AcknowledgmentHussain T, Plunkett B, Ejaz M, Espley RV and Kayser O (2018) Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata. Front. Plant Sci. 9:537.doi: 10.3389/fpls.2018.00537

For further details:This study was supported by the DISCO under the grant agreement 613513. We thank Mr. Sven Buijssen for providing the computationl resource, LiDong HPC. We also thank Dr. Felix Stehle for ESI-MS/MS and Dr. Pawel Rodziewicz for GC/MS analysis. Thanks to Dr. Richard ESpley for providing the Radula material and expressionl analysis.

Transcriptomic analysis of Radula marginata revealed identification of genes for cannabinoid-like compounds

Tajammul Hussain and Oliver KayserDepartment of Technical Biochemiestry, Faculty of Bio-Chemical Engineering, Technical University Dortmund

tajammul.hussain@tu-dortmund.de; oliver.kayser@tu-dortmund.de

Poster presented: "Emerging Trends in Natural Product Biotechnology" at Technical University Dortmund on 20-21 September 2018

This is the first transcriptomic study for the Radulaceae family. We captured, assembled and annotate the R.marginata transcriptome. Identification of the precursor genes for the cannabinoid-like compound, validationof these genes through quantitative real time expressional analysis, characterization of the central precursorand finally detection of compound from metabolomics approach lead us to speculate that the cannabinoid-likepathway is likely to be conserved in lower and high land plants with the exception of first intermediate.However, these findings require further experimental work to confirm this proposed novelty. Overall, this studywould serve as a reference transcriptomic resource for Radulaceae family to further explore especially Radulamarginata. Moreover, this study also proposes R. marginata as an alternate to Cannabis sativa forcannabinoid-like compounds.

1. Toyota, M. et al. New bibenzyl cannabinoid from the New Zealand liverwort Radula marginata. Chem.Pharm. Bull. (Tokyo). 50, 1390–1392 (2002).2. Park, B. H. & Lee, Y. R. Concise synthesis of perrottetinene with bibenzyl cannabinoid. Bull. KoreanChem. Soc. 31, 2712–2714 (2010).3. Russo, E. B. Beyond Cannabis: Plants and the Endocannabinoid System. Trends Pharmacol. Sci. 37,594–605 (2016).

Similarity % Transcripts %

30-50 1.7650-59 13.9360-69 23.3770-79 24.8880-89 21.59

90-100 14.47

Sequence similarityB

E-value Transcripts %

0 ~ 1e-100 1.921e-100 ~ 1e-60 6.091e-60 ~ 1e-45 13.831e-45 ~ 1e-30 23.311e-30 ~ 1e-15 29.771e-15 ~ 1e-5 21.70

E-value distributionC

Marchantia polymorpha

Physcomitrella patens

Sorangium cellulosum

Pseudonocardia

Selaginella moellendorffi

i

Pseudonocardia autotrophica

Vitis vinife

ra

Nelumbo nucifera

Glycine max

Gossypium raim

ondii

Picea sitchensis

Theobroma cacao

Elaeis guineensis

Klebsormidium fla

ccidum

Gossypium hirsutum

Nicotiana ta

bacum

Eucalyptus grandis

Phoenix dactylifera

Noccaea caerulescens

Zea mays

Musa acuminata

Daucus carota

Gossypium arboreum

Num

ber o

f HS

P h

its

(Tho

usan

ds)

A

2

4

6

8

10

0

Species distribution

BLAST analysis: A)Blast species distribution of closely related bryophyte species againstNCBI non redundant (nr) database with an E-value of 1e -03. B) Sequence similarity and C)E-valuedistribution of BLAST results.

a) BLAST homology search

Gene Ontology analysis A) Gene ontology functional annotation for biologicalprocess (BP), Molecular function (MF) and Cellular component (CC) B)Overall annotationresults from BLAST, InterProScan (IPR), Gene ontology (GO) and KEGG pathway databases.

b) Gene ontology term and intrproscan mapping

iii) Identification of precursor genes for the bioynthesis ofcannabinoid-like compound and validation by qPCR

Candidate precursor genes for cannabinoid biosynthesis in Radula marginata: A)Identification of candidate genes for cannabinoid biosynthesis in R. marginata. GPP transcripts as well as all the upstream genes were identified and transcriptomic expression of identified genes was determined from FPKM B) Relative expression of candidate gene by real time quantitative PCR (RT-qPCR).

