Metabolomics : SCBT 33312

Lecture 6, 7, 8 & 9

Plant Metabolomics
Discovering future cures from phytochemistry

G. Prabhakaran 1
 Learning Outcome

Lecture 6 & 7

1. Primary metabolites Vs Secondary metabolites

2. Classification of Secondary Metabolites

3. Function / Role of Secondary Metabolites

4. Priority herbal plants in Malaysia

Lecture 8 &9

5. Secondary metabolites from plant cell cultures

6. Strategies for the production of plant secondary metabolites

7. Phytochemistry

8. Plant Metabolomics
2
Too few genes, too many metabolites?
Plant Metabolism

10
 METABOLISM
Basic metabolism refers to the anabolic and catabolic
processes required for respiration, nutrient assimilation, and
growth /development; i.e. those processes required for cell
maintenance and proliferation.

Secondary metabolism refers to compounds present in

specialized cell’s that are not necessary for the cells survival
but are thought to be required for the plants survival in the
environment.

􀂄 Primary metabolite – Cell survival

􀂄 Secondary metabolite – Plants survival

11
 1. Primary metabolites Vs Secondary metabolites

Primary metabolite – Cell survival Secondary metabolite – Plants survival

13
Role of Secondary Metabolites

14
 15
Classification of Secondary Metabolites ………..
 2. Classification of Secondary Metabolites

1) According to carbon skeletal type. This was found to be

“far too cumbersome for practical use.”

2) According to biogenesis or biosynthetic origin. This is the

most widely used system of SM classification.

Using a biosynthetic classification system, largest

groups of Secondary Metabolites:

1. Terpenoids
2. Alkaloids and other nitrogen compounds ATP
ATP
3. Phenolics
16
https://www.slideshare.net/Thirusangu/secondary-metabolites-49723653?from_action=save
 ATP

Alkaloids and other nitrogen compounds, Terpenoids and Phenolics

17
~ 50,000 metabolites Croteau et al., 2000
. segregation 
Secondary metabolites are derived from primary metabolites 26
1

4
3
Alkaloids
• Best known nitrogen-
containing metabolites

• Low content offset by

biological potency

• Found in 20% angiosperms

• Most familiar and addictive.

Cocaine, morphine, THC,
caffeine, nicotine

• Insecticide

• Bitter tasting, affect cell

membrane function, cause
difficulties in digestion.

• Camptothecin, Vinblastine for

anticancer

• Generally affect nervous

system.

THC : Marijuana 32
• Camptothecin is an indole alkaloid, derived from
tryptophan.
• Has anticancer and antiviral activity
• Two CPT analogues have been used in cancer
chemotherapy, topotecan and irinotecan.

33
34
How does camptothecin work?

• Chromosomes are highly

complex (supercoil, knot…)

• DNA replication, gene

transcription, and DNA
recombination require
unwinding of chromosomes.
Topoisomerases can help.

35
Camptothecin is DNA topoisomerase I inhibitor

1. TOPO I binds to DNA

2. Makes a nick in one
strand
3. Unwind the DNA strand
once
4. Re-ligate DNA

Camptothecin binding of
TOPO I leads to DNA
fragmentation !

36
Terpenoids or Isoprenoids
 Terpenoids
Terpenoids (isoprenoids)

• Common biosynthic origin

from isopentenule and
dimethyulallyl
purophosphates
• generally lipophilic, plant
oils
• leaf glandular trichomes,
bud exudates, bark resins
• Flavonoids, aromatics,
carotenoids, chlorophylls
• May mimic insect growth
hormones
• Include Cardiac Glycosides
which affect the heart.
• Toxic to most fungi and
insects 38
 --- Artemisinin

Latex is a natural polymer of isoprene 

42
44
45
46
 Taxol

• Taxol is a terpenoid
• "the best anti-cancer agent” by National
Cancer Institute
• Has remarkable activity against advanced
ovarian and breast cancer, and has been
approved for clinical use. 47 47
 Where does taxol come from?
Taxol is found in the bark of the tree.

Problems:

• Pacific yew tree is very slow-growing.

