction of subcellular localization.

2.6. Functional Verification of Candidate Genes in N. benthamiana

The pEAQ-HT plasmid was used for the transient expression of heterologous proteins in N. benthamiana [37]. Full-length open reading frames of candidate TAs biosynthesis genes of A. luridus were recombined into the expression vector pEAQ-HT, and the detailed methods for vector construction were the same as previously described [28]. Gene-specific primers are listed in Supplementary Table S1. The verified recombinant plasmids were successfully transformed into A. tumefaciens GV3101 using the freeze–thaw method. The positive colonies were cultured overnight in the YEP medium containing antibiotics, and the collected bacteria were re-suspended in MMA buffer (10 mM MES pH = 5.6, 10 mM MgCl2, 150 μM acetosyringone), with each strain adjusted to an optical density (OD600) of 0.3. After incubation at room temperature for 2 h, the relevant Agrobacterium suspensions were mixed in an equal volume and then infiltrated into the leaves of N. benthamiana using a 1 mL syringe. The leaves were harvested 4 days later and lyophilized to determine the dry weight of the leaves.

2.7. Quantification of TAs by LC-MS

An amount of 25 mg dry powder of freeze-dried N. benthamiana leaves was extracted with 1 mL of extract solution (20% methanol + 0.1% formic acid). After centrifuges, the supernatant was filtered through a 0.22 μm needle filter. The filtrate was diluted 50 times with extract solution and detected by LC-MS. The TAs content in leaf extract was measured using an Orbitrap Exploris 120 LC-MS (Thermo Scientific, Pittsburgh, PA, USA) according to the methods described previously [6].

3. Results

3.1. Transcriptome Sequencing and Quality Assessment

To identify the genes involved in the biosynthesis of TAs, transcriptome sequencing was carried out in six tissues of A. luridus, namely the secondary root (SR), primary root (PR), stem, leaf, flower, and fruit, and the quality of the sequencing is shown in Table 1. After quality control, a total of 115.77 Gb of Clean Data was obtained, and the Clean Data of each sample was greater than 5.7 Gb, the GC content was about 42%, and the Q30 was above 94%. Overall, the database was of high quality and could be used for the subsequent analysis.

3.2. The Identification of Genes Involved in the Biosynthesis of TAs

Since the genes involved in the TAs biosynthesis of A. belladonna have all been identified [6], we performed a homologous gene search in A. luridus by using a combination of the BLAST search and HMM methods, with the functional tested sequences in A. belladonna as a reference. We manually corrected the faulty automatic annotation and classified all homologous genes into 13 enzyme families. The amino acid sequences of TAs biosynthesis genes identified in A. luridus and those in A. belladonna are highly conserved, with identity values exceeding 90% (Table 2; Figure 1). The conservation of these genes across species within the same lineage likely reflect their shared evolutionary history and functional importance in TAs biosynthesis.

3.3. Analysis of Physical and Chemical Properties and Subcellular Locations

The 13 candidate TAs biosynthesis genes of A. luridus were analyzed, as shown in Table 3. The length of the coding DNA sequence (CDS) ranged from 801 bp (AlTRI) to 2118 bp (AlMPO), and the predicted proteins ranged from 266 to 705 amino acids (AA) in length. The prediction of subcellular localization showed that AlODC, AlAT4, and AlLS were located in the plasma membrane, and the other ten proteins were located in the cytoplasm.

3.4. Expression Analysis Based on RNA-Seq

The accumulation of secondary metabolites was usually tissue-/organ-specific. As reported, the TAs biosynthesis gene identified at each biosynthesis step of A. belladonna and D. stramonium was highly expressed in the roots [6]. To examine the tissue expression pattern, transcriptome data of the secondary root (SR), primary root (PR), stem, leaf, flower, and fruit of A. luridus were used for the expression analysis. The results (Figure 2) show that all 13 candidate TAs biosynthesis genes from A. luridus were highly or specifically expressed in the root, especially in the secondary root. This was consistent with the report that TAs were synthesized in the roots of medicinal plants from Solanaceae [39].

