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. 2022 Oct 19;851:146981. doi: 10.1016/j.gene.2022.146981

Whole transcriptome analysis identifies full-length genes for neoandrographolide biosynthesis from Andrographis alata, an alternate source for antiviral compounds

Tanuja 1, Madasamy Parani 1,⁎
PMCID: PMC9578972  PMID: 36270458

Abstract

Andrographolide and related compounds are effective against several viral diseases, including dengue, COVID-19, influenza, and chikungunya. Andrographis paniculata is the primary source for these compounds, but its availability is limited. A. alata is a potential alternative source, and neoandrographolide (NAG) is the major antiviral compound in this species. Since molecular studies in A. alata are scarce, we sequenced its leaf transcriptome to identify the full-length genes involved in neoandrographolide biosynthesis. We assembled 13.6 Gb RNA-Seq data and generated 81,361 transcripts with 1007 bp average length and 1,810 bp N50. The transcripts were categorized under biological processes (2,707), cellular components (678), and molecular functions (2,036). KEGG analysis mapped 975 transcripts to the secondary metabolite pathways. Among the 420 transcripts mapped to terpenoids and polyketides pathways, 142 transcripts were related to the biosynthesis of andrographolide and its derivatives. After a detailed analysis of these transcripts, we identified 32 full-length genes coding for all the 22 enzymes needed for andrographolide biosynthesis. Among them, 15 full-length genes were identified for the first time from Andrographis species. These full-length genes and the transcripts shall serve as an invaluable resource for the metabolic engineering of andrographolides and neoandrographolide in Andrographis and other species.

Keywords: Andrographolide, Andrographis, Andrographis alata, Neoandrographolide, Secondary metabolite, Transcriptomics

Abbreviations: UGT, UDP-glucotransferase; GO, Gene Ontology; EC, Enzyme Commission; RIN, RNA integrity number; DXS, 1-deoxy-d-xylulose-5-phosphate synthase; GGPPS, Geranylgeranyl pyrophosphate synthase; FPPS, Farnesyl-pyrophosphate synthase; HMGS, Hydroxymethylglutaryl-CoA synthase; HMGR, Hydroxymethylglutaryl-CoA reductase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; FTase, Farnesyltransferase; ICMT, Protein-S-isoprenylcysteine O-methyltransferase; KS, Ent-kaurene synthase; GGPP, Geranylgeranyl diphosphate; IPPI, Isopentenyl diphosphate isomerase; CDS, Coding sequence; MVA, Mevalonic acid; MEP, Methyl 2-C-methyl-d-erythritol-4-phosphate; DMAPP, Dimethylallyl pyrophosphate; AACT, Acetyl-CoA C-acetyl transferase; MVK, Mevalonate kinase; PMK, 5-phosphomevalonate kinase; MDD, Mevalonate diphosphate decarboxylase; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; MCS, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; CPS, ent-copalyl diphosphatase synthetase; CPR, NADPH:cytochrome P450 reductase; RT-PCR, Reverse transcriptase-polymerase chain reaction; qRT-PCR, Quantitative Reverse transcriptase-polymerase chain reaction

1. Introduction

Plant-derived andrographolides have potent antiviral activity against diverse viruses. The therapeutic value of these compounds was proved in several mechanistic studies reported over the past two decades. Experiments in Chikungunya virus, Influenza A virus, Zika virus, Hepatitis B virus, Dengue virus, Human immunodeficiency virus, Epstein-Barr virus, Hepatitis C virus, and Herpes simplex virus type-1 showed that these compounds could reduce the expression of viral RNA and proteins (Wintachai et al., 2015), inhibit viral DNA replication (Chen et al., 2014, Lee et al., 2014, Yuan et al., 2016), restrict the viral particle multiplication (Li et al., 2020), reduce virus-induced cell mortality (Yu et al., 2014), increase CD4 + T cell count (Calabrese et al., 2000), and inhibit the expression of viral lytic proteins (Lin et al., 2008). In silico studies showed that andrographolides and neoandrographolide are promising candidates for treating SARS-CoV-2 (Murugan et al., 2021, Sa-Ngiamsuntorn et al., 2021). Therefore, extracts from the plants producing these compounds are used as a general-purpose therapy against viral infections and fever. During viral epidemics and pandemics like the recent one due to SARS-CoV-2, the demand for these plants increases dramatically. Meeting such a demand is challenging because these plants are obtained mainly from wild collections.