• Harvesting taxol from the bark kills

the tree

• To treat one cancer patient

requires 60 pounds of Yew tree
Taxus brevifolia Nutt. bark, the equivalent of three, one
Pacific Yew hundred year old trees
48 48
 Phenolics

• Common biosynthetic
origin from
phenylalanine

• Aromatic

• Ionize in presence of a
base

• Used in spices

• Tannins are used in

tanning leather

49
 SMs and Mankind
• Taxol
• According to The National Cancer – A chemical discovered in the pacific
Society, Tropical Forests could contain Yew Tree is now the first drug of
20 ‘superstar’ drugs Anticancer drugs. choice in several tumorous cancers
including Breast Cancer.
• Of the 150 most commonly prescribed
drugs in the United States, 57% contain
at least one major active compound
derived from compounds in nature.

• While 25% of all Western

Pharmaceuticals are derived from
rainforest ingredients, less than 1% of
these tropical tress and plants have been
tested by scientists Vinblastine

• The U.S. National Cancer Institute has - A chemical discovered in the

identified 3000 plants that are active
against cancer cells.
Madagascar Periwinkle . First drug of
choice in many forms of Leukemia and
has increased survival rate of childhood
leukemia by 80%
50
51
4

Global herbal market

$65 billion

Malaysia
RM 4.55 Billion

http:// 52
www.pnc.upm.edu.my/upload/dokumen/20170731172828DISCOVERING_FUTURE_CURES_from_PHYTO
MALAYSIAN

53
54
“What is a Weed - A Plant whose Virtues have yet to
be Discovered” Robert Frost

Amazonia curare Chondrodendron tomentosum (left) and

Madagascar periwinkle Catharanthus roseus (middle), the
botanical source of tubocurarin (muscle relaxant) and
vincristine (anticancer), respectively 56
 Labisia pumila and its chemical markers:

Chemical Marking Kachip Fatimah

Used as traditional medicine by the indigenous communities in the

Malay archipelago, for treating pre- and post-partum complications,
menstrual disorders, dysentery, rheumatism and flatulence and as a
health tonic to regain vigor
57
Metabolomics : SCBT 33312

Lecture 8 & 9

Secondary metabolites from plant cell cultures

Part 1 Part 2 Part 3

Strategies for the Techniques for Other Strategic

production of Taxol, increasing yields of Approaches for over
plant sec. metabolites sec. metabolites in production of Plant
plant cell culture Sec. Metabolites

60
G. Prabhakaran
 Learning Outcome

1. Secondary metabolites from plant cell cultures

2. Strategies for the production of plant secondary metabolites

3. Phytochemistry

4. Plant Metabolomics

61
 Secondary metabolites are derived from primary metabolites

63
Alkaloids and other nitrogen compounds, Terpenoids and Phenolics
• Primary metabolites run $1 to $2 per pound

• Secondary metabolites run up to several hundred thousand $ / pound

65
*

* Terpenoids 66
Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative
agent,Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has
67
developed several potential routes of transmission within and between different host species . 
MALDI : Matrix Assisted Laser Desorption Ionization
69
 STRATEGIES FOR INCREASING PRODUCTION OF
ARTEMISININ
• Three different approaches: 1) nontransgenic, 2)
transgenic plants, and 3) heterologous transgenic systems.

• Nontransgenic approaches include selective breeding,

alteration of nutrient / environmental conditions, use of in
vitro cultures, and exploitation of a plant’s natural defense
system through elicitation.

• Transgenic A. annua plants using a variety of different

genes. The most recent approach for increasing artemisinin
is via heterologous systems, insertion of key artemisinin
biosynthetic genes into organisms other than A. annua.
 Elicitors are molecules that stimulate number of
defense responses in plants. 

• Treatment with elicitors derived from

phytopathogens can be an effective strategy to
increase the yield of specific metabolites obtained
from plant cell cultures.

• Understanding how plant cells perceive microbial

elicitors and how this perception leads to the
accumulation of secondary metabolites, in terms
of quantity and of quality of the compounds.

Ex: salicylic acid spray for tomatoes. Jasmonic acid for grapevine
71
72
 Taxus brevifolia)

Taxol, a complex diterpene

(Chemical precursor for taxol)

73
 Taxol: an example
• Taxol is a unique anticancer drug from the
bark of the Pacific Yew (Taxus breviola)

Taxol is a terpenoid
 Taxol Facts
• Very effective treatment against ovarian cancer,
breast cancer, melanoma, and colon cancer .

• Stops cell division, thus blocking cancer. It does

this by interfering with microtubule function.
Microtubules are responsible for pulling
apart the sets of chromosomes in mitosis.
 Taxol Needs
• It is estimated that 250 kg of pure Taxol are
needed to treat cancer in the USA. This would
require the bark of about 360,000 trees per
year!

