Cloning and molecular analysis of two new sesquiterpene cyclases
from
Artemisia annua
L.
Els Van Geldre
a, Isabel De Pauw
a, Dirk Inze´
b, Marc Van Montagu
b,
Elfride Van den Eeckhout
a,*
aLaboratory for Pharmaceutical Biotechnology,Uni6ersity of Ghent,Harelbekestraat72,9000Ghent,Belgium bLaboratory for Genetics,Uni6ersity of Ghent,K.L.Ledeganckstraat35,9000Ghent,Belgium
Received 5 April 2000; received in revised form 15 June 2000; accepted 15 June 2000
Abstract
Artemisia annuaL. is the only source of artemisinin, a new promising antimalarial drug (Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J. 92 (1979) 811). Our efforts are focused on the overproduction of this valuable medicine by genetic engineeredA.annuaplants. Therefore, we decided to isolate the gene(s) encoding sesquiterpene cyclase(s) inA.annuaas a first step in improving artemisinin yield. Four partial genomic clones,gASC21,gASC22,gASC23 andgASC24, were isolated through polymerase chain reaction (PCR) with degenerated primers based on homologous boxes present in sesquiterpene cyclases from divergent sources. Intron – exon organisation of those partial genomic clones was analysed and it was shown that A. annua contains a gene family for sesquiterpene cyclases. Based ongASC21, gASC22, gASC23 andgASC24 sequences, the full-length cDNA clonescASC34 andcASC125 were subsequently isolated by rapid amplification of cDNA ends PCR. The derived amino acid sequences of both full-length clones show high homology with sesquiterpene cyclases from plants. Reverse transcription-PCR analysis revealed transient and tissue specific expression patterns for cASC34 and cASC125, in contrast to the constitutively expressed 8-epicedrol synthase, a previously reported sesquiterpene cyclase fromA.annua. BothcASC34 andcASC125 could only be detected in flowering plants when artemisinin concentration is at highest. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Terpene cyclases; Artemisinin; Malaria
www.elsevier.com/locate/plantsci
1. Introduction
Artemisia annua L., a member of the Composi-tae family, has been used for decennies in the treatment of fever and malaria [1]. Analysis of the active compounds responsible for these pharmaco-logical activities led to the discovery of a sesquiter-pene lacton, called artemisinin or qinhaosu [1]. This molecule possesses a different mode of action in comparison with existing antimalarial drugs (e.g. quinine) and is active against chloroquine
resistant forms of Plasmodium falciparum [2].
Based on this secondary plant metabolite, new semi-synthetic drugs for the treatment of malaria, e.g. artemether, arteether and artesunate, are pro-duced [1]. Unfortunately, only very low amounts Abbre6iations: A, adenine; aa, amino acid(s); bp, base pair(s); C,
cytosine; CAD, cadinene synthase; cDNA, DNA complementary to RNA; dNTP, deoxyribonucleotide triphosphate; FPP, farnesylpy-rophosphate; G, guanine; M, A/C; PCR, polymerase chain reaction; pI, isoelectric point; R, A/G; RT-PCR, reverse transcription-poly-merase chain reaction; RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; S, C/G; Taq, Thermus Aquaticus Polymerase; TEAS, 5-epi-aristolochene synthase; T, thymidine; VS, vetispiradiene synthase; Y, C/T.
The nucleotide sequence data reported in this article have been submitted to the GenBank/EMBL Database under Accesssion Num-bers AJ271792, AJ271793, AJ276412, AJ276413, AJ276414, AJ276415.
* Corresponding author. Tel.: +32-9-2648053; fax: + 32-9-2206688.