The Andrographis genus contains 29 species, including A. paniculata, A. alata, A. gracilis, A. affinis, A. longipedunculata, A. elongata, and A. macrobotrys (Gnanasekaran and Murthy, 2015). Genes coding for NADPH: cytochrome P450 reductase, ent-copalyl diphosphate synthase, geranylgeranyl pyrophosphate synthases (GGPPS), and UDP-glucotransferase (UGT) were cloned from A. paniculata and functionally characterized (Lin et al., 2017, Misra et al., 2015, Wang et al., 2019, Srivastava et al., 2021). Chromosome-level genome sequence and transcriptome data are also available for A. paniculata (Liang et al., 2020). In other research areas also, A. paniculata is well-studied among the Andrographis species. The greater scientific interest in A. paniculata stems from the fact that this is the species predominantly used for therapeutic purposes. However, considering the frequent viral epidemics and increasing acceptance of plant-based therapy, exploring other potential sources for these compounds would be desirable. Ethanol extract from A. alata was reported to have antipyretic and anti-inflammatory activities in male albino rats (Balu et al., 1993, Balu and Alagesaboopathi, 1993). A. alata leaves are an excellent source of neoandrographolide with 33.21 mg/g dry weight (Dalawai et al., 2021). In vitro regenerated A. alata plants contained 32.21 mg/g dry weight neoandrographolide (Kadapatti and Murthy, 2021). However, research on A. alata is minimal, with just eight publications since 1993. Cloning the genes coding for the relevant enzymes will help increase the yield of andrographolides and related compounds in the resident species and transfer the biosynthetic pathway to new species. As of now, the GenBank database of NCBI contains only 413 protein-coding sequences from Andrographis species. Several genes in the terpenoid pathway in general and andrographolide biosynthesis, in particular, remain to be characterized. RNA-Seq is a promising method for large-scale gene identification in a cost-effective and high throughput manner (Kumar et al., 2012). In the current study, we have assembled the leaf transcriptome of A. alata and identified several genes involved in the biosynthesis of neoandrographolide.

2. Methods

2.1. Plant collection and RNA isolation

Seedlings of A. alata were collected from the surrounding lakes near Potheri, Chennai, Tamil Nadu. The seedlings were transplanted into pots containing garden soil, and grown in a greenhouse at 23–25 °C temperature, 16 h light, and 50 % relative humidity. A botanist taxonomically identified the plants. Mature leaves from a 2-month-old plant were harvested, frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using RNAiso plus reagent (TaKaRa, Japan) according to the manufacturer’s protocol. DNase I (Qiagen, GmbH, Germany) was used to remove DNA contamination and the RNeasy MinElute Clean-up Kit was used to purify DNase treated RNA (Qiagen, GmbH, Germany). Intact rRNA bands without degradation or DNA contamination were observed in the purified total RNA samples. RNA concentration and quality were estimated using the Qubit RNA BR assay kit (Invitrogen, California, USA) and NanoDrop spectrophotometer. The RNA integrity was checked using Bioanalyzer 2100 (Agilent Technologies, California, USA). Total RNA (550 ng/µl) with 260/280 ratio of 1.9 and RNA integrity number (RIN) of 8.5 was used for cDNA library construction.

2.2. cDNA library construction and sequencing

The cDNA library was prepared from 1 µg of total RNA according to the Illumina TrueSeq mRNA library preparation kit protocol (https://protocols.hostmicrobe.org/legacy-protocols/truseq-mrna-library-prep). In brief, mRNA was purified from the total RNA using poly-A enriched Oligo-dT magnetic beads. The purified mRNA was fragmented, and first-strand cDNA synthesis was performed by reverse-transcription with Superscript II reverse transcriptase and random hexamers. Second strand cDNA synthesis was done in the presence of DNA polymerase 1, and cDNA size selection was performed using magnetic beads. The 3′ ends of double-stranded cDNAs were end-repaired and adenylated using an end repair and A-Tailing mix. The adapters were ligated to the fragmented cDNAs and enriched for adapter-ligated fragments by PCR amplification. Finally, validation of the library was done using bioanalyzer 2100 (Agilent Technologies, USA). The RNA-Seq library was quantified using the Qubit dsDNA BR assay kit for Qubit3.0 fluorimeter and sequenced in the Illumina NextSeq500 platform (Illumina, USA). The sequence data were submitted to NCBI Sequence Read Archive (SRA) database (Accession No. SRR18210043 and Bio project ID PRJNA811695).

2.3. Processing of raw data and de novo assembly

The following steps were performed with the raw paired-end sequence data generated from the Illumina NextSeq500 platform. First, raw read quality checks were performed using Fastqc (Andrews, 2010). Adapters sequences and low-quality reads (<30 phred score) were removed using Trimmomatic (Bolger et al., 2014). Processed reads with bases quality score > 30 were used for transcriptome assembly using Trinity assembler (Grabherr et al., 2011). The transcripts were clustered using Cd-hit-est (Li and Godzik, 2006) to remove redundant transcripts and produce unique transcripts.