• Obviously Taxol would be very expensive by

this method (approximately $200,000 to
$300,000 per kg).

anticancer drug taxol (generic name paclitaxel)

 Strategies for the production of plant
secondary metabolites

Taxol is a very good target for biotechnology

1. tissue culture of bark cells

2. fungus produces taxol
3. alternative species
4. genetic engineering
5. chemical synthesis

http://tru.uni-sz.bg/tsj/Vol.%2013,%20N%202,%202015/H.N.Badi.pdf
Overview of the Taxol
biosynthetic pathway.
Biosynthesis involves 19 steps .

78
80
 1) tissue culture of bark cells

• Many cells from different bark tissues from different

trees were screened.

• There are at least 25 fold differences in production. It

was found to be secreted into the medium thus
facilitating collection.

• So far 1 to 3 mg of taxol are produced per liter of cell

culture. This is equivalent to about 25 g of bark.
82
 2) fungus produces taxol
• It was found that a endophytic fungus that
colonizes yew trees also produces taxol

• Fungal culture technology which is better

developed than plant cell culture technology
could be an important source for taxol
production

Endophytic fungus :
Taxomyces andreanae and Pestalotiopsis microspora
 3) alternative species
• Some researchers found that the European Yew
(Taxus baccata) produces a precursor to taxol.

• This precursor can then be converted to an analog of

taxol in the laboratory.

• The precursor is used for chemical synthesis of taxol.

https://www.researchgate.net/publication/223596238_Production_of_the_
anticancer_drug_taxol_in_Taxus_baccata_suspension_cultures_A_review
 4) genetic engineering
• Other scientists are trying to identify and clone
the genes which produce taxol

• This will enable them to scale up production in

cell culture.

• Scientists have developed a form of root culture

using Agrobacterium rhizogenes, the cause of
hairy root disease.
 5) Chemical synthesis
Taxol is a terpenoid

• Chemical synthesis formidable

• 3 different ways to synthesize taxol are now

known

• Some take up to 13 steps

• Cost per patient still expensive; about $20,000

 Secondary metabolites from plant cell cultures

Types of secondary metabolites (SMs)

1. pharmaceuticals from plant products (e.g., alkaloids,

analgesics, cancer-fighting chemicals)

2. plant-derived flavors and colorings (where natural

compounds are necessary/desirable

SMs made in:

– batches (small to medium-scale)

– bioreactors (large-scale production) 88

 Constraints in production of Secondary
metabolites from plant cell cultures

• Features of Plant Sec metabolites

– low molecular wt
– synthesis (in plant cell) is tightly regulated

• A few products are commercially produced

– shikonin, ginseng compounds, berberine
– more are not produced because of two main problems
• low yields in in vitro culture
• feedback inhibition from SMs stored intracellularly

89
• Transport mechanisms in cultivated plant cells

– cells that transport SMs to vacuolar space for

storage
• final yield limited to storage capacity of medium

– cells that secrete SMs into the medium

• final yield limited to properties of the medium
• production is more easily manipulated, but
separation of culture medium from cultures is
required

90
• Outlook for SM production in plant cell cultures (where
plant cell systems may be preferred over whole
plants)

– rare and endangered plants of pharmaceutical interest

– plants that grow too slowly to match a sudden demand

(e.g., Taxol present only in the bark of Taxus brefolia)

– some cell cultures can be made to produce a single

compound, e.g., sanguinarine from poppy cell culture

91
Part 2

Secondary metabolites from plant cell cultures

• Techniques for increasing yields ( 4 major techniques)

1 –
two-phase systems
• strategy – to provide an artificial site for SM accumulation
• example – sanguinarine from cultures of California poppy
cells
– sanguinarine is an alkaloid that acts as an agent against dental
caries
• cells are grown in a 2-liter air-lift bioreactor
• this bioreactor uses 2 phases: an aqueous culture phase and
a silicone oil extraction phase
• bioreactor is sparged thru a central draft tube; gas bubbles
are disengaged thru a top layer of silicone fluid

92
*Two-phase systems, immobilized cell culture, hairy root culture & biotransformation
• advantages (of this two-phase system)

– SM product is isolated rapidly

– SM product can be localized in a reduced

volume

– elicitation can be incorporated

» biosynthetic enzymes induced

» increase in SM production results
93
 Secondary metabolites from plant cell cultures

2 – immobilized cell culture (2 approaches)

1. entrapment in alginate beads

2. adherence of cells to a porous membrane
in both cases, the objectives:
– control of growth (high nutrient levels first,
then elicitation or starvation)
– release of SM into the medium where it can
be harvested