3.5. RT-PCR Validation

To verify the expression profiles characterized by transcriptome data, candidate TAs biosynthesis genes were subjected to a RT-PCR analysis in different tissues. The results (Figure 3) show that all 13 candidate genes (AlODC, AlPMT, AlMPO, AlPYKS, AlCYP82M3, AlTRI, AlAT4, AlPPAR, AlUGT1, AlLS, AlCYP80F1, AlHDH, and AlH6H) for TAs biosynthesis were highly expressed in the roots (PR and SR) and lowly expressed in the stems, leaves, flowers, and fruits, and the expression trend was intensively consistent with the transcriptome data, indicating that the tissue expression pattern analysis is reliable.

3.6. De Novo Synthesis of TAs in N. benthamiana

To verify that the 13 candidate genes are responsible for the biosynthesis of TAs, we established a transient expression system in the leaves of N. benthamiana, a species that does not naturally produce TAs, utilizing the pEAQ vector system, which enables high-level expression of recombinant proteins [37]. Leaves infiltrated with Agrobacterium were used to analyze the content of TAs by LC-MS. In the LC-MS detection system, the retention times (RTs) of the hyoscyamine standard, anisodamine standard, and scopolamine standard were 6.99 min, 6.25 min, and 6.10 min, respectively, and the corresponding m/z values were 290.1757, 306.1706, and 304.1548, respectively.
The results (Figure 4) show that hyoscyamine, anisodamine, and scopolamine could be detected when 13 candidate TAs biosynthesis genes of A. luridus were co-expressed in N. benthamiana cells, while neither hyoscyamine nor scopolamine was detected in the vector controls. Based on the results of transient co-expression, the biosynthetic pathway of TAs in A. luridus was mapped (Figure 5). Therefore, we could draw a conclusion that the TAs biosynthesis genes identified in A. luridus were validated to be involved in the biosynthesis of TAs, and the de novo synthesis of TAs was realized in N. benthamiana for the first time.