2.4. Functional annotation and pathway enrichments

Functional annotation of transcripts was conducted by sequence similarity comparison to non-redundant databases of NCBI with an E value threshold ≤ 1e-5. The resultant annotated transcripts were used for Gene Ontology (GO) annotation and Kyoto Encyclopedia of Gene and Genomes analysis. Blast2GO software (Conesa et al., 2005) was used for KEGG pathway analysis, and the retrieved GO terms were classified into biological processes, cellular components, and molecular functions. The transcripts were assigned with unique Enzyme Commission (EC) numbers and used to map KEGG pathways.

2.5. Reverse transcriptase-polymerase chain reaction (RT-PCR)

Seven genes coding for the enzymes in the neoandrographolide biosynthesis were selected for RT-PCR analysis. First-strand cDNA was synthesized using 1 µg RNA, 0.5 µM oligo-dT primer, and 200 units Superscripts II reverse transcriptase (Invitrogen, USA). Gene-specific primers for the selected genes were designed using Primer 3 plus software (https://www.primer3plus.com/) to amplify 906 to 2386 bp cDNA fragments (Supplementary Table 1). PCR reactions contained Taq 2X Master Mix RED (Ampliqon, Denmark) to 1X concentration, 0.5 μM primers, and 15 ng cDNA. PCR amplification steps comprised of initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 52–59 °C for 30 sec, and extension at 72 °C for 1 min, final extension at 72 °C for 5 min, and hold at 4 °C.

2.6. Quantitative-RT-PCR (qRT-PCR)

Seven genes coding for the enzymes in the neoandrographolide biosynthesis were selected for qRT-PCR analysis. Elongation factor 1-alpha was selected as the housekeeping gene. Gene-specific primers targeting 115 to 213 bp regions in the 3′UTR of the transcripts (Supplementary Table 2) were designed using Primer 3 plus software (https://www.primer3plus.com/). First-strand cDNA was synthesized using 1 µg RNA, 0.5 µM oligo-dT primer, and 200 units Superscripts II reverse transcriptase (Invitrogen, USA). The qPCR reactions (10 μl) in triplicate containing 5 μl TB Green Premix Ex Taq II 2X mix (Takara, Japan), 0.5 μM primers, and 15 ng of cDNA were performed in QuantStudio 5 Real-time PCR machine (Thermo Scientific, USA). The qRT-PCR amplification cycle comprised of an initial denaturation at 95 °C for 2 min, 40 cycles of amplification at 95 °C for 30 sec, and 57 °C for 30 sec, a melting curve analysis at 95 °C for 15 sec, 57 °C for 1 min, and 95 °C for 15 sec.

3. Results

3.1. Transcriptome sequencing and de novo assembly

We generated 97 million paired-end (2 x150 bp) reads from the leaves of A. alata. After removing low-quality reads (<Q30), 81 million high-quality reads were obtained for de novo transcriptome assembly. The assembled transcriptome contained 81,361 transcripts with 1,007 bp average length and 1,810 bp N50 value. The assembly statistics of the transcriptome are provided in Table 1 . The transcript size varied from 200 bp to 16,682 bp. About 47 % of transcripts (38,718) were smaller than 500 bp. Transcripts of size 501–1,000 bp, 1,001–2,000 bp, and 2,001–5,000 bp were 18 % (14,680), 20 % (16,013), and 14 % (11,330), respectively. The overall size distribution of the transcripts is shown in Fig. 1 .

Table 1.

Statistics of A. alata transcriptome sequencing and assembly.

Particulars Numbers
Number of raw reads (Millions) 97
Number of clean reads (Millions) 81
Number of bases > Q30 (Gb) 8.9
Mapped reads (%) 95
Number of assembled transcripts 81,361
Average length (bases) 1,007
Median contig length (bases) 542
Max length (bases) 16,682
Min length (bases) 200
Contig N50 (bases) 1,810
GC content (%) 47
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Fig. 1.

Fig. 1

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Size distribution of the transcripts assembled from A. alata.

3.2. Functional annotation

The de novo assembled transcripts were functionally annotated using NR, GO, KEGG, and UniProt databases. Among the assembled transcripts, 57 % (46,764) showed significant homology with other proteins, and 43 % (34,597) did not show significant similarity with any sequence in the database. About 85 % (39,728) of the annotated transcripts matched existing gene models, and 15 % (7,036) were classified under uncharacterized protein. Regarding the top hit species, the top hits were found with Sesamum indicum (37.2 %), followed by Erythranthe guttata (8 %) and Nicotiana tabacum (0.8 %). The top hit species distribution is shown in Fig. 2 . According to the GO analysis, the transcripts were classified into three main domains: biological process, cellular components, and molecular function. GO analysis assigned 38,069 transcripts to one or more GO categories; 2,707, 679, and 2,037 categories under biological processes, cellular components, and molecular functions. The top 20 categories from each domain are represented in Fig. 3 . Most of the transcripts related to the biological process belonged to the oxidation–reduction process (2,418), followed by protein phosphorylation (1,424) and transmembrane transport (1,077). Under cellular components, integral components of the membrane (7,566), cytoplasm (2,652), and membrane (2,317) were the predominant categories. Under molecular functions, ATP binding (3,826), metal ion binding (2,051), and DNA binding (1,038) were amongst the top three major classes.

