Oxidosqualene cyclases from Withania somnifera Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases From Withania somnifera (L.) Dunal Niha Dhar 1 , Satiander Rana 1 , Sumeer Razdan 1 , Wajid Waheed Bhat 1 , Aashiq Hussain 2 , Rekha S. Dhar 1 , Samantha Vaishnavi 3 , Abid Hamid 2 , Ram Vishwakarma 4 , Surrinder K. Lattoo 1* From the Divisions of 1 Plant Biotechnology, 2 Cancer Pharmacology, 4 Medicinal Chemistry, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi-180001, India 3 School of Biotechnology, Shri Mata Vaishno Devi University, Katra-182320, India *Running title: Oxidosqualene cyclases from Withania somnifera To whom correspondence should be addressed: Dr. Surrinder K. Lattoo, Plant Biotechnology, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, Tel.: +919419203465; Fax: +91-191-2569019; E-mail: [email protected]Keywords: Isoprenoid; metabolic engineering; gene regulation; mass spectrometry; western blotting; Withania somnifera; oxidosqualene cyclases; Schizosaccharomyces pombe; elicitor; withanolides Background: Pharmacological investigations position withanolides as important bioactive molecules demanding their copious production. Results: Differential transcriptional and translational expression of three oxidosqualene cyclases leads to redirection of metabolic fluxes. Conclusion: Negative regulator channelizes substrate pool towards cycloartenol synthase at sub-dividing junction leading to enhanced withanolide production. Significance: Understanding regulatory role of oxidosqualene cyclases on withanolide accumulation could serve as prognostic tool for metabolic engineering. ABSTRACT Oxidosqualene cyclases (OSCs) positioned at a key metabolic sub-dividing junction execute indispensable enzymatic cyclization of 2, 3- oxidosqualene for varied triterpenoid biosynthesis. Such branch-points present favourable gene targets for redirecting metabolic flux towards specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS) and cycloartenol synthase (WsOSC/CS) having open reading frames of 2289 bp, 2268 bp and 2277 bp were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses and kinetic studies confirmed their mono- functionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript-profiling under methyl jasmonate (MeJA), gibberellic acid (GA 3 ) and yeast extract (YE) elicitations displayed differential 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M114.571919 The latest version is at JBC Papers in Press. Published on April 25, 2014 as Manuscript M114.571919 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on June 16, 2020 http://www.jbc.org/ Downloaded from
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Oxidosqualene cyclases from Withania somnifera
Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases From Withania somnifera (L.) Dunal
Niha Dhar1, Satiander Rana
1, Sumeer Razdan
1, Wajid Waheed Bhat
1, Aashiq Hussain
2, Rekha S.
Dhar1, Samantha Vaishnavi
3, Abid Hamid
2, Ram Vishwakarma
4, Surrinder K. Lattoo
1*
From the Divisions of 1Plant Biotechnology,
2Cancer Pharmacology,
4Medicinal Chemistry,
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi-180001, India 3School of Biotechnology, Shri Mata Vaishno Devi University, Katra-182320, India
*Running title: Oxidosqualene cyclases from Withania somnifera
To whom correspondence should be addressed: Dr. Surrinder K. Lattoo, Plant Biotechnology, CSIR -
Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, Tel.: +919419203465; Fax: +91-191-2569019; E-mail: [email protected]
Keywords: Isoprenoid; metabolic engineering; gene regulation; mass spectrometry; western blotting; Withania somnifera; oxidosqualene cyclases; Schizosaccharomyces pombe; elicitor;
withanolides
Background: Pharmacological investigations
position withanolides as important bioactive molecules demanding their copious
production.
Results: Differential transcriptional and
translational expression of three oxidosqualene
cyclases leads to redirection of metabolic
fluxes.
Conclusion: Negative regulator channelizes substrate pool towards cycloartenol synthase at
sub-dividing junction leading to enhanced
withanolide production.
Significance: Understanding regulatory role of
oxidosqualene cyclases on withanolide
accumulation could serve as prognostic tool for metabolic engineering.
ABSTRACT
Oxidosqualene cyclases (OSCs) positioned at
a key metabolic sub-dividing junction execute
indispensable enzymatic cyclization of 2, 3-
oxidosqualene for varied triterpenoid
biosynthesis. Such branch-points present
favourable gene targets for redirecting
metabolic flux towards specific secondary
metabolites. However, detailed information
regarding the candidate OSCs covering
different branches and their regulation is
necessary for desired genetic manipulation.