4. Discussion

Pharmaceutical TAs, including scopolamine and hyoscyamine, are derived primarily from Solanaceae species and have been found in more than ten species, such as A. belladonna, D. stramonium, B. arborea, Hyoscyamus niger, A. luridus, Hyoscyamus muticus, A. acutangulus, etc. [4]. Since the scopolamine biosynthetic pathway was fully analyzed in 2021 [5], breakthroughs have been obtained on related research in recent years. The de novo production of hyoscyamine and scopolamine was achieved in yeast [17]. In 2023 and 2024, the whole genomes of six TAs-producing Solanaceae species, including A. belladonna, D. stramonium, A. tanguticus, B. arborea, M. caulescens, and A. acutangulus, were sequenced, assembled, and annotated. Additionally, the evolution of tropane alkaloid biosynthesis was thoroughly studied [6,7,19,20]. The biosynthetic pathway of TAs has existed since the origin of Solanaceae, and in subsequent diversification and evolution, the pseudogenization or multiple loss of TAs biosynthesis genes resulted in the inability to synthesize TAs [6,7]. Transcription factor AaWRKR11 from A. acutangulus was successively reported, which can transcriptionally activate the expression of H6H and therefore regulate TAs biosynthesis [20]. Recently, the identification of a mitochondrion-localized BAHD acyltransferase from A. belladonna, 3β-tigloyloxytropane synthase (TS), which catalyzes 3β-tropanol and tigloyl-CoA to form 3β-tigloyloxytropane, enriched the knowledge of TAs biosynthesis [40].
With the identification of AbTRI and AbMPO, all genes involved in scopolamine biosynthesis have been identified in A. belladonna [6]. In this study, we used the functional identified TAs biosynthesis gene from A. belladonna as a reference and conducted homologous gene research in the transcriptome data of different tissues of A. luridus. The biosynthesis genes of TAs identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to those of A. belladonna. The results of our study indicate that the two species may share the same biosynthetic pathway for TAs biosynthesis. It is further suggested that the TAs biosynthetic pathway is conserved across TAs-producing plant species.
The metabolic engineering of TAs has been extensively reported and reviewed in detail by Srivastava et al. [2]. The overexpression of NtPMT in hairy root cultures of Datura metel and Scopolia parviflora could increase the contents of hyoscyamine and scopolamine in transgenic hair roots [41,42]. When DsTRI was overexpressed in hairy root cultures of A. belladonna, both hyoscyamine and scopolamine production were improved [43]. Yun et al. overexpressed the HnH6H in A. belladonna plants, which greatly promoted the transformation of hyoscyamine into scopolamine, resulting in almost all scopolamine in the leaves of the transgenic plants, with the highest content of scopolamine exceeding 1% dry weight [44]. The scopolamine and anisodamine contents were remarkably elevated in the root cultures of S. lurida overexpressing SlH6H/HnH6H [1]. Previously, A. luridus was reported with a high yield of TAs, especially hyoscyamine [1]. The functional identification of the TAs biosynthesis genes of A. luridus facilitates the screening of enzymes with high catalytic activity. However, there are still a lot of interesting studies worth conducting, such as studies to determine the in vitro enzyme activity of identified genes, the identification of rate-limiting steps, etc.
Transient protein expression in a heterologous system is very useful in many areas of research. Tobacco, as a plant expression system, has the advantages of large leaves, easy injection, easy sampling, etc., and is widely used in the study of protein interaction, transcription regulation, subcellular localization, etc. Wang et al. achieved the de novo synthesis of cocaine by transiently expressing EnCYP81AN15 and EnMT4, as well as four identified functional enzymes (EnPKS1, EnATR, EnMecgoR, and EnCS15), in N. benthamiana [28]. Similarly, the simultaneous transient expression of AbPMT, AbMPO, AbPYKS, and AbCYP82M3 in N. benthamiana can detect the production of tropinone [11]. In this study, we employed a similar approach to verify the functions of identified genes. This marks the systematic and comprehensive elucidation of the biosynthetic pathway of TAs within A. luridus.
The heterologous synthesis of tropane alkaloids and their precursors in other organisms has been reported. The de novo synthesis of tropine, hyoscyamine, and scopolamine in yeast and the de novo synthesis of cocaine in N. benthamiana have both been achieved [17,28,45]. Littorine can be synthesized by co-expressing AbUGT1 and AbLS in N. benthamiana leaves when tropine and phenyllactate are added [15]. In this study, a total of 13 TAs biosynthesis genes identified in A. luridus were co-expressed in N. benthamiana, and scopolamine was detected in leaf extract, indicating that these 13 genes were involved in the biosynthesis of TAs in A. luridus. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also show the realization of the de novo synthesis of TAs in N. benthamiana for the first time. This study provides new ideas for the identification of TAs biosynthetic pathways in other TAs-producing plants and provides more functional genes for the efficient production of TAs by metabolic engineering or synthetic biotechnology.

5. Conclusions

Based on homologous gene retrieval, genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to those from A. belladonna, and they were confirmed to be involved in TAs biosynthesis by co-expression in N. benthamiana. The functional identification of the TAs biosynthesis genes of A. luridus facilitates the screening of enzymes with high catalytic activity.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14112460/s1, Table S1: Primers used in this study. Table S2: The CDS of TAs biosynthesis genes identified in A. luridus.

Author Contributions

Formal Analysis, Investigation, and Validation, M.W.; Conceptualization, Funding Acquisition, Project Administration, Resources, and Supervision, Z.L.; Writing—Original Draft, M.W. and Z.L.; Writing—Review and Editing, J.Z., F.Q. and F.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the NSFC project (Grant No. 32101224), the National Key Research and Development Program of China (Grant No. 2022YFD1201600), the Fourth National Survey of Traditional Chinese Medicine Resources, Chinese or Tibet Medicinal Resources Investigation in Tibet Autonomous Region (State Administration of Chinese Traditional Medicine 20191217-540124 and 20200501-542329), the Doctoral Research Innovation Project of Chongqing (CYB22115), and the Experimental Technology Research Project of Southwest University (SYJ2024028).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author(s). Raw data of RNA-seq is available at National Genomics Data Center (https://ngdc.cncb.ac.cn/?lang=zh, BioProject: PRJCA031299).