Fig. 2.

Fig. 2

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Top hit species based on the similarity search to the non-redundant database of NCBI.

Fig. 3.

Fig. 3

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Histogram showing GO term assigned to A. alata transcripts under biological process, cellular component, and molecular function.

3.3. Analysis of metabolic pathways

The assembled transcripts were mapped to the KEGG database to determine their involvement in various metabolic pathways. We mapped 19,169 transcripts to 150 KEGG pathways. Most transcripts were mapped to the purine metabolism pathway (1,986). The other pathways with a significantly higher number of transcripts were thiamine metabolism (1,800), starch and sucrose metabolism (714), drug metabolism (426), and glycolysis (419). Transcripts mapped to the top ten pathways are represented in Fig. 4 . The KEGG pathways are classified under five groups. The highest number of transcripts were mapped to metabolism (16,053), further classified into 11 subgroups. More than 50 % of the transcripts under metabolism were mapped to the metabolism of carbohydrates (3,752), cofactors and vitamins (2,866), and nucleotides (2,173). We mapped 975 transcripts to the metabolism of terpenoids, polyketides, and other secondary metabolites. Details of the transcripts mapped to the 11 subgroups under the metabolism category are given in Fig. 5 .

Fig. 4.

Fig. 4

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The distribution of A. alata transcripts among the top ten pathways in KEGG pathway analysis.

Fig. 5.

Fig. 5

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The distribution of A. alata transcripts among the 11 metabolic pathways in KEGG pathway analysis.

3.4. Terpenoid backbone biosynthesis pathway

Andrographolides and related compounds are part of the terpenoids and polyketides pathway. We mapped 420 transcripts to this pathway. Terpenoid backbone biosynthesis is the major pathway under the metabolism of terpenoids and polyketides (Fig. 6 ). We found 158 transcripts encoding 26 enzymes involved in synthesizing terpenoids and polyketides (Fig. 7 ). The highest number of transcripts (23) encoded for 1-deoxy-d-xylulose-5-phosphate synthase (DXS), followed by geranylgeranyl pyrophosphate synthase (GGPPS) and farnesyl-pyrophosphate synthase (FPPS) with 16 transcripts. Hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), farnesyltransferase (FTase), and Protein-S-isoprenylcysteine O-methyltransferase (ICMT) enzymes had 5 to 10 transcripts. Enzymes in the terpenoid backbone pathway and the number of transcripts coding for these enzymes are given in Table 2 . We also identified 20 transcripts involved in the diterpenoid pathway, and five of them encoded Abieta-7,13-dien-18-ol hydroxylase. Ent-kaurene synthase (KS) and Gibberellin 3-beta-dioxygenase were encoded by three and two transcripts, respectively. The number of transcripts coding for the different enzymes in the diterpenoid backbone pathway is given in Table 3 .

Fig. 6.

Fig. 6

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The distribution of A. alata transcripts among the metabolic pathways of different terpenoids and polyketides in KEGG pathway analysis.

Fig. 7.

Fig. 7

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KEGG analysis of the A. alata transcriptome showing the transcripts coding for the enzymes of the terpenoid biosynthesis pathway. Enzyme Commission number of the enzymes are given inside the boxes.

Table 2.

Enzyme involved in terpenoid biosynthesis and the number of transcripts identified from A. alata for each enzyme.