E-mail address: [email protected] (E. Van den Eeckhout).
of artemisinin can be isolated from the aerial
parts of A. annua (between 0.01 and 0.6% dry
weight) [3 – 5]. The limited supply of this
valu-able compound from A. annua L. prompted
re-searchers to develop alternate means of
production. Many attempts have been under-taken to overproduce artemisinin through cell or tissue cultures [6 – 9]. However, so far, only very limited success has been obtained. Also, total chemical synthesis of artemisinin was revealed to be not economically interesting due to stereo-chemical problems [10]. Therefore, all efforts are focused on the overproduction of this valuable
compound by genetic engineered A. annua
plants. Such plants can be obtained through ge-netic improvement of the biosynthetical pathway
of artemisinin by sense and/or antisense
tech-niques [11]. An Agrobacterium tumefaciens
-medi-ated transformation procedure has already been established in our laboratory [11 – 13]. In the biosynthesis of artemisinin, very little is known about enzymes responsible for the production of the different intermediates. At one of the impor-tant branchpoints farnesylpyrophosphate (FPP) is cyclisised by the action of a sesquiterpene cy-clase to amorpha-4,11-diene, which leads to se-quentially to artemisinic acid, dihydroartemisinic acid and artemisinin by a series of oxidation steps and nonenzymatic conversions [3,14 – 17] (Fig. 1). Thus, the cyclase reaction establishes an
important stereochemical framework upon which all other chemical modifications take place [17]. Several publications report on the fact that this cyclisation step is a putative regulatory point, probably rate limiting [17,18]. The accumulation of artemisinic acid and dihydroartemisinic acid in absence of any intermediates en route from FPP also supports this hypothesis [14]. Farnesyl diphosphate synthase, which produces FPP, has
already been cloned from A. annua [19], but this
enzymatic reaction is probably not rate limiting for the production of artemisinin.
Therefore, we decided to isolate the gene(s)
encoding sesquiterpene cyclase(s) in A. annua as
a first step in improving artemisinin yield. In
1992, tobacco 5-epi-aristolochene synthase
(TEAS) was cloned from elicitor-treated tobacco cell cultures [20]. Based on the TEAS sequence
information, vetispiradiene synthase from
Hyoscyamus muticus [17] and (+)-delta-cadinene
synthase from Gossypium arboreum [21] were
cloned. During the course of this work, the
iso-lation of 8-epicedrol synthase from A. annua was
reported [22,23]. The role of 8-epicedrol synthase in A. annua and artemisinin biosynthesis is un-certain, since 8-epicedrol cannot be detected by
gas chromatography – mass spectrometry in A.
annua extracts and the conversion of epicedrol to the putative artemisinin bioprecursor artemisinic acid would require rearrangements that do not seem plausible [22,23].
This paper reports the isolation of two other members of the sesquiterpene cyclase gene family from A. annua. Both full-length isolated genes are new potential candidates involved in the rate-limit-ing cyclisation step of artemisinin biosynthesis, thereby enabling a molecular approach for im-provement of artemisinin production.
2. Materials and methods
2.1. Materials
Restriction endonucleases were purchased from Boehringer Mannheim (Mannheim, Germany) and Biolabs (New England). Taq DNA polymerase was purchased from Perkin Elmer (Norwalk, CT, USA). ABI PRISM™ Ready Reaction Dye De-oxy™ Terminator Cycle Sequencing Kit and ABI PRISM™ dRhodamine Terminator Cycle
Se-quencing Ready Reaction Kit with AmpliTaq®
DNA Polymerase FS were from Applied Biosys-tems. Dynabeads mRNA Direct Kit was from Dynal (Oslo, Norway). The SMART RACE PCR Kit was from Clontech (Palo Alto, CA, USA). Superscript II Reverse Transcriptase was from Gibco BRL (Life Technologies). Gene Images ran-dom priming labeling module and CDP-star detec-tion module was from Amersham Life Science
(Little Chalfont, Buckinghamshire, UK). Es
-cherichia coli strain JM109 was used as bacterial host for pGem-T-Easy recombinant plasmids. Wizard SV DNA extraction, dNTPs, SP6 Promo-tor primer, Reverse Transcription System and
pGem-T-Easy vector were purchased from
Promega (Madison, USA). M13/pUC universal
sequencing primer 17-mer was from Boehringer Mannheim.
2.2. Plant materials
Seeds of A. annua of West Virginia origin were
kindly provided by the Walter Reed Army Insti-tute of Research (Washington). Plants were grown in an experimental greenhouse using Hg- and
Na-vapor lamps 16 h/day and at a temperature of
22°C and a relative humidity of 40%.