94
 Secondary metabolites from plant cell cultures

– hairy root culture – culture of fast-growing roots by:

• inoculation with Agrobacterium rhizogenes (esp. useful
where differentiated cells are required for SM production)
• transfer of T-DNA causing hairy roots, then elimination of
Agrobacterium by antibiotic
• (transformed) hairy roots are first sub cultured to solid,
then liquid medium

The transfer DNA (abbreviated T-DNA) is the transferred DNA of the

tumor-inducing (Ti) plasmid of some species of bacteria such as
Agrobacterium tumefaciens and Agrobacterium rhizogenes. It derives
its name from the fact that the bacterium transfers this DNA fragment
into the host plant's nuclear DNA genome.
95
Many secondary metabolites are produced in roots

• Scientists have developed a form of root culture

using Agrobacterium rhizogenes, the cause of
hairy root disease.

• Cells transformed with some of the bacteria’s

DNA, causes the cells to be more sensitive to the
hormones they produce. The cells form into
roots. These roots grow very fast and produce
the secondary metabolites that ordinary roots
produce.
 Hairy root culture
• Hairy root culture, also called transformed root culture, is a type of 
plant tissue culture that is used to study plant metabolic processes or to
produce valuable secondary metabolites, often with plant 
genetic engineering.

• A naturally occurring soil bacterium Agrobacterium rhizogenes that

contains root inducing plasmids (also called Ri plasmids) can infect plant
roots and cause them to produce a food source for the bacterium, opines,
and to grow abnormally.

• The abnormal roots are particularly easy to culture in artificial media

because hormones are not needed, and they are * neoplastic, with
indefinite growth.
• The neoplastic roots produced by A. rhizogenes infection have a high
growth rate (compared to untransformed adventitious roots), as well as
genetic and biochemical stability.

* Tumour = an abnormal growth of tissue

Hairy root cultures 

(i) are suitable for production of all root derived

biochemicals,

(ii) are faster growing, higher producing and much easier to

maintain than untransformed root cultures,

(iii) present no risk of instability common with cell cultures, 

(iv) in some cases a portion of the product is released in

the medium, and

(v) provide opportunities for isolation of stable high

producing hairy root lines by exploiting somaclonal
variations. Hairy root cultures are quite promising,
particularly in case of tropane alkaloids.
100
– hairy root culture (contin)

• SM is released into the medium, then harvested

• primary advantage – a simple system w/o
problems inherent with cell suspensions
• limited to spp. susceptible to Agrobacterium
rhizogenes

101
Root cultures are often better than cell cultures

• Roots often secrete the metabolites into the

surrounding medium, making it easy for collection.

• Charcoal can be added to the medium, the

metabolites are absorbed by the charcoal, and this
stimulates even higher production of the metabolite.

102
 Some secondary metabolites produced in
cell and root culture

• L-DOPA: a precursor of catecholamines, an important

neurotransmitter used in the treatment of Parkinson’s
disease
• Shikonin: used as an anti-bacterial and anti-ulcer agent
• Anthraquinone: used for dyes and medicinal purposes

• Opiate alkaloids: particularly codeine and morphine

for medical purposes
• Berberine: an alkaloid with medicinal uses for cholera
and bacterial dysenterry
• Valepotriates: used as a sedative
• Ginsenosides: for medicinal purposes
103
104
105
4 – Biotransformation

• chemical conversion of an exogenously supplied

substance by living cell cultures

• example: addition of hydroquinone (inexpensive

precursor) into liquid culture medium containing
vinca cells results in efficient production of arbutin
(a skin depigmentation agent)

106
 Compounds commercialized from Plant Cell Culture Technology

Compound Plant Use

 Shikonin ` Lithospermum Pigment

erythrorhizon
 Ginseng Panax ginseng Health tonic
 Taxol Taxus baccta Anti-Cancer
Drug
 Vincristine & C. roseus Anti-Cancer
Vinblastin Drug
 Berberin Coptis japonica Anti-malarial
 Other Strategic Approaches for over
production of Plant Secondary Metabolites

1. Screening & Selection of a Variety (over producer)

2. Mass propagation by Tissue Culture techniques ( Cell suspension

cultures, Hairy root cultures, etc)

3. Modern Breeding Techniques ( Mutation breeding, Marker

Assisted Breeding, Metabolites finger printing etc)