S. No. Enzyme code Name of the enzyme No. of transcripts
1 ec:2.2.1.7 1-deoxy-d-xylulose-5-phosphate synthase (DXS) 23
2 ec:2.5.1.29 Geranylgeranyl diphosphate synthase (GGPPS) 19
3 ec:2.5.1.10 Farnesyl-diphosphate synthase (FPPS) 19
4 ec:2.5.1.1 Dimethylallyltranstransferase (DMATT) 16
5 ec:2.3.3.10 Hydroxymethylglutaryl-CoA synthase (HMGS) 10
6 ec:1.17.7.1 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (Ferredoxin) (HDS) 7
7 ec:1.1.1.34 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) 8
8 ec:1.1.1.88 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) 8
9 ec:2.5.1.58 Protein farnesyltransferase (PFT) 6
10 ec:2.1.1.100 Protein-S-isoprenylcysteine O-methyltransferase. 6
11 ec:2.7.4.26 Isopentenyl phosphate kinase (IPK) 5
12 ec:2.3.1.9 Acetyl-CoA C-acetyltransferase (AACT) 4
13 ec:1.14.14.145 Abietadienol hydroxylase 4
14 ec:5.3.3.2 Isopentenyl-diphosphate Delta-isomerase (IPPI) 3
15 ec:1.1.1.267 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) 3
16 ec:1.3.1.83 Geranylgeranyl diphosphate reductase. (GPPS) 3
17 ec:2.7.4.2 Phosphomevalonate kinase (PMK) 2
18 ec:4.1.1.33 Diphosphomevalonate decarboxylase (DMD) 2
19 ec:2.5.1.30 Heptaprenyl diphosphate synthase (HepPPS) 2
20 ec:1.17.7.4 4-hydroxy-3-methylbut-2-enyl-diphosphate reductase (HDR) 2
21 ec:1.8.3.5 Prenylcysteine oxidase. 1
22 ec:2.7.1.36 Mevalonate kinase (MVK) 1
23 ec:2.7.1.148 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (CMK) 1
24 ec:2.7.7.60 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT). 1
25 ec:4.6.1.12 2-C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase (MCS) 1
26 ec:1.1.1.216 Farnesol dehydrogenase (FDL) 1
TOTAL 158
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Table 3.

-. Enzyme involved in diterpenoid biosynthesis and the number of transcripts identified from A. alata for each enzyme.

S. No. Enzyme code Name of the enzyme No. of transcripts
1 ec:1.14.11.15 Gibberellin 3-beta-dioxygenase (GA3OX) 3
2 ec:1.14.11.13 Gibberellin 2-beta-dioxygenase (GA2OX) 2
3 ec:1.14.14.145 Abieta-7,13-dien-18-ol hydroxylase 5
4 ec:4.2.3.19 Ent-kaurene synthase (KS) 4
5 ec:5.5.1.13 Ent-copalyl diphosphate synthase (CPS) 1
6 ec:1.6.2.4 NADPH: Cytochrome p450 reductase (CPR) 3
7 UDP-glycosyltransferase 73AUI (UGT73AUI) 1
8 UDP-glycosyltransferase 86CII (UGT86CII) 1
Total 20
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3.5. Full-length genes involved in neoandrographolide biosynthesis

At least 22 enzymes are required to synthesize geranylgeranyl diphosphate (GGPP) and convert it to neoandrographolide. From the leaf transcriptome of A. alata, we identified 130 transcripts coding for these 22 enzymes. After a detailed analysis of these transcripts, we identified full-length genes for all the enzymes required for the biosynthesis of neoandrographolide. Three full-length genes each were identified for 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), geranylgeranyl pyrophosphate synthase (GGPPS), and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) enzymes. Two genes each were identified for acetyl-CoA C-acetyl transferase (AACT), 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), isopentenyl diphosphate isomerase (IPPI), and farnesyl pyrophosphate synthase (FPPS). Single genes encoded all other enzymes. The coding sequence (CDS) of the full-length genes ranged between 720 and 2,451 bp and encoded 240 to 817 amino acids. Sequences of all the full-length genes were submitted to the GenBank under the accession numbers ON049353 to ON049382 and ON211623 to ON211625. Details of the full-length genes involved in neoandrographolide biosynthesis identified from A. alata are given in Table 4 .

Table 4.

Details of the genes identified from A. alata involved neoandrographolide biosynthesis.