2.3. Isolation of nucleic acids
Genomic DNA was isolated from plant leaves
according to the extraction procedure from Stacey and Isaac [24]. Plasmid DNA samples were pre-pared by an alkaline lysis method with the Wizard DNA Purification System (Promega) according to the manufacturer’s instructions. Total RNA was isolated with the Trizol reagent according the pro-cedures of Life Technologies. mRNA could be isolated with Dynabeads from Dynal (Oslo, Norway).
2.4. Oligonucleotide primer design
Comparison of the amino acid sequences from sesquiterpene cyclases isolated from divergent
sources Nicotiana tabacum, H. muticus and G.
arboreum, revealed highly conserved boxes for which degenerate primers were designed (PILEUP and LINEUP from GCG package). These boxes were used to design degenerate primers by back-translation from the respective amino acid se-quences. At places of three- and fourfold base redundancy, inosines were incorporated to prevent high degeneracy of the primers. One forward
primer (5%-TAYCAYTTYGARATIGARGA-3%)
and one reverse primer (5%
-GARTAYTGIG-GTTCRAARTAIACICC-3%) (R=A, G; Y=C,
T) were thus selected with respective anealing tem-peratures of 62°C and 68°C calculated with
[2°*(A+T)+4°*(C+G)].
2.5. Polymerase chain reactions
The primers were subsequently used in a Hot Start polymerase chain reaction (PCR) on
ge-nomic DNA from A. annua. (10 mM Tris – HCl
(pH 8.3), 25°C, 50 mM KCl, 200mM dNTP, 1.2 U
Taq polymerase and 20 pmol of each primer in a
total volume of 50 ml; concentrations of MgCl2
ranged from 1.5 to 4.5 mM). Cycling conditions were set as follows: 94°C for 1 min, 50°C for 1 min and 72°C for 1 min for a total of 35 cycles. This PCR resulted in a predominant 900 base pair (bp) product as detected by agarose gel electrophoresis and ethidium bromide staining. This product was sequenced after TA-cloning in pGem-T-Easy Vec-tor System (Promega) according to the manufac-turer’s instructions. Recombinant plasmids were
isolated after amplification in E. coli JM109 and
analysed for insert by blue/white screening and
2.6. Nucleotide sequencing and analysis of nucleic acids
Double-stranded forms of template DNA were used in dideoxynucleotide cycle sequencing reac-tions. Both sense and antisense strands of the TA-cloned PCR products were sequenced with
vector-specific primers (M13/pUC universal
se-quencing primer 17-mer or SP6 Promotor primer) or ABI PRISM™ Ready Reaction Dye Deoxy™
Terminator Cycle Sequencing Kit and ABI
PRISM™ dRhodamine Terminator Cycle
Sequenc-ing Ready Reaction Kit with AmpliTaq® DNA
Polymerase FS (Applied Biosystems) using an
auto-mated fluorescence-based system ABI373 or
ABI310 (Applied Biosystems), respectively. DNA sequences and the deduced amino acid sequences were analyzed and aligned with related sequences using programs available through the Genetics Computer Group package (University of Wiscon-sin).
2.7. Southern blot
Twenty micrograms of genomic DNA were
blot-ted onto a+charged nylon membrane (Boehringer
Mannheim) according to standard procedures [25]. Prehybridisation and hybridisation were performed at 55°C. The probe, fluoresceine labeled, consisted of the partially purified cDNA sequence already
isolated inA.annua(Gene Images Random priming
labeling and CDP-star detection).