4. Genetic Engineering Methods

5. Metabolic Engineering

6. Extraction & Purification of Secondary metabolites

108
What is Metabolic Engineering?

Metabolic engineering is referred to as

the directed improvement of cellular
properties through the modification of
specific biochemical reactions (or) the
introduction of new ones, with the use of
recombinant DNA technology
Major Approaches :
1. Pathway and co-factor engineering

2. Cofactor manipulations
Plant Metabolomics: An Indispensable System Biology Tool for
Plant Science
 Glucose
PEP
Pathway and co-factor engineering
Pyruvate
Glucose-6P
ATP

ADP
Fructose 1,6-diP

Glyceraldehyde-3 P Dihydroxyacetone-P
Metabolic
Pi + NAD+
NADH
Molecular Biology Techniques evolution
Glycerate 1,3-diP
ADP
ATP
PCR, RE digest, ligation, transformation,
CO2
PEP ADP knockout, etc.
CO2 PYK ATP LDH

OAA PYC Pyruvate Lactate

NADH
Citrate NADH NAD+
Insert/Knockout
Malate NAD+ H2
aceB PFL
Acetyl -CoA Formate CO2

Fumarate glyoxylate
aceA Isocitrate
Acetyl - CoA
PTA
Acetyl-P
ACK
Acetate Genes/pathways Potential
NADH
NAD+
aceA 2NADH
ADH
ADP ATP strains Controller
Controller
2NAD+

Succinate Temperature
Ethanol
Metabolic thermocouple

Gene Targets Engineering/systems pH

probe

Identified biotechnology Cycle Pumps

3 N HNO3
3 N NaOH
Shake
Gas outlet

Flask/Bioreactor 0.2m filters

Mass flow

Experiments controller

Fresh Medium Air

1L Glass Vessel
Waste tank

Modeling
Bioreactor NBS BioFlo110

Analysis

metabolomics proteomics transcriptomics

Figure 1: Metabolic engineering strategies to enhance the flux through a
pathway. The various strategies used to improve the flux from a cellular
intermediate to the desired product is shown. This includes enhancing pathways
leading to the formation of intermediates (shown in yellow) and pathways which
are rate limiting (B to C). Additionally branched chain pathways and feed back
controls need to be blocked.
Go to publication
https://
www.researchgate.net/figure/Metabolic-engineering-strategies-to-enhance-the-flux-th
 Succinic acid production

Simplified central anaerobic pathway in E. coli Resulting pathway design

Glucose
Glucose PEP
PEP
Pyruvate
Pyruvate
Biomass Glucose-6P
Glucose-6-P ATP
ATP
ADP ADP
Fructose 1,6-diP Fructose 1,6-diP
NAD+
NAD+
NADH
NADH
Glycerate-1,3-diP
ADP Glycerate 1,3-diP
CO2
ATP ADP
OAA Pepc
PEP ATP
ADP
ATP CO2 PEP
ADP
Ldh
Pyruvate Lactate CO2 Pyk ATP
2NADH NADH NAD+
OAA Pyc Pyruvate
NADH CO2
2NAD+ Formate CoA
NAD + Citrate Pfl H2
Malate
aceB
CO2 Acetyl-CoA Formate
Pta aceA Isocitrate
H2 Acetyl- CoA Acetyl-P Fumarate glyoxylate Acetyl-CoA
ADP
2NADH Ack NADH
Succinate ATP aceA
2NAD + NAD+
Glyoxylate pathway
Acetate Fermentative
Ethanol pathway Succinate (no NADH requirements)

NADH limitation
Max theoretical Yield Experimental yield= 1.6 mol/mol
1 mole/mole of glucose
 Example: lycopene production in E.coli
500 136.0% (2.4) 136.7% (2.4)
Lycopene production is limited
by NADPH availability

Specific lycopene production

400

( m g/g biomass)
Glucose 300
PEP
Pyr 2 NADP+ 2 NADPH CO2 200
G6P P5P
100

F6P
ATP GAP S7P 0
ADP pHL621 LB 2YT
Medium
GAP
NADP+ MG1655 (pDHC29/pK19-lyco) MBS100 (pHL621/pK19-lyco)
NAD+ E4P
GAPA GAPC F6P
NADH NADPH
ADP F6P GAP WT mutant
ATP
3-PG
NADH NAD+
PEP Pyr AcCoA Acetate
CO2 ADP ATP
ADP ATP

OAA ICT
NADP+
CO2
NADH NADPH CO2
NAD+
AKG
Mal ATP
NAD+

FADH2
ADP NADH Lycopene is a carotenoid. Lycopene useful in preventing or treating
CO2
FAD+
Suc
prostate cancer. Lycopene is a powerful antioxidant,
Cofactor engineering, a subset of metabolic engineering, is defined as
the manipulation of the use of cofactors in an organism’s metabolic
pathways.

