SECTION 3 FUNGAL GENETICS AND PLANT METABOLOMICS

Section 3 Metabolomics
SECTION 3 PLANT METABOLOMICS
Section leader R. Verpoorte
GENERAL INTRODUCTION
Plants produce a wide range of secondary metabolites. These plant
specific compounds represent very important quality traits of plants.
They are involved in defense against pests and diseases and responsible
for color, taste, flavor and health effects of our food. Many secondary
metabolites are economically important fine chemicals such as drugs,
flavors, fragrances, dyes, antioxidants and insecticides.
The Pharmacognosy group has as mission:
‘To find, produce and determine the role of economically important
secondary metabolites in plants and unravel their biosynthesis, and to
create an international academic ambiance in which students can be
trained in multidisciplinary projects related to these goals to become
creative and independent researchers’.
In terms of research this is translated to projects on understanding the
role of secondary metabolism in plants, on finding new leads for drug
development, and on the biosynthesis of secondary metabolites, with the
aim to improve their production in plants, plant cell cultures or
microorganisms by using genetic engineering. For mapping biosynthetic
pathways, a metabolomics approach is developed. The metabolic
engineering project is carried out in close collaboration with the Section
Plant Physiology (Prof.Dr. J. Memelink, Dr. H.J.M. Linthorst). The
section has established a platform for metabolic profiling, which, among
others, is applied as a tool in studying plant-insect and plant-fungal
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interactions, together with the sections Plant Physiology and Plant
Ecology (Dr. P.G.L. Klinkhamer, Prof.Dr. E. van der Meijden). NMRspectrometry
is the major tool for these metabolomics projects.
In connection with the educational activities the department has
particularly many MSc- and PhD-students from 2nd and 3rd world
countries working on projects related to the exploration of their very
rich biodiversity.
Research themes:
1. Leadfinding
2. Cannabis
3. Isolation Natural Products
4. Metabolomics
1. Leadfinding
In this project the effect of various plant extracts are studied for
antimicrobial activity and in particular the effect against wood rot fungi.
Several active compounds were isolated and identified from saw dust of
tropical hardwoods. In a joint project with some industrial partners and
Indonesia we are working on the isolation of compounds that have an
anti-obesitas effect. In collaboration with the division of Medicinal
Chemistry (LACDR) we work on the isolation of compounds which
have activity on the adenosine or opioid receptors.
2. Cannabis
Studies on medicinal cannabis are performed in an STW-project with
three Dutch companies, Feyecon, Farmalyse and Enzyscreen, and the
Delft University of Technology. Our research includes the isolation of
cannabinoids by using centrifugal partitioning chromatography. All
major cannabinoids can now be produced in larger (gram) quantities and
are made available commercially. The production will be further
developed for GMP-conditions in the STW project. In collaboration
with Enzyscreen bacterial strains have been found that can produce
novel cannabinoids derivatives.
Studies on the inhalation and the oral administration of medicinal
cannabis tea have resulted in improved protocols, which will be very
helpful for patients using the legal standardized medicinal cannabis.
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Section 3 Metabolomics
3. Isolation Natural Products
A large grant from Mibiton enabled the department to invest in
centrifugal partitioning chromatography (CPC) and LC-MS equipment.
This grant was obtained in close collaboration with several companies
(Feyecon, Farmalyse, Xenobiosis, Holland Biodiversity and
Enzyscreen) that recently have combined their complementary expertise
and marketing efforts in a new start-up company PRISNA (Product
Isolation from Nature). The CPC will be used, among others, for the
isolation of cannabinoids and hop bitter compounds. For the latter we
developed a completely novel large scale, food compatible separation
method for the cis- and trans-iso-alpha acids, the compounds
responsible for the bitter taste of beer.
4. Metabolomics
In the past 7 years we have developed NMR as a tool for metabolomics
studies. The NMR offers the great advantage of a very reproducible
method for metabolome analysis, which is very easy to quantify, not
requiring any calibration curves as for all compounds the 1 H-NMR
signals are only dependent on the molar concentration. In the mean time
we have built up a library of both 1D and 2D spectra of more than 500
common plant metabolites all run under strictly defined conditions.
Moreover we have more than 20,000 spectra of plants all recorded
following standard protocols, which can thus be used for data-mining.
This database concerns mainly plant and plant cell cultures of
Catharanthus roseus, and plants such as Arabidopsis, Brassica rapa
(IOP Genomics project with Universities of Wageningen and
Groningen), Senecio, tobacco, vanilla, Ilex species (including herba
mate), tomatoes (ERA-PG project), and wine and grapewines (ERA-PG
project) for studies of functional genomics, biosynthesis and resistance
against pests and diseases. Metabolomics for quality control and the
pharmacological activities is applied on some medicinal plants, e.g.