2.8. RNA analysis
In order to isolate corresponding cDNA se-quences, new degenerate primers based on the partial genomic sequences were designed. One
for-ward (5%-AAGCMCTTCSCTYTGGTTCMGA-3%)
and one reverse primer (5%
-CCAAAARTAR-CMTTCAACYATTCT-5%) were selected with the
programOLIGO™ (M=A or C; S=C or G; Y=C
or T). Dnase-treated RNA isolated from leaves and flowers harvested on different stages during plant development (from young cotyledons to flowering plants) was denatured for 2 min at 90°C and subsequently transcribed to cDNA with AMV Re-verse Transcriptase during 60 min at 42°C. This reaction could be used immediately for PCR with 20 pmol of both primers and 2.5 U Taq in 10 mM
Tris – HCl (pH 8.3), 25°C, 50 mM KCl and 200mM
dNTP. The reaction conditions were 94°C during 30 s, 53°C during 30 s and 72°C during 1 min for a total of 40 cycles. For detection of specific transcripts by
reverse transcriptase (RT)-PCR exact primers (5%
-GGCTATAAACTTGCGCTAGTAG-3% and 5%
-CCGGATCCTCATGTAATGATGGCATC-3%for
8-epicedrol synthase; 5%
-CCTCACCAACGATG-TAGAAG-3% and 5%
-A-GGTAGATTGTTTGGG-ACATC-3% for cASC34; 5%
-TCTGTATGAAG-CGGCATTTATG-3% and 5%
-GTCTCGAACATA-AGGAAGCTTA-3%for cAS-C125) were designed
on three isolated sequences and used for PCR with the following cycling conditions: 1 min at 94°C, 1 min at 60°C and 1 min at 72°C for 30 cycles. Repeating the experiment twice with newly isolated RNA from fresh plant samples compensated the lack of an internal constitutively expressed control.
2.9. Rapid amplification of cDNA ends PCR
Both 5% rapid amplification of cDNA ends
(RACE) and 3%RACE PCR were performed with
gene-specific primers designed against partial se-quences obtained through RT-PCR with the SMART-RACE PCR kit (Clontech) according to the manufacturer’s instructions. Primer sequences
were as follows: forcASC32, 5%RACE 5%
-AGGTA-GATTGTTTGGGACATC-3% and nested primer
5%-CTCAAATATGTTGCCTCGTACAGC-3%; 3%
-RACE 5%-CCTCACCAACGATGTAGAAG-3%;
for cASC125, the primers used for 5%-RACE were
5%-GTCTCGAACATAAGGAAGCTTA-3% and
nested primer 5%
-CCCTCATAAATGCCGCTTC-ATACAG-3%, and 3%-RACE was performed with
5%-TCTGTATGAAGCGGCATTTATG-3%.
3. Results and discussion
3.1. Cloning of partial genomic clones gASC21, gASC22, gASC23 and gASC24
Molecular comparisons of plant terpene cyclases have revealed a striking level of sequence similarity [17,26,27]. After comparison of the amino acid
sequences of sesquiterpene cyclases isolated fromN.
tabacum[20],G.arboreum[21] andH.muticus[17], different degenerate primers were subsequently designed against conserved boxes highlighted by this comparison. RT-PCR with these primers
Table 1
Identities (%) and similarities (%) (in parentheses) obtained through the BLASTX/BEAUTY search for gASC21–gASC24, cASC34 and cASC125 with corresponding amino acid sequences of sesquiterpene cyclases from divergent sourcesa
CAD1A VS GS
TEAS 8-epicedrol CAD VS1
synthase
G.arboreum H.muticus L.esculentum
N.tabacum A.annua G.hirsutum S.tuberosum
51 (68) 46 (62) 56 (74) 60 (72)
aCAD, Cadinene synthase; VS, vetispiradiene synthase.
L. plants remained unsuccessful (data not shown). Therefore, analysis of genomic DNA has been performed. This resulted in a specific PCR product that appeared to be a mixture of four different
genomic fragments (gASC21, gASC22, gASC23
and gASC24), respectively, 841, 821, 788 and 841
bp in length. Alignment of the deduced amino acid sequences for these four different partial genomic clones revealed high identities and similarities with other cloned sesquiterpene cyclases, as shown in Table 1. These results clearly suggested that we cloned partial genomic sequences encoding
differ-ent sesquiterpene cyclases from Artemisia annua
(see Table 2 for identities of four partial clones on nucleotide level). Moreover, the fact that four different but clearly homologous sequences have been cloned suggested that we are dealing with a complex gene family of sesquiterpene cyclases in A. annua L., which has also been confirmed by a Southern blot (data not shown). This was not
surprising since H. muticus contains a small gene
family of approximately six to eight genes [17],
and 12 – 15 epi-aristolochene synthase like genes
are present in the tobacco genome [20]. From G.