In cofactor engineering, the concentrations of cofactors are changed

in order to maximize or minimize metabolic fluxes. This type of
engineering can be used to optimize the production of a metabolite
product or to increase the efficiency of a metabolic network.

The use of engineering single celled organisms to create lucrative

chemicals from cheap raw materials is growing, and cofactor engineering
can play a crucial role in maximizing production.

Cofactors are non-protein compounds that bind to proteins and are

required for the proteins normal catalytic functionality.

Cofactors can be considered “helper molecules” in biological activity, and

often affect the functionality of enzymes. Cofactors can be both organic and
inorganic compounds.
Plant metabolic engineering
Metabolic Engineering of the Terpenoid and Indole
Pathways in Catharanthus roseus hairy roots

Catharanthus roseus (Madagascar Periwinkle)

 Produce a wide range of secondary metabolites
 Ajmalicine and Serpentine – hypertension
 Vinblastine and Vincristine – anticancer drugs used to treat
lymphomas and leukemia

vinblastine vincristine
121
 Not all cell types produce the desired
metabolite

• Vinblastine is an anti-cancer (antineoplastic or

cytotoxic) chemotherapy drug. This chemical
is classified as a plant alkaloid

• Within a specific cultivar of Catharanthus

roseus, 62% of the clones produced the desired
metabolite
• whereas in another only 0.3% produced the
metabolite
Terpenoid Indole Alkaloid Pathway Pyruvate + G3P
DXS
Chorismate 1-Deoxy-D-Xylulose-5-Phosphate
AS Mevalonate
2-C-Methyl-D-erythitol-4-phosphate
Anthranilate
DMAPP IPP
Indole
Pathway GPP
Tryptophan
Geraniol Terpenoid
TDC enzyme G10H
Tryptamine 10-Hydroxygeraniol Pathway
STR enzyme Loganin
Secologanin
Strictosidine

Ajmalicine Catharanthine
Tabersonine

Serpentine Lochnericine Vindoline Vinblastine

Vincristine
123
Hörhammericine Refers to specific Genes
 Terpenoid Indole Alkaloid pathway

• Vinblastine and Vincristine and their semi-

synthetic derivatives are important anticancer
drugs used clinically. Low product yields have
propelled research to increase TIA biosynthesis.

• Overexpression of one indole gene (ASalpha)

and one terpenoid gene (DXS) showed the
importance of increasing the flux through each
pathway.

124
125
 Clone Generation with metabolically engineered
Terpenoid Indole Alkaloid pathway genes
Metabolic engineering + Hairy root culture = overproduction of Vinblastine

Plasmid Adapt to
Agrobacterium
Construction Liquid
in E. coli Media
(24 weeks)
Transgene

Ri

Infection

Sterile Grown (6 weeks) Selection Media

Plants (6 weeks)
(5 weeks) 126
Metabolic engineering + Hairy root culture = overproduction of Vinblastine

Hairy root culture

Produce useful
compounds that
exhibit desirable
biological functions

127
Metabolite production is frequently higher
in cell cultures
• Berberine production from Coptis japonica is
about 5% of dry weight after 5 years of root
growth, which equals 0.17 mg/g per week.

• Whereas in selected cell lines it can be 13.2% of

the dry weight in cell culture after 3 weeks,
which is about 44 mg/g/week or about 250
times higher.

Berberine is for Diabetes, dyslipidemias and cardiovascular diseases

 The economics of large-scale plant cell
culture favor only a few products at the
present time

This is because it usually takes 10 years of research

to produce a product. This requires that a product
sell for at least $400 per kg to make it economically
worthwhile.
 3. Phytochemistry

Ethnobotany is the study of the relationship between plants and people & their culture.

130
Andrographis paniculta : andrographolide 131
132
Standardized plant extraction protocols applied in GC-MS and LC-MS metabolic profiling
 4 Plant Metabolomics  

6 steps:
1- sampling (storage)
2- metabolite extraction (standardisation, reproducibility)
3- biochemical analysis (GC-MS, LC-MS, NMR)
4- data pre-processing (base line correction….)
5- data visualisation and mining (PCA, data bases)
6- integration of data (metabolic pathways, genome..)