S. No. Name of the gene mRNA size (bp) ORF size (bp) ORF size (aa) GenBank Accession number
1 Acetyl- CoA C- acetyl transferase 2 (AACT 2) 1728 1248 415 ON049353
2 Acetyl- CoA C- acetyl transferase 1 (AACT 1) 1654 1209 402 ON049354
3 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) 1874 1383 460 ON049355
4 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2 (HMGR2) 3757 1671 556 ON049356
5 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1) 2107 1788 595 ON049357
6 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1A (HMGR1a) 2420 1680 559 ON049358
7 Mevalonate kinase (MVK) 1629 1161 386 ON049359
8 5-phosphomevalonate kinase (PMK) 2056 1521 506 ON049360
9 Mevalonate diphosphate decarboxylase (MDD) 1585 1260 419 ON049361
10 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (CMK) 1583 1197 398 ON049362
11 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) 1953 1437 478 ON049363
12 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT) 1307 921 306 ON049364
13 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS) 1025 720 239 ON049365
14 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) 2808 2238 745 ON049366
15 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 (HDR1) 1729 1380 459 ON049367
16 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 2 (HDR2) 1932 1392 463 ON049368
17 Isopentenyl diphosphate isomerase I (IPPI 1) 1092 894 297 ON049369
18 Isopentenyl diphosphate isomerase II (IPPI 2) 1283 864 287 ON049370
19 Geranyl pyrophosphate synthase (GPS) 1976 1260 419 ON049371
20 Farnesyl pyrophosphate synthase 1 (FPPS 1) 1531 1047 348 ON049372
21 Farnesyl pyrophosphate synthase 2 (FPPS 2) 1436 1029 342 ON049373
22 Geranylgeranyl pyrophosphate synthase 1 (GGPPS 1) 1521 1107 368 ON049374
23 Geranylgeranyl pyrophosphate synthase 2 (GGPPS 2) 1436 1044 347 ON049375
24 Geranylgeranyl pyrophosphate synthase 3 (GGPPS 3) 2365 969 322 ON049376
25 Copalyl/labdadienyl diphosphate synthase 2 (CPS2) 2822 2451 816 ON049377
26 Kaurene synthase 1 (KS1) 2745 2391 796 ON049378
27 1-deoxy-d-xylulose 5-phosphate synthase 1 (DXS 1) 2909 2160 719 ON049379
28 1-deoxyxylulose 5-phosphate synthase Chloroplastic 2A (DXS 2A) 2638 2163 720 ON049380
29 1-deoxy-d-xylulose-5-phosphate synthase 2 (DXS 2) 2624 2208 735 ON049381
30 UDP-glycosyltransferase 73 (UGT73) 1809 1365 454 ON049382
31 NADPH-cytochrome P450 reductase 2 (CPR2) 2637 2136 711 ON211623
32 Cytochrome P450 76A1 (CYP76A1) 1817 1647 548 ON211624
33 UDP-glycosyltransferase 86 (UGT86) (partial) 1301 1247 416 ON211625
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3.6. Reverse-transcriptase validation

The genes coding for the MCS, IPPI2, MCT, MVK, FPPS2, HDR2, HMGR1A, and DXS2A enzymes in the neoandrographolide biosynthesis pathway were selected for validating the transcript assembly and gene expression. RT-PCR amplification of full-length genes yielded the cDNA fragments of the expected size ranging from 906 bp to 2368 bp (Fig. 8 ) indicating correct de novo transcript assembly. In qRT-PCR, all the genes selected from the neoandrographolide biosynthesis pathway showed a high level of expression as indicated by the Cq value ranging between 20.9 and 25.5 (Fig. 9 ).

Fig. 8.

Fig. 8

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Agarose gel electrophoresis of the RT-PCR amplified cDNA fragments of 1) 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS), 2) 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), 3) Mevalonate kinase (MVK), 4) Farnesyl pyrophosphate synthase 2 (FPPS2), 5) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 2 (HDR2), 6) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1A (HMGR1a), and 7) 1-deoxyxylulose 5-phosphate synthase Chloroplastic 2A (DXS2A). M denotes 1 Kb ladder (New England Biolabs, USA).

Fig. 9.

Fig. 9

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Analysis of the expression of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS), Isopentenyl diphosphate isomerase II (IPPI 2), Mevalonate kinase (MVK), Farnesyl pyrophosphate synthase 2 (FPPS2), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 2 (HDR2), 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1A (HMGR1a), and 1-deoxyxylulose 5-phosphate synthase Chloroplastic 2A (DXS2A) genes involved in the neoandrographolide biosynthesis by qRT-PCR. The Elongation Factor 1 (EF1) is a housekeeping gene, which was used as a control. All the genes were analyzed in triplicate.

4. Discussion

The antiviral property of Andrographis species is mainly due to andrographolide and related diterpenoid compounds (Wintachai et al., 2015, Chen et al., 2009). Although Andrographis paniculata is the primary source for these compounds, increasing demand for this species necessitates searching for alternate sources. Another species of Andrographis, A. alata, is rich in neoandrographolide and shows antipyretic, anti-inflammatory, and antiviral properties (Balu et al., 1993, Balu and Alagesaboopathi, 1993, Dalawai et al., 2021). However, compared to A. paniculata, research on A. alata is extremely limited. The present study focused on sequencing the whole transcriptome from A. alata leaves to generate a large-scale genomics resource and to identify the full-length gene involved in neoandrographolide biosynthesis. De novo transcriptome assembly using next-generation sequencing is a fast and economical tool for generating expressed sequence data for non-model plants, particularly for those species which do not have genome sequences yet (Tanuja et al., 2021, Bhambhani et al., 2017). This is the first time a transcriptome is assembled for A. alata. It had a higher number of transcripts and a greater N50 value than the leaf and root transcriptomes of A. paniculata reported earlier (Garg et al., 2015, Patel et al., 2020). The assembled transcriptome showed 95 % alignment with the quality-filtered reads. Based on the top-hit species distribution, most of the A. alata transcripts matched with Sesamum indicum. This may be because the GenBank database contains a significantly higher number of gene sequences from Sesamum (66,404 entries) than Andrographis (447 entries), and both species belong to the same order, Lamiales.