Ephedra, Ginseng, Gingko and licorice preparations.
By NMR in a single operation both primary and secondary metabolites
can be determined. Particularly 2D-NMR spectrometry methods have
been shown to be very useful for simplifying the spectra (2D-J-resolved
projected spectra) and structure elucidation in complex extracts (e.g.
2D-J-resolved, HMBC, HMQC, COSY). In the coming years we will
collaborate with the LUMC to further build up our library of natural
products NMR spectra and exchange experience in field of NMR-based
metabolomics in various organisms. In a collaborative project with
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Qiagen, supported by a European grant, we will develop standardized
methods for sample preparation in metabolomics (the pre-analytical
phase).
In retrobiosynthetic studies we have shown that salicylate and 2,3-
dihydroxybenzoic acid in Catharanthus roseus are derived from the
isochorismate branch of the chorismate pathway rather than from the
phenylalanine branch. Though both compounds are formed immediately
after elicitation, the total incorporation levels in both differ, showing
that the development of fluxomics is important to really understand the
regulation of biosynthetic pathways. The development of fluxomics for
plants will thus be a major target for the coming period.
PERSONNEL
Prof.dr. R (Rob) Verpoorte
Professor UL
Dr H.K. (Hye Kyong) Kim
Postdoc UL
Dr Y.H. (Young) Choi
Postdoc UL
Dr. T. (Teus) Luyendijk
Postdoc, Phytoconsult
Dr. F. (Federica) Maltese
Postdoc, EU scholarship
Dr. A. (Alfi) Khatib
Postdoc, STW scholarship
Dr. F. (Frank) van der Kooy
Postdoc, STW scholarship
Dr. A. (Alba) Sargoni
Postdoc, own funding
Dr. A. (Annelies) Schulte
Postdoc, STW scholarship
Dr. M. (Marianne) Verberne Postdoc, STW scholarship
Prof.dr. D-X. (De-Xiu) Zhao Visiting Professor
Dr. M.E. (Maria) Cazar
Postdoc, own funding
Dr. M. (Margarida) Fortes
Postdoc, own funding
Dr. U.K. (Umesh) Patil
Postdoc, Boyscast
fellowship
N.R. (Ria) Mustafa
grad.student, own funding
P. (Hwan) Sumthong grad.student, Thai Government
L (Lien) Le Thi
grad.student, Vietnamese
Government
I.J. (Isvett) Flores Sanchez
grad. student, CONACYT scholarship
S.B. (Sanimah) Simoh
grad. student, Malaysian Government
H.T. (Hien) Dao
grad. student, Vietnamese
Government
G (Ghulam) Hasnain
grad. student, Pakistan University
M. (Muhammad) Jahangir grad. student, own funding
I. (Ibrahim) Bayoumi grad. student, Ford
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Section 3 Metabolomics
Foundation fellowship
R. (Roman) Romero grad. student, scholarship
Universidad de Los Andes
T. (Tony) Palama sandwich PhD, Government
La Réunion
B. (Barbora) Pomahacova sandwich PhD
N.D. (Nancy) Yuliana
grad. Student, PSE
scholarship
M.N. (Nadeem) Akram
grad. student, NUFFIC
M. (Muzamal) Iqbal grad. student, NUFFIC
A. (Ali) Kashif grad. student, NUFFIC
M.T. (Tayyab) Akhtar
grad. student, NUFFIC
M.Y. (Yahya) Mushtaq
grad. student, NUFFIC
L.C. (Leen) Verhagen
researcher, own funding
M. (Marica) Fraccaroli sandwich PhD
C. (Claudia) Lopez grad. student, own funding
E. (Esther) Fernandez grad. student, own funding
P. (Pablo) Sanchez grad. student, own funding
S. (Simon) Bordage grad. student, own funding
S. (Sytze) Elzinga analist, STW
J.P. (Anneke) Poels
administrative staff
EU
STW
UL
PSE
European Union
Netherlands Technology Foundation
Universiteit Leiden
Phytochemical Society of Europe
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COLLABORATIONS
National
- Delft, TU Delft, Botanische tuin, Apparatenbout, Prof.dr. G.J. Witkamp
- Den Haag, Bureau Medicinale Cannabis, Ministerie VWS
- Holland biodiversity, B.V.
- Wageningen, Genetwister, B.V.
- Leiden, TNO, dr. Mei Wang, dr. H. Korthout
- Leiden, NMR-afdeling LIC
- Leiden, Enzyscreen
- Leiden, Prof.dr. L.M. Deelder, LUMC
- Oostvoorne, Prisna BV
- Weesp, Feyecon BV
- Zaandam, Farmalyse
International
- Dept. Pharmacognosy, Faculty of Pharmacy, Uppsala, Sweden, Prof.dr. L.