arboreum, several cadinene synthase genes have already been cloned, indicating that this organism also contains a gene family for sesquiterpene
cy-clase [21]. Solanum tuberosumappears also to
con-tain different veti-spiradiene synthase genes [28]. Comparison of intron – exon organisation of
gASC21,gASC22,gASC23 and gASC24 with
cor-responding sequences of TEAS from N. tabacum
and vetispiradiene synthase from H. muticus
re-vealed that intron positions (Fig. 2) and sizes (not
shown) of theA.annuaclones were consistent with
earlier reported results [17,20]. This correlates with the fact that plant sesquiterpene cyclases have
highly conserved intron – exon organisation of ge-nomic DNA [17]. This conservation can even be extended to the diterpene cyclases [29].
3.2. Detection of sesquiterpene cyclase transcripts
Based upon the four partial genomic sesquiter-pene cyclase sequences, a new set of degenerated primers was designed for RT-PCR. mRNA
iso-lated from leaves and flowers ofA.annuaL. plants
at different moments of development served as a template for RT-PCR. We detected a specific product both in leaves and flowers of flowering
plants (data not shown). Also, in Fragaria 6esca
L., a partial sequence encoding sesquiterpene cy-clase was isolated along with seven other ripening-induced cDNAs by differential screening of a cDNA library [30]. The author states that the absence of a detectable signal in commercial strawberry either suggests that sesquiterpene cy-clase does not play an important role in the ripen-ing of the fruits or that the transcript levels are too low in commercial strawberries [30]. It is possible
that this phenomenon also takes place inA.annua,
and that sesquiterpene cyclase transcripts are only detectable in flowering plants, when expression levels are highest.
Table 2
Fig. 2. Alignment of sesquiterpene cyclases isolated from divergent plant sources (PILEUP GCG package, University of Wisconsin). cASC34 and cASC125, new sesquiterpene cyclases isolated from A. annua;gASC21 –gASC24, corresponding AA sequences deduced from respective partial genomic sequencesgASC21 –gASC24; Aa, A.annua 8-epicedrol synthase; Hmut,H.
muticus VS; Ntab, N. tabacumTEAS; Garb, G. arboreumCAD. Conserved amino acid sequences used for primer design are
marked with a frame., Positions of introns in partial genomic sequencesgASC21 –gASC24;, introns in TEAS and VS. The
Two partial cDNA sequences were obtained
(cASC34-p and cASC125-p), of 465 and 462 bp,
respectively. Comparison of the deduced amino
acid sequences revealed that gASC22 and
cASC34-p were identical. The corresponding
ge-nomic sequence of cASC125-p, however, has not
yet been isolated. cASC125-p showed only
identi-ties between 49 and 61% and similariidenti-ties between 62 and 77% with the four partial genomic se-quences. Identities and similarities with
sesquiter-pene cyclases cloned fromN. tabacum,H.muticus,
G. arboreum, Gossypium hirsutum, S. tuberosum and Lycopersicon esculentum were comparable
with those obtained for the gASC21 to gASC24
(identities ranging from 45 to 8% and 46 to 58%; similarities between 65 and 81% and 65 and 78%,
for cASC34-p and cASC125-p, respectively).
These values clearly indicate that cASC34-p and
cASC125-p are partial cDNA sequences encoding
two different members of theA.annuagene family
for sesquiterpene cyclases. The fact that, for
cASC125-p, the corresponding genomic sequence
escaped isolation thus far can be due to the high degeneracy of the primers used for PCR on
ge-nomic A. annua DNA. Out of ten partial genomic
sequences isolated and analyzed, we identified four different sesquiterpene cyclases. In addition, it could also be possible that not all members of the sesquiterpene cyclase gene family are active at the
flowering time of A. annua or that some members
of the gene family stay below detection limits. It is not clear at which moment of plant development the artemisinin biosynthetical pathway is acti-vated. Studies on influences of elicitors on artemisinin production have been established, but the influence on enzymes related to this biosynthe-sis has not been analyzed. Analybiosynthe-sis of artemisinin yield estimated at different stages of development reveals a positive correlation between plant age and artemisinin yields, without any peak produc-tion at any moment of the plant development [3,31 – 33]. This probably means that enzymes re-lated to artemisinin biosynthesis are active during a large part of plant development, but probably stay below detection limits at mRNA level. RNA transcripts of previously isolated sesquiterpene
cy-clases from N. tabacum, H. muticus and G. ar
-boreum could only be detected after induction by elicitor treatment [17,20,21], and UV treatment was necessary to detect sesquiterpene cyclase protein in pepper [26].