Secondary metabolites from plants are significantly used in the pharmaceutical industries and traditional medicine. Understanding the biosynthetic pathways, regulation, and the genes involved will help improve yield and metabolic engineering. Terpenoids constitute the largest class of structurally diverse secondary metabolites and are categorized based on the number of 5-carbon isoprene units: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (>C40). Neoandrographolide (NAG), a diterpenoid compound, is the major secondary metabolite responsible for the antiviral property of A. alata (Murugan et al., 2021). Geranylgeranyl pyrophosphate (GGPP) is the precursor from which several important diterpenoids, including NAG, are synthesized. GGPP is synthesized from the mevalonic acid (MVA) pathway and methyl 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the cytosol and plastids, respectively (Srivastava and Akhila, 2010). These two pathways are divergent till the synthesis of dimethylallyl pyrophosphate (DMAPP), which is converted to GGPP through a common pathway. In the MVA pathway, the synthesis of DMAPP from acetyl-CoA involves seven enzymes; acetyl-CoA C-acetyl transferase (AACT), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDD), and isopentenyl diphosphate isomerase (IPPI). We identified 12 full-length genes coding for these seven enzymes. HMGR is a rate-limiting enzyme in the MVA pathway, and its expression levels in the leaves of A. paniculata correlated with andrographolide content. ApHMGR gene coding for 595 amino acids was cloned from A. paniculata and expressed in Escherichia coli (Srinath et al., 2019). We identified the same gene from A. alata, annotated as AaHMGR1, coding for 595 amino acids with 99 % identity to ApHMGR. In addition, we identified two other full-length HMGR genes, AaHMGR1a (559 aa) and AaHMGR2 (556 aa). HMGR1 had 68 and 70 % identity with HMGR1a and HMGR2. Three splice variants of the HMGR2 gene were identified. Two genes each for AACT and IPPI and one gene each for HMGS, MVK, PMK, and MDD were also identified for the first time from A. alata.

In the MEP pathway, the synthesis of DMAPP from glyceraldehyde 3-phosphate involves seven enzymes, which are 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (CMK), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR). In this study, we identified 11 genes coding for these seven enzymes. DXS controls flux to the MVP pathway, and it is a rate-limiting enzyme. Expression levels of the DXS gene correlated with accumulation of andrographolide. DXS gene was cloned from A. paniculata (ApDXS) and expressed in E. coli (Srinath et al., 2021). We identified a full-length AaDXS1 gene that showed 99 % identity with ApDXS. However, AaDXS1 (GenBank Acc. No. ON049379) codes for 719 amino acids, whereas ApDXS codes for 691 amino acids. Further analysis showed that the ApDXS gene (GenBank Acc. No. MG271749.1) has a single base pair deletion at 410th position, which changed the longest open reading frame to start from 323rd position (coding for 691 amino acids) instead of 240th position (coding for 719 amino acids). Additionally, we identified two more full-length DXS genes; one is cytosolic (AaDXS2, GenBank Acc. No. ON049381), and the other is chloroplastic (AaDXS2a, GenBank Acc. No. ON049380). We also got 16 splice variants for the AaDXS1 gene. HDR gene was cloned from A. paniculata (ApHDR), and its expression was 5.5 to 16-fold higher in the leaves than in the roots (Shailaja et al., 2021). We identified two full-length HDR genes, AaHDR1 (459 amino acids) and AaHDR2 (463 amino acids), with 88 % and 82 % identity to ApHDR. Besides, for the first time, we identified full-length genes for DXR, MCT, CMK, MCS, and HDS from Andrographis.

In a common pathway, DMAPP synthesized from MVA and MEP pathways is converted into GGPP by geranyl pyrophosphate synthase (GPS), farnesyl pyrophosphate synthase (FPPS), and geranylgeranyl pyrophosphate synthase (GGPPS). For the first time, we identified one full-length gene for GPS and two for FPPS from Andrographis, along with four splice variants of FPPS2. There are at least four GGPPS genes in A. paniculata, and only ApGGPPS2 was reported to be involved in andrographolide biosynthesis (Wang et al., 2019). We also identified a full-length GGPPS2 gene from A. alata (AaGGPPS2), showing 99 % identity with ApGGPPS2.