Acknowledgments

We thank Xiaozhong Lan, Min Chen, and Chunxian Yang for critically reviewing the study proposal. We thank Lingjiang Zeng for helping with the LC-MS analysis and Wentang Chen for helping with data collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 02460 g001
Figure 1. VISTA [38] sequence conservation plot between TAs biosynthesis genes from A. luridus using A. belladonna as reference.
Figure 1. VISTA [38] sequence conservation plot between TAs biosynthesis genes from A. luridus using A. belladonna as reference.
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Figure 2. Tissue expression patterns of candidate TAs biosynthesis genes of A. luridus based on RNA-Seq. SR: secondary root; PR: primary root.
Figure 2. Tissue expression patterns of candidate TAs biosynthesis genes of A. luridus based on RNA-Seq. SR: secondary root; PR: primary root.
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Figure 3. RT-PCR validation of candidate TAs biosynthesis genes of A. luridus in different tissues. PR: primary root; SR: secondary root.
Figure 3. RT-PCR validation of candidate TAs biosynthesis genes of A. luridus in different tissues. PR: primary root; SR: secondary root.
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Figure 4. Detection of hyoscyamine, anisodamine, and scopolamine in leaf extract by LC-MS. (A,D,G) Extracted ion chromatograms (EICs) for hyoscyamine, anisodamine, and scopolamine, respectively, produced by enzymes co-expressed in N. benthamiana leaves, compared with corresponding authentic standard. Vector control was used as negative control. (B,C) MS data of hyoscyamine produced by enzymes co-expressed in N. benthamiana leaves (B) compared with fragmentation of hyoscyamine standard (C). (E,F) MS data of anisodamine produced by enzymes co-expressed in N. benthamiana leaves (E) compared with fragmentation of anisodamine standard (F). (H,I) MS data of scopolamine produced by enzymes co-expressed in N. benthamiana leaves (H) compared with fragmentation of scopolamine standard (I). (J–L) Relative abundance of LC-MS ion for hyoscyamine (J), anisodamine (K), and scopolamine (L) produced in N. benthamiana within three biologically independent samples.
Figure 4. Detection of hyoscyamine, anisodamine, and scopolamine in leaf extract by LC-MS. (A,D,G) Extracted ion chromatograms (EICs) for hyoscyamine, anisodamine, and scopolamine, respectively, produced by enzymes co-expressed in N. benthamiana leaves, compared with corresponding authentic standard. Vector control was used as negative control. (B,C) MS data of hyoscyamine produced by enzymes co-expressed in N. benthamiana leaves (B) compared with fragmentation of hyoscyamine standard (C). (E,F) MS data of anisodamine produced by enzymes co-expressed in N. benthamiana leaves (E) compared with fragmentation of anisodamine standard (F). (H,I) MS data of scopolamine produced by enzymes co-expressed in N. benthamiana leaves (H) compared with fragmentation of scopolamine standard (I). (J–L) Relative abundance of LC-MS ion for hyoscyamine (J), anisodamine (K), and scopolamine (L) produced in N. benthamiana within three biologically independent samples.
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Figure 5. The biosynthetic pathway of TAs in A. luridus. ODC, ornithine decarboxylase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; PYKS, polyketide type III synthase; CYP82M3, tropinone synthase; TRI, tropinone reductase I; AT4, phenylalanine aminotransferase; PPAR, phenylpyruvic acid reductase; UGT1, phenyllactate UDP-glycosyltransferase; LS, littorine synthase; CYP80F1, littorine mutase; HDH, hyoscyamine dehydrogenase; H6H, hyoscyamine 6β-hydroxylase.