134
 GC/MS and plant metabolomics
• Huge challenge

– plant genomes contain

20,000-50,000 genes,
– currently 50,000
metabolites identified
– number set to rise
~200,000

• Current plant
metabolomics uses
metabolic profiling
through GC-MS of plant
extracts.

Fiehn O et al. 2000 Nature Biotechnology, 18, 1157-1161.

137
 GC/MS and plant metabolomics
• Fiehn et al.

• GC-MS quantifies 326

distinct compounds in
Arabidopsis thaliana leaf
extracts
– chemical structure to half of
these.
– PCA separates 4 genotypes
– GC-TOF-MS now detected &
characterised ~1000
metabolites.
• Since used these data
bases to identify metabolic
cliques

Fiehn O et al. 2000 Nature Biotechnology, 18, 1157-1161.

138
Schematic diagram
indicating the work flow
from biological sample
to metabolite functional
assignment. Two main
routes are given, which
either follow a targeted
or a non-targeted
metabolome analysis.
NMR, nuclear
magnetic resonance;
MS, mass
spectrometry; PC,
principal component.
Use of metabolomics to analyse plants perturbed by genetic or
environmental changes.

Last, Daniel and Shachar-Hill Nature Reviews Molecular Cell Biology 8, 167–174 (February 2007) |
doi:10.1038/nrm2098
Mass spectrometry as a quantitative tool in plant
metabolomics
Quantification of metabolites from plant extracts

142
The most common modes of acquiring LC/MS data are : 

(1) Full scan acquisition resulting in the typical total ion current plot (TIC) 

(2) Selected Ion Monitoring (SIM) 

(3) Selected Reaction Monitoring (SRM) or multiple reaction monitoring (MRM). 

MRM and SRM are essentially the same experiment.

http://www.ionsource.com/tutorial/msquan/intro.htm
MS/MS analysis of Olanzapine. (A) UHPLC-MS chromatogram of standard (olanzapine)
and internal standard (olanzapine-d3) (B) MS/MS Spectrum of both standard
(olanzapine ) and internal standard (olanzapine-d3)
Spiking the standard and internal standard and extraction of metabolite from matrix
(Sera, Saliva, Water, cell extract and Urine)

https://www.ncbs.res.in/research-facilities/ms-metabolomics-capabilities-expertise
Approach for Production of Secondary metabolite

145
146
PHB: Polyhydroxybutyrate

147
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 A schematic representation of a plant metabolomics platform using a systems biology approach.

Oksman-Caldentey K et al. PNAS 2004;101:9949-9950

150
©2004 by National Academy of Sciences
 A schematic representation of a plant metabolomics
platform using a systems biology approach.

1. The platform is divided into multiple analytical

approaches aimed at increased coverage of the
metabolome.

2. Data processing methods enable us to track both

known and unknown metabolites, which can then be
integrated from across multiple platforms.

3. Multivariate analyses are used to find trends in data

and select most relevant unknown compounds for
further identification.
151
Non-Aqueous Fractionation (NAF): A Method Allowing the Application
of Metabolomics Technologies to Subcellular Compartments 

Stephan Kueger et al .,2012

Non-Aqueous Fractionation (NAF): A Method Allowing the Application
of Metabolomics Technologies to Subcellular Compartments 

The entire non-aqueous fractionation (NAF) procedure can be divided into

experimental- and computational-driven analyses.

The experimental part starts with the fast quenching of metabolism by snap-
freezing of plant material in liquid nitrogen.

The frozen leaf material is homogenized and freeze-dried in a pre-cooled

lyophilizer. The dried powder is resuspended in a non-aqueous mixture of
tetrachloroethylene (TCE) / heptane and sonicated on ice. The obtained
suspension is poured through a nylon sieve and loaded on a linear density
gradient. After 50-min centrifugation at 5000 g and 4°C equilibrium
distribution of the fractionated particles is reached. Fractions are taken from
the top of the gradient and separated into 10 1-ml aliquots.