3.3. Cloning of cASC34 and cASC125
Using cASC125-p and cASC34-p as a probe to
screen a cDNA library of A. annua, we did not
obtain any clone containing the corresponding full-length sequence of the respective probe used, but we cloned a full-length sequence encoding another member of the sesquiterpene cyclase fam-ily (unpublished results). By the time we were
performing expression experiments with the
protein encoded by this gene, two other research groups reported the isolation of 8-epicedrol
syn-thase from A.annua [22,23], which appeared to be
exactly the same sequence as our full-length clone. But this sesquiterpene cyclase is apparently not involved in artemisinin biosynthesis [22,23].
Since we were unable to clone the corresponding
full length sequence of cASC34-p and cASC125-p
from our cDNA library, 5%RACE and 3%RACE
PCR for the full-length cloning of both genes was performed.
For cASC34, a sequence of 2026 bp was
ob-tained containing 38 bp of the untranslated 5%end
with an ORF encoding a 549 amino acid (aa) protein of 64.2 kDa and a calculated isoelectric point (pI) of 5.28. Two AATAAT – poly-adenyla-tion signals were found in the 338 bp untranslated
3% sequence (positions, 1876 and 2003 bp).
The cASC125 sequence contained an ORF of
1734 bp encoding a 577 aa protein with a molecu-lar weight of 67.4 kDa and a pI of 5.50. This
sequence has 60 bp at the 5%untranslated end and
an untranslated 3% sequence of 48 bp. Here, we
found the poly-adenylation signal AATTAA at 1820 bp.
Comparison of deduced amino acid sequences
from cASC34 andcASC125 with cloned
sesquiter-pene cyclases from divergent sources revealed high similarities and identities as shown by Table 1 and Fig. 2.
3.4. RNA analysis for three sesquiterpene cyclases from A. annua
Fig. 3 shows the expression in leaves and
flow-ers of 8-epicedrol synthase, cASC34 andcASC125
during plant development. We were able to demonstrate that 8-epicedrol synthase is expressed during the whole plant development in leaves and
flowers, but that cASC125 is only present in
Fig. 3. RNA analysis for 8-epicedrol synthase (A), cASC34 (B) andcASC125 (C).
artemisinin biosynthesis. The enzymes related to this biosynthetical pathway are possibly more ac-tively transcribed with as a consequence that their mRNA transcripts are detectable during this mo-ment of plant developmo-ment. Further experimo-ments are necessary, and bacterial expression analysis of
cASC34 andcASC125, in order to determine their
exact enzymatic activity, will be performed in the
near future. On the other hand, if cASC34 or
cASC125 do not possess amorpha-4,11-diene
ac-tivity, the search for the gene(s) encoding a protein with this enzymatic activity will be continued by
use of the partial genomic sequences gASC21,
gASC23 and gASC24 reported here.
Acknowledgements
The authors thank Prof. L. Gheysen and all members of the Nematode Group (Laboratory of Genetics, University of Ghent, Belgium) for assis-tance during the course of this work, Christophe Gilot (Laboratory of Genetics, University of Ghent, Belgium) for synthesis of primers, and Ardiles Diaz Wilson and Villarroel Raimundo (Laboratory of Genetics, University of Ghent,
Bel-gium) for sequencing analysis of cASC34 and
cASC125. Prof. Chappell is gratefully
acknowl-edged for the gift of pBSK-TEAS, used in a preliminary stage of our experiments, and Dr D.L.
Klayman for providing Artemisia annua L. seeds.
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