Bohlin
- Dept. Pharmacognosie, Faculté de Pharmacie, Reims, France, Prof. M.
Zèches
- Dept. Pharmacognosie, Faculté de Pharmacie, Luik, Belgium, Prof.dr. L.
Angenot, Dr. M. Frédérich
- Dept. Agriculture, Federal Univerity of Santa Catarina, Florianopolis,
Brazil, Prof.dr. M. Maraschin
- Dept. Organic Chemistry, UNESP, Araraquara, Brazil, Prof.dr. V. Bolzani
- Botanical Gardens USP, Sao Paulo, Brazil, Prof.dr. M. Braga
- Dept. Plant Biotechnology, CINVESTA, Mexico City, Mexico, Dr. A.
Ramos
- Dept Pharmacology, Faculty of Medicine, Chiang Mai, Thailand, Prof.dr.
Ampai Panthong
- Dept. Pharmaceutical Sciences, Naresuan University, Phitsanulok,
Thailand, Prof.dr. I. Kornkanok
- CSIC, Madrid, Spain, Dr. A. Gonzales
- Dept. Pharmacognosie, Faculté du Pharmacie Amiens, France, Prof.dr. M.
Fliniaux, Dr. F. Mesnard
- Royal School of Pharmacy, Copenhagen, Denmark, Dr. D. Staerk, Prof.dr.
J. Jarwozewski
- Vlaams Instituut Biotechnologie, Gent, België, Prof.dr. D. Inze, Dr. A.
Goossens
- Center for Rhizosphere Biology, Colorado state University, Fort Collins,
USA. Prof.dr. J.M. Vivano
- UMR CIRAD, Université de La Réunion, France, Dr. H. Kodja
- Adaptogen, St.Petersburg, Russia, Prof.dr. V.G. Makarov, Dr. A. Shikov
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Section 3 Metabolomics
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam,
Germany, Dr. J. Kopka, Dr. A.R. Fernie
- Dipartimento scientifico e tecnologico, Universitá degli studi di Verona,
Italy, Prof.dr. M. Levi
- Lab of Biotechnology and Plant Molecular Biology, ICAT, FCUL, Lisbon,
Portugal, Dr. A.M. Fortes
- School of Natural Products Studies, Pharmaceutical Technology, Kolkata,
India, Dr. P.K. Mukherjee
GRANTS
- Pakistan government: 2 PhD students
- Venezuela government: 1 PhD student
- Chinese government: 1 visiting professor (6 months)
- Thai government: 1 PhD Antifungal natural products
- Vietnamese government: 1 PhD Metabolomics Medicinal Plants
- Mexican government: 1 PhD Biosynthesis cannabinoids
- Malaysian government: 1 PhD student
- EU Marie Curie grant: 1 postdoc
- EU Erasmus: sandwich PhD student Czech Republic
- EU Leonardo da Vinci, Spain: 2 researchers (6 months)
- STW: 1 postdoc and 0.5 technician
- STW valorization grant: 1 postdoc (3 months)
- Government Portugal: 1 postdoc (6 months)
- EVD: grant for organizing Russian Mission and meeting in Leiden on
Medicinal Plants
- Government La Réunion: 1 sandwich PhD
- PSE: 1 PhD student
- NUFFIC: 5 PhD students
PUBLICATIONS
Abdel-Farid, I.B., H.K. Kim, Y.H. Choi and R. Verpoorte (2007)
Metabolic characterization of Brasica rapa leaves by NMRspectroscopy.
J. Agric. Food. Chem 55: 7936-7943
El-Sayed, M. and R. Verpoorte (2007) Catharanthus terpenoid indole
alkaloids: biosynthesis and regulation. Phytochem. Rev. 6: 277-305
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El-Seedi, H.R., N.S. Abd El-Hamid, U. Goransson, and R. Verpoorte
(2007) Cyclopeptide alkaloids. Phytochem. Rev. 6: 143-165
Hazekamp, A., K. Bastola, H. Rashidi, J. Bender and R. Verpoorte
(2007) Cannabis tea revisited: A systematic evaluation of the
cannabinoid composition of cannabis tea. J. Ethnopharmacol. 113: 85-
90
Khatib, A., E.G. Wilson, H.K. Kim, M. Supardi, Y.H. Choi, R.