The aim of the present study, therefore, was
to characterize members of OSC superfamily
from Withania somnifera (Ws), a medicinal
plant of immense repute known to synthesize
a large array of biologically active steroidal
lactone triterpenoids called withanolides.
Three full length OSC cDNAs, β-amyrin
synthase (WsOSC/BS), lupeol synthase
(WsOSC/LS) and cycloartenol synthase
(WsOSC/CS) having open reading frames of
2289 bp, 2268 bp and 2277 bp were isolated.
Heterologous expression in
Schizosaccharomyces pombe, LC-MS analyses
and kinetic studies confirmed their mono-
functionality. The three WsOSCs were found
to be spatially regulated at transcriptional
level with WsOSC/CS being maximally
expressed in leaf tissue. Promoter analysis of
three WsOSCs genes resulted in identification
of distinct cis-regulatory elements. Further,
transcript-profiling under methyl jasmonate
(MeJA), gibberellic acid (GA3) and yeast
extract (YE) elicitations displayed differential
1
http://www.jbc.org/cgi/doi/10.1074/jbc.M114.571919The latest version is at JBC Papers in Press. Published on April 25, 2014 as Manuscript M114.571919
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
WsOSC/BS showed a rise which hinted towards the decrease in the total substrate availability for
WsOSC/CS but at protein level WsOSC/BS
expression declined with increasing time intervals. Thus possibly substantiating the drop
in WS-3 concentration due to decreased
WsOSC/CS protein availability.
Interestingly, microbe-derived exogenous YE
elicitor played a role of negative regulator for the
two competitive OSCs of WsOSC/CS
(WsOSC/BS and WsOSC/LS) at both protein and mRNA level. While as WsOSC/CS showed no
change in its transcript or protein expression in
response to YE. However, there was significant
increase in withanolide concentration with YE in comparison to MeJA treatment. The down
regulation of WsOSC/BS and WsOSC/LS is
possibly indicative of differential channelling of common substrate among the three branch OSCs.
Plausibly, this leads to rearrangement of
metabolic fluxes wherein bulk of 2, 3- oxidosqualene substrate pool shifts towards
WsOSC/CS leading to much improved
withanolide yields.
In continuum to our previous studies covering characterization of withanolide biosynthetic
genes, the present investigation has validated
OSCs covering three branches of an important
metabolic junction. Further, for homologous
intensification of withanolides, these results in
totality could be useful to reveal various underlying signal transduction pathways as
indicated by elicitations in corroboration with
cis-regulatory motifs. Specific transcription factors along with the biosynthetic genes can
become prospective targets for pathway
engineering. Plausibly, the characterization and
validation of WsOSCs seems important for strategising the enhanced production of
withanolides.
Acknowledgements - This work was supported by
a grant from the Council of Scientific and Industrial Research (CSIR), Government of
India, New Delhi under Network Project BSC-
0108. N.D., S. Rana, S. Razdan, A. Hussain and
W.W.B. are highly thankful to CSIR, Government of India, New Delhi for Senior Research
Fellowship (CSIR-SRF). The authors are grateful
to Prof. Asis Datta (National Institute of Plant Genome Research, New Delhi, India) for
providing Schizosaccharomyces pombe and
pDS472a. We are also thankful to. R.K. Khajuria
and A.P. Gupta (Indian Institute of Integrative medicine Jammu, India) for facilitating LC-MS
analyses. This manuscript represents institutional
communication number IIIM/1568/2013.
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FIGURE 3. Predicted three dimensional models and ligand binding sites for WsOSCs: Ribbon
model display of the 3-D structures of (A) WsOSC/BS, (B) WsOSC/LS and (C) WsOSC/CS as
predicted by Phyre2
web server, using crystal structure of human OSC (Protein Data Bank (PDB) ID: 1w6ka) as template for modelling of all the three proteins. Predicted ligand binding sites (highlighted
in blue at the core of the structure) in (D) WsOSC/BS, (E) WsOSC/LS and (F) WsOSC/CS as predicted
by 3DLigandSite web server.