For the analysis of marker activity and metabolite profiling, aliquots of each
fraction are dried and then extracted in the respective buffer or solvent by
strong vortexing or shaking in a pre-cooled Retsch mill. The computational
phase of the work includes the validation, classification, visualization, and
interpretation of the obtained data (.Klie et al., 2011).
In plant metabolome the subcellular localization of metabolites and their exchange
between subcellular compartments pose the real challenge
Protoplast Fraction
Non .Aq. Fraction
1) Protoplast Fraction 2) Non Aqueous Fraction
Linear density gradient centrifugation
Differential centrifugation
Differential centrifugation
 Metabolomics Facts - Technologies
Complexity –
• Plants contain (not all in each plant) an estimated >200,000 different compounds

Technical complexity –

• Polar (water-soluble) and non-polar (lipid-soluble) metabolites. Stereo-isoforms

may be difficult to distinguish, absolute amount may be low.

Technologies -
• NMR (nuclear magnetic resonance, MRI) – metabolite fingerprints for compounds
with non-zero magnetic moments (best: 1H, 13C, 19F, 31P). 1H-NMR can be a
problem > low “chemical shift dispersion” unless one uses powerful magnets.
Provides good fingerprint of most metabolites. Examples follow.

• FT-IR (Fourier-transform infrared spectroscopy) measures vibrations of functional

groups / polar bonds. IR-radiation interacts with compounds. Recorded is
absorption and its intensity. The spectrum is compared with a database.

• MS (mass spectrometry) combined with chromatography [LC or GC] most widely used,
particularly productive for LMW compounds (peptides as well). In GC/MS the
sample must become volatile, which requires derivatization. In LC/MS, without
derivatization, compound groups must be “selected” (size, chemical properties)
by the choice of columns or isolation procedures.

Vicki Malone: Plant metabolomics. BioTeach J., Fall2004, pp. 92-99 [www.bioteach.ubc.ca]
 Technologies that depend on the determination of mass,
often combined with chromatography
• GC/MS – Gas Chromatography + Mass Spectrometry
relatively low cost
high separation efficiency
separation of several hundred compounds per run
compounds must be derivatized to become volatile
derivatization (may) equal disturbance, increased variance

• LC/MS – Liquid Chromatography + Mass Spectrometry

separation even better than GC/MS when use in tandem
allows for enrichment of classes of compounds
selection of compound class from column used or extraction
no derivatization necessary

Both rely on the comparison of unknowns with reference substances

Both are ideal for sugars, organic acids, sugar alcohols, amino acids & fatty acids
- i. e., molecular masses of up to several hundred.
(hexoses ~ 180; Glu1P – 336; oleic – 282; verbascose - 828)
Both can be used for secondary product analysis, but for defined compound classes
LC/MS is the preferred tool.
 METABOLITE DATABASES
The Scripps Research Institute maintains a metabolite mass spectral database.
The Human Metabolite Database acts as an electronic repository for identification of small molecule metabolites. 
The Human Natural Products Database information on formulas, masses, descriptions of endogenous metabolites.
The Golm Metabolome Database public access to mass spectra libraries, metabolite profiling experiments and other
information related to metabolomics.
The Spectral Database for Organic Compounds SDBS access to of spectra of organic compounds (NMR, MS, IR).

METABOLIC PATHWAYS
Sigma Aldrich clickable metabolic pathway map.
The Nicholson minimaps an overview of major individual metabolic pathways.
MetaCyc a database of nonredundant, experimentally elucidated metabolic pathways (<300 organisms). 
KEGG pathways, molecular interaction networks, metabolic & regulatory pathways, molecular complexes.
ExPASy biochemical and metabolic pathways.

INFORMATION ON METABOLITES AND BIOFLUIDS

Frontiers in Bioscience information on properties of metabolites, reference values in biological fluids.
ChemFinder database of chemical structures, physical properties, and hyperlinks.
Lipidbank for Web  a database system offering information on lipids.
The LIPID LIBRARY a series of web documents serving lipid analysts. 
The LIPID MAPS seeks to identify and measure the amounts of all lipids within a cell.
Lipids Online, online resource on atherosclerosis, dyslipidemia and lipid management.
LIPID DATA BASE  a convenient gateway to the world of  lipids and related materials.
LIPIDOMICS EXPERT PLATFORM an established by the European Lipidomics Initiative
SOCIETIES, GROUPS, COMPANIES
The Metabolomics Society, new website partly under construction, may become a useful resource.
The Fiehn metabolomics lab at UCDavis.
The Bioanalytical Sciences Group at the University of Manchester.
The Analytical Biosciences Group at Leiden University.
Check the Hannelore Daniel an extensive introduction to nutritional metabolomics.
Companies: Lipomics -  Metabolon - Metabometrix - Metanomics - Phenomenome -Surromed - Chenomx.

http://www.nugo.org/metabolomics/13187