GGPP is the common precursor for several diterpenoids compounds, including andrographolides. The committing enzyme that diverts GGPP to ent-labdane-related diterpenoids is ent-copalyl diphosphatase synthetase (ent-CPS). Misra et al. (Misra et al., 2015) cloned ent-CPS2 gene from A. paniculata (Ap-ent-CPS2), coding for 816 amino acids that converts GGPP to ent-copalyl diphosphate (ent-CPP) (Misra et al., 2015). The ent-CPS2 gene from A. alata (Aa-ent-CPS2) also encoded 816 amino acids and showed 99 % identity with ApCPS2. Shen et al. (2015) have cloned and functionally characterized another CPS2 gene with 832 aa and 57 % identity with Aa-ent-CPS2, but we did not find this gene in our study (Shen et al., 2016). The enzyme ent-kaurene synthase (KS1) converts ent-CPP to ent-copalol [8(17),13-ent-labdadiene-15,16,19-triol]. In E. coli, co-expression of ent-CPS2 from A. paniculata and KS1 from rice and wheat produced ent-copalol (Shen et al., 2016). In our study, we got one KS1 gene from A. alata (AaKS1), which coded for 796 amino acids with 99 % similarity to the KS1 from A. paniculata (ApKS1, Accession No. KY979588.1). Cytochrome P450 76 (CYP76), along with NADPH:cytochrome P450 reductase 2 (ApCPR2), converts ent-copalol to andrograpanin (Lin et al., 2017). We identified full-length CPR2 and CYP76 genes from A. alata (AaCPR2 and AaCYP76). In the final step, UDP-glycotransferase (UGT) enzyme glycosylates andrograpanin to produce neoandrographolide. Sun et al. (2019) functionally characterized recombinant UGT73AU1, UGT71, UGT76, UGT85, and UGT88 from A. paniculata and reported that ApUGT37AUI could convert andrograpanin to neoandrographolide (Sun et al., 2019). However, an extensive functional characterization of 23 UGTs from A. paniculata revealed that UGT86CII could be the most probable enzyme, and it showed relatively higher catalytic activity than UGT73AUI (Srivastava et al., 2021). A. alata transcriptome contained 103 transcripts coding for 21 UGTs, including a full-length gene for UGT73AUI (AaUGT73AUI) and a partial gene for UGT86CII (AaUGT86CII). The proposed pathway of neoandrographolide biosynthesis based on the previous reports and the current study is given in Fig. 10 .

Fig. 10.

Fig. 10

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A schematic representation of proposed pathways for the biosynthesis of neoandrographolide. The numbers given in the bracket indicate the number of genes identified for each enzyme. Abbreviations: AACT: Acetyl- CoA C- acetyl transferase; HMGS: 3-hydroxy-3-methylglutaryl coenzyme A synthase; HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase; MVK: Mevalonate kinase; PMK: 5-phosphomevalonate kinase; MDD: Mevalonate diphosphate decarboxylase; IPPI: Isopentenyl diphosphate isomerase; DXS: 1-deoxy-d-xylulose-5-phosphate synthase; DXR: 1-deoxy-d-xylulose 5-phosphate reductoisomerase; MCT: 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK: 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; MCS: 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; GPS: Geranyl pyrophosphate synthase; FPPS: Farnesyl pyrophosphate synthase; GGPPS: Geranylgeranyl pyrophosphate synthase; CPS: Copalyl/labdadienyl diphosphate synthase; KS: Kaurene synthase; CPR: NADPH-cytochrome P450 reductase; CYP: Cytochrome P450; UGT: UDP-glycosyltransferase.

NAG is a potential antiviral compound, and A. alata is the primary source of this compound. In this study, we identified one to three full-length genes for all the 22 enzymes involved in the synthesis of NAG, starting from acetyl CoA and d-glyceraldehyde 3 phosphate. Among the 32 full-length genes reported in this study, 15 were identified for the first time from Andrographis species. Interestingly only three genes showed splice variants. We also identified several transcripts related to the biosynthesis of other secondary metabolites. The full-length genes and transcripts identified from this study will provide valuable information for understanding the biosynthesis of NAG and other metabolites in A. alata and serve as an excellent genomics resource that will help genetic manipulation and metabolic engineering.

Funding

This study was financially supported by SRM-DBT Partnership Platform for Con-temporary Research Services and Skill Development in Advanced Life Sciences Technologies (Order No. BT/PR12987/INF/22/205/2015.

Data Availability Statement: The data was submitted to NCBI SRA database with Accession Number SRR18210043. Full length gene was submitted under the GenBank accession numbers ON049353 to ON049382 and ON211623 to ON211625.

CRediT authorship contribution statement

Tanuja: Conceptualization, Methodology, Investigation, Writing – original draft. Madasamy Parani: Conceptualization, Writing – review & editing, Project administration, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Edited by Takashi Gojobori

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.gene.2022.146981.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (19.2KB, docx)

Data availability

The authors are unable or have chosen not to specify which data has been used.

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Associated Data

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Supplementary Materials

Supplementary data 1
mmc1.docx (19.2KB, docx)

Data Availability Statement

The authors are unable or have chosen not to specify which data has been used.

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