FIGURE 4. Phylogenetic tree of WsOSC/BS, WsOSC/LS and WsOSC/CS. Phylogenetic analysis
was performed using the ClustalW program and MEGA 5 software based on the neighbour-joining method. OSCs grouped into three sub-groups namely OSC1- Cycloartenol synthase, OSC2- Lupeol
synthase and OSC3- β-amyrin synthase. WsOSC/BS, WsOSC/LS and WsOSC/CS clustered with their
respective subgroups. 23 protein sequences used for analysis were from subsequent plant species: Solanum lycopersicum (Solanum lycopersicum BS, NCBI Reference Sequence: NP_001234604.1,
FIGURE 5. Identification of Withania somnifera β-amyrin, lupeol and cycloartenol synthases by
use of the yeast heterologous expression system. Extracted ion chromatogram (EIC) of standards β-
amyrin, lupeol and cycloartenol and S. pombe cells transformed with expression constructs (A)
pDS472aB, (B) pDS472aL, (C) pDS472aC and empty vector (D) pDS472a. The mass spectrometry (MS) data of (E) pDS472Ab, (F) β-amyrin standard, (G) pDS472aL, (H) lupeol standard, (I) pDS472aC
and (J) cycloartenol standard. Fragmentation pattern of (K) β-amyrin, (L) pDS472aB, (M) lupeol (N)
pDS472aL (O) pDS472aC and (P) cycloartenol.
FIGURE 6. SDS-PAGE profile of purified recombinant proteins. SDS-PAGE (10%) of purified
recombinant proteins from S. pombe transformed with pDS472aB, pDS472aL and pDS472aC. Lane 1;
Purified recombinant GST-fused WsOSC/BS, Lane 2; Purified recombinant GST fused WsOSC/LS, Lane 3; Standard protein marker, Lane 4; Purified recombinant GST-fused WsOSC/CS.
FIGURE 7. Kinetic study of WsOSC/BS, WsOSC/LS and WsOSC/CS. Michaelis–Menten plot of (A) β-amyrin synthase, (WsOSC/BS) (B) lupeol synthase (WsOSC/LS) and (C) cycloartenol synthase
(WsOSC/CS) with 2,3- oxidosqualene. Kinetic parameters Km and Vmax were obtained by fitting the
data in the Michaelis–Menten equation by non-linear regression analysis using GraphPad Prism 5 software
FIGURE 8. Tissue-specific real-time expression analysis. Quantitative estimation of the expression
of (A) WsOSC/LS, (B) WsOSC/BS and (C) WsOSC/CS in leaf, roots, stalk and berries of Withania somnifera. Data were compared and analysed with analysis of variance (ANOVA). Values are means,
with standard errors indicated by bars, representing three independent biological samples, each with
three technical replicates. Differences were scored as statistical significance at *p<0.05 and **p<0.01 levels.
FIGURE 9. Transcript profiles of WsOSCs in response to elicitor treatments. (A) Time courses
of WsOSC/BS, WsOSC/LS and WsOSC/CS expression in micropropagated Withania somnifera elicited
by methyl jasmonate (MeJA; 0.1 mM), gibberellic acid (GA3; 0.1 mM) and yeast extract (YE;
0.1% w/v). β-actin was kept as endogenous control. (B) Densitometric quantification of WsOSC/BS, WsOSC/LS and WsOSC/CS band intensities for the different treatments and controls (ethanol and
water). Experiments were performed in triplicate with similar results; error bars indicate ± standard
deviation of the mean. IOD, integrated optical density; A.U., arbitrary units.
FIGURE 10. Western immunoblot of WsOSCs in response to elicitor treatments. Time courses of
WsOSC/BS, WsOSC/LS and WsOSC/CS protein expression in micropropagated Withania somnifera
elicited by ( A) methyl jasmonate (MeJA; 0.1 mM), ( B) gibberellic acid (GA3; 0.1 mM) and (C)
yeast extract (YE; 0.1% w/v). β-actin was kept as endogenous control.
FIGURE 11. Time-course effect of elicitor treatments on accumulation of withanolides.
Withanolide accumulation in response to (A) 0.1mM methyl jasmonate (MeJA), (B) 0.1 mM gibberellic
acid (GA3) and (C) 0.1%w/v yeast extract (YE) at different time courses. Variation in three key
withanolides - withanolide A (WS-1), withanone (WS-2) and withaferine A (WS-3) was confirmed by HPLC analysis at 6, 12, 24 and 48 h. All values obtained were means of triplicate with standard errors.
Time-course accumulation of WS-1, WS-2 and WS-3 was statistically significant at p<0.01 level.
FIGURE 12. Southern blot analysis of (A) WsOSC/BS, (B) WsOSC/LS and (C) WsOSC/CS: Withania somnifera genomic DNA was digested with SpeI (non-cutter), ScaI and EcoRI (single-
cutter) for WsOSC/BS, with SalI (non-cutter), NcoI and EcoRV (single-cutter) for WsOSC/LS and
with XbaI, XhoI (non-cutter), HindIII and DraII (single cutter) for WsOSC/CS, separated on 0.8 % agarose gel, blotted onto a nylon membrane and hybridized with DIG-labelled ORF of WsOSC/BS,