Figure 5. The biosynthetic pathway of TAs in A. luridus. ODC, ornithine decarboxylase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; PYKS, polyketide type III synthase; CYP82M3, tropinone synthase; TRI, tropinone reductase I; AT4, phenylalanine aminotransferase; PPAR, phenylpyruvic acid reductase; UGT1, phenyllactate UDP-glycosyltransferase; LS, littorine synthase; CYP80F1, littorine mutase; HDH, hyoscyamine dehydrogenase; H6H, hyoscyamine 6β-hydroxylase.
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Table 1. Transcriptome sequencing quality results of different tissues from A. luridus.
Table 1. Transcriptome sequencing quality results of different tissues from A. luridus.
SamplesClean ReadsClean BasesGC Content% ≥ Q30Total Reads
SR-121,416,9636,412,990,51442.12%96.15%42,833,926
SR-221,508,4996,439,821,85643.19%95.27%43,016,998
SR-321,400,3036,406,596,65042.29%94.68%42,800,606
Flower-122,920,4356,861,817,63642.60%94.50%45,840,870
Flower-224,844,6337,438,750,73442.58%94.81%49,689,266
Flower-321,680,9466,491,325,06642.59%95.08%43,361,892
Fruit-120,698,8626,196,316,81642.78%95.10%41,397,724
Fruit-219,288,5605,774,477,71042.13%94.98%38,577,120
Fruit-321,871,0946,547,980,30842.22%94.86%43,742,188
Leaf-122,519,0976,743,921,39242.33%94.69%45,038,194
Leaf-223,490,5397,034,719,03242.21%94.92%46,981,078
Leaf-320,035,2075,998,901,81642.07%94.78%40,070,414
PR-119,990,6425,984,977,53242.96%94.25%39,981,284
PR-221,249,3556,359,057,55842.94%94.91%42,498,710
PR-320,825,1446,233,208,31042.87%94.63%41,650,288
Stem-121,476,1366,423,857,12642.19%95.02%42,952,272
Stem-220,458,6516,125,644,13042.01%94.69%40,917,302
Stem-321,037,9416,296,989,81042.03%94.74%42,075,882
Table 2. Amino acid sequence identity of TAs biosynthesis genes from A. belladonna and A. luridus.
Table 2. Amino acid sequence identity of TAs biosynthesis genes from A. belladonna and A. luridus.
NameIdentity (%)
A. luridus vs. A. belladonna
ODC97.67%
PMT97.34%
MPO90.35%
PYKS97.19%
CYP82M397.30%
TRI90.48%
AT497.16%
PPAR91.84%
UGT193.28%
LS92.65%
CYP80F195.47%
HDH98.63%
H6H90.41%
Table 3. Physical and chemical properties and subcellular locations of candidate TAs biosynthesis genes of A. luridus.
Table 3. Physical and chemical properties and subcellular locations of candidate TAs biosynthesis genes of A. luridus.
Gene NameLength of CDSNum. of AAMW (Da)pISubcellular Location
AlODC129343046,526.935.61Plasma Membrane
AlPMT101733837,176.545.73Cytoplasmic
AlMPO211870579,090.016.38Cytoplasmic
AlPYKS117939243,261.087.15Cytoplasmic
AlCYP82M3156051959,463.586.69Cytoplasmic
AlTRI80126628,652.856.45Cytoplasmic
AlAT4127242347,057.535.89Plasma Membrane
AlPPAR102934238,254.445.92Cytoplasmic
AlUGT1147649154,927.145.54Cytoplasmic
AlLS143147653,931.696.07Plasma Membrane
AlCYP80F1152150657,674.27.19Cytoplasmic
AlHDH109836538,951.975.26Cytoplasmic
AlH6H103534438,883.495.02Cytoplasmic
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Wen, M.; Zeng, J.; Qiu, F.; Zhang, F.; Liao, Z. De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus. Agronomy 2024, 14, 2460. https://doi.org/10.3390/agronomy14112460

AMA Style

Wen M, Zeng J, Qiu F, Zhang F, Liao Z. De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus. Agronomy. 2024; 14(11):2460. https://doi.org/10.3390/agronomy14112460

Chicago/Turabian Style

Wen, Mengling, Junlan Zeng, Fei Qiu, Fangyuan Zhang, and Zhihua Liao. 2024. "De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus" Agronomy 14, no. 11: 2460. https://doi.org/10.3390/agronomy14112460

APA Style

Wen, M., Zeng, J., Qiu, F., Zhang, F., & Liao, Z. (2024). De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus. Agronomy, 14(11), 2460. https://doi.org/10.3390/agronomy14112460

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