A

FP

KM

100

80

60

40

20

0

120

Rm_DXS

Rm_DXR

Rm_ISPD

Rm_ISPE

Rm_ISPF-1

Rm_ISPF-2

Rm_ISPG

Rm_ISPH

Rm_GPPS

Rm_STS

B

Rm_GPPS

Rel

ativ

e ex

pre

ssio

n

1.0

0.8

0.6

0.4

0.2

0.0

1.2

Rm_DXS

Rm_DXR

Rm_ISPD

Rm_ISPE

Rm_ISPG

Rm_ISPH

Rm_STS

i) de novo transcriptomic assembly Paired-end Illumina Hiseq-2000 platform generated 30 million raw reads for each pair. Raw reads filtered and quality trimmed. Trinity was used for de novo assembly of raw reads and resulted in 87,460 transcripts with a median contig size of 303 bp, and a maximum of 23,874 bp (Table 1).

RESULTS

Table 1: Sequencing Summary

ii) Annotation of assembled trasncriptomeTo annotate, all the assembled transcripts (87460) were BLASTed against NCBI publically availabale "nr" and "nt" databases (a) , followed by scanning with InterProScan (IPR) for protein signatures, assigning Gene ontology (GO) term (b) and finally map against KEGG database (c) for active pathways in Radula marginata.

c) KEGG Pathway mapping

Identified enzymes for respective pathway category

Metabolism 2464

No. of pathways

15

14

01

12

17

02

09

20

14

07

17

11

01

02

02

144

No. of unigenes

21158

2309

1573

3491

879

1456

941

5917

524

595

227

248

241

37

256

Pathway category

Carbohydrate metabolism

Amino acid metabolismBiosynthesis of antibiotics (Global and overview maps)

Metabolism of cofactors and vitamins

Lipid metabolism

Energy metabolism

Xenobiotics biodegradation and metabolism

Nucleotide metabolism

Metabolism of other amino acids

Biosynthesis of other secondary metabolites

Glycan biosynthesis and metabolism

Metabolism of terpenoids and polyketides

Translation

Immune systemSignal transduction

323

275

164

135

114

113

104

97

62

53

48

29

22

12

03

1554

No. of enzymesMain category type

Genetic Information Processing

Environmental Information Processing

Organismal Systems

Terpenoid backbone biosynthesis map00900 12 84 ec:2.7.4.2 Kinaseec:4.1.1.33 Decarboxylaseec:1.17.7.1 Synthase(ferredoxin)ec:2.2.1.7 Synthaseec:2.7.1.148 5'-diphospho-2-C-methyl-D-erythritolkinaseec:4.6.1.12 2-4-cyclodiphosphatesynthaseec:5.3.3.2 Delta-isomeraseec:2.3.1.9 C-acetyltransferaseec:2.3.3.10 Synthaseec:1.1.1.267 Reductoisomeraseec:2.1.1.100 O-methyltransferase

The liverwort Radula marginata belongs to the bryophyte division of land plants. Bryophytes are an early-diverged group of land plants. The division comprising of around 20,000 species and are divided into three phyla: mosses, hornworts and liverworts. Among them liverworts are the most abundant consisting of 6000-8000 species. Radulales is an order of subclass Jungermanniidae in this phylum. Radulaceae is the only family in this order, comprises Radula genus and 283 species. Radula marginata is of significant interest since perrottetinene and perrottetinenic acid 1,2 had been reported. These compounds are structural analogs to tetrahydrocannabinol (Δ9-THC), a psychopharmacological compound in Cannabis sativa L. Recently, agonistic activity of these compounds with cannabinoid receptor 1 (CB1) 3 further confirmed the potential of this specie in cannabinoid-based pharmaceutical industry. Therefore genetic-level understanding of secondary metabolic pathways that lead to the synthesis of cannabinoid-like natural compounds would be desirable.

INTRODUCTION

gene identification

cDNA library preparation

Next generation sequencing

de novo assembly

RNA extractionSample collection from native habitate in New Zealand

Illumina- Highseq 2000

Methodology

Conclusion and future prospects References