Verpoorte (2007) NMR assignment of iso--acids from isomerised
extracts of Humulus lupulus L. cones. Phytochem. Anal. 18: 371-377
Korthout, H. and R. Verpoorte (2007) Vanilla. In Flavours and
Fragrances. Chemistry, Bioprocessing and Sustainability. Springer-
Verlag Berlin, Heidelberg, pp 203-217
Mustafa, N.R. and R. Verpoorte (2007) Phenolic compounds in
Catharanthus roseus. Phytochem. Rev. 6: 243-258
Prasad Bastola, K., A. Hazekamp and R. Verpoorte (2007) Synthesis
and spectroscopic characterization of cannabinolic acid. Planta Med. 73:
273-275
Roytrakul, S. and R. Verpoorte (2007) Role of vacuolar transporter
proteins in plant secondary metabolism: Catharanthus roseus cell
culture. Phytochem. Rev. 6: 383-396
Sanchez-Sampredo, A., H.K. Kim, Y.H. Choi, R. Verpoorte and P.
Corchete (2007) Metabolomic alterations in elicitor treated Silybum
marianum suspension cultures monitored by nuclear magnetic
resonance spectroscopy. J. Biotechnol. 130: 133-142
Verberne, M.C., K. Sansuk, J.F. Bol, H.J.M. Linthorst and R. Verpoorte
(2007) Vitamin K1 accumulation in tobacco plants overexpressing
bacterial genes involved in the biosynthesis of salicylic acid. J.
Biotechnol. 128: 72-79
Verpoorte, R., Y.H. Choi and H.K. Kim (2007) NMR-based
metabolomics at work in phytochemistry. Phytochem. Rev. 6: 3-14
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Section 3 Metabolomics
Verpoorte, R. (2007) Introduction. In: Applications of plant metabolic
engineering. R. Verpoorte, A.W. Alfermann and T.S. Johnson (Eds.).
Springer, Dordrecht, pp. XI-XXI
Zarate, R. and R. Verpoorte (2007) Strategies for the Genetic
Modification of the Medicinal Plant Catharanthus roseus (L.) G. Don.
Phytochem. Rev. 6: 475-491
Zhao, J. and R. Verpoorte (2007) Manipulating indole alkaloid
production by Catharanthus roseus cell cultures in bioreactors: From
biochemical processing to metabolic engineering. Phytochem. Rev. 6:
435-457
IN PRESS
Abdel-Farid, I.B., Y.H. Choi, H.K. Kim, C.A.M.J.J. van den Hondel, E.
van der Meijden and R. Verpoorte (2008) The role of secondary
metabolites in Arabidopsis and Brassica in the interaction with fungi.
Curr. Top. Plant Biol. in press
Angelova, N., H.–W. Kong, R. Van der Heijden, S.–Y. Yang, Y.H.
Choi, H.K. Kim, M. Wang, T. Hankemeier, J. Van der Greef, G. Xu and
R. Verpoorte (2008) Recent methodology in the phytochemical analysis
of ginseng. Phytochem. Anal. in press
Jahangir, M., H.K. Kim, Y.H. Choi and R. Verpoorte (2008)
Metabolomic response of Brassica rapa submitted to pre-harvest
bacterial contamination. Food Chem. 107: 362-368
Mosleh Arany, A., T.J. De Jong, H.K. Kim, N.M. Van Dam, Y.H. Choi,
R. Verpoorte and E. Van Der Meijden (2008) Glucosinolates and other
metabolites in leaves of Arabidopsis thaliana from natural populations
and their effects on a generalist and a specialist Herbivore. Chem. Ecol.
in press
Mustafa, N.R., H.K. Kim, Y.H. Choi and R. Verpoorte (2008) Single
step purification of salicylic acid from Catharanthus roseus cell culture
(plant material) by anion exchange for NMR analysis. J. Liq.
Chromatogr. Rel. Tech. in press
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Simoh, S., Huub J.M. Linthorst, R. Verpoorte (2008) Host-bacterium
interactions in Agrobacterium tumefaciens-mediated plant
transformation: Mechanism of action and Agrobacterium/plant factors
involved. Curr. Top. Plant Biol. in press
Zárate, R. and R. Verpoorte (2008) Metabolic Engineering of Plant
Cellular Metabolism: Methodology, Advances and Future Directions.
Wei Zhang (Editor), Progress in Bioproducts and Biotechnological
Applications, Springer. In press
DISSERTATIONS
N.R. Mustafa. Retrobiosynthetic study of salicylic acid in Catharanthus
roseus cell suspension cultures. PhD Thesis, Leiden University, 23 May
2007. Promotor: Prof.Dr. R. Verpoorte.
P. Sumthong. Antimicrobial compounds as side products from the
agricultural processing industry. PhD-Thesis, Leiden University, 19 June
2007. Promotor: Prof.Dr. R. Verpoorte.
A. Hazekamp. Cannabis, Extracting the medicine. PhD-Thesis, Leiden
University, 5 September 2007. Promotor: Prof.Dr. R. Verpoorte.
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