Transcript Protein Glycosylation - Texas A&M University

Science of Foods for Health
Biosynthesis
Dr. Hisashi Kiowa
Texas A&M University
Biosynthesis and Engineering of Plant
Natural Products
• What are natural products?
• How natural products are produced in plants
• Bioactivity of natural products
• Engineering natural products biosynthesis--case studies
Natural Products are often called as secondary
metabolites
Secondary metabolites
Primary metabolites
• Not essential for plants
• Complicated regulation of the biosynthesis
– Can be both constitutive and inducible
– Tissue specificity
• Bioengineering
– Complex chemical structure (difficult though not
impossible to chemically synthesize)
– Beneficial for human use
Nature may not be the best source of Natural Products
Accumulation of natural products can take years
In vitro system to produce Natural
Products
Copis plants
Biosynthesis in roots
Products are transferred
to leaves to be stored
Berberine crystals
5-6 years
5% Dry Weight
Selected alkaloidproducing cells
3 weeks
8-10% Dry Weight
Tissue culture system has been used to study
secondary metabolite synthesis
Induction of shikonin biosynthesis
Pigments has been used for
cosmetics industry
Red beet hairy root culture
Topics for natural products
1. Terpenoid (isoprenoid)
2. Alkaloids (N-containing basic compound)
3. Phenylpropanoids and Phenolics
Lignins
Flavonoids
Terpenoids
• Lipid molecules
– estimated 22,000 different types
– ~ 400 in tobacco alone
– MOST DIVERSE GROUP OF PLANT CHEMICALS
• Essential compounds
– membrane sterols, carotenoids, groups on chlorophyll,
heme a, UQ & PQ, cytokinins, abscisic acid gibberellins
– function in photosynthesis, respiration, growth &
development, membrane architecture
• Secondary metabolites (natural products)
– volatiles (essential oils) - major plant odours
– tree resins & polyterpenes - insect- & rot-proof
– phytoalexins
– important in response to environment
Reasons for the greater diversity in plan
terpenoids
• Mode of connections of C5 units
• Length of connected C5 units
• Diverse cyclization reaction
•
Various secondary modifications
What is the C5 precursor of terpenoids?
OPP
isopentenyl PP (IPP, C5)
Diversity of terpenoids first arises from diverse way of
joining multiple isopentanes (C5 unit).
Architecture of terpenoids
Hemiterpenes: isoprene
Monoterpenes: volatile essences of flowers
essential oil
Sesqiterpenes: essential oil
phytoalexins
Diterpenes:
phytol (chlorophyll side chain)
gibberellin, resin, taxol
Sesterterpenes:
Triterpenes:
brassinosteroids
membrane sterols
Tetraterpenes: carotenoid pigments
Acyclic and Cyclic Natural Terpenes:
IPP synthesised by 2 different pathways
MVA pathway
MEP pathway
Archaebacteria,
fungi and animals
Most eubacteria
(cytosol and ER)
(plastid)
Monoterpenes
Diterpenes
Cytokinins
Abscisic acid
Gibberellins
Carotenoids
Phytol
Plastoquinone
Tocopherols
Brassinosteroids
Phytosterols
Phytoalexins
Prenyl groups
Ubiquinone (mitos)
• Most organisms only use one of the two pathways for the biosynthesis of
their precursors.
• Plants use both the MEP pathway and the MVA pathway for isoprenoid
biosynthesis, although they are localized in different compartments
Non-mevalonate pathway functions in plastids
chlorophyll
thiamine
pyridoxol
DXS
DXR
CDP-ME
synthase
carotenoid
CDP-ME
kinase
ME-cPP
synthase
HMBPP
synthase
IDP/DMAPP
synthase
fosmidomycin
Formation of parent carbon skeltons
Cytokinins
Substituted tRNA
bases
Prenyln Isopentenyl pyrophosphate (IPP)
Geranyl PP
(C10; GPP)
Sterols
Triterpenes
Coupling
Squalene
Farnesyl PP
C30
(C15; FPP)
Phytol
Gibberellins
Phytoene
C40
Cyclase
Monoterpenes
Cyclase
Sesquiterpenes
Geranylgeranyl PP Cyclase
Diterpenes
(C20; GGPP)
Coupling
PT
Carotenoids
C45-50
PT
Abscisic acid
Sidechains of
PQ & UQ
Polyterpenes
(C30,000)
PT
Rubber
Terpene
synthase/cyclase
Isopentenyl pyrophosphate (IPP)
Geranyl PP
(C10; GPP)
Farnesyl PP
(C15; FPP)
Cyclase
Monoterpenes
Cyclase
Sesquiterpenes
Geranylgeranyl PP Cyclase
Diterpenes
(C20; GGPP)
Limonene synthase as a prototypical monoterpene synthase
next slide for mechanism
Oil grand EST project yielded
identification of several
biosynthetic enzymes
Plant Physiology, 120, 879
Modification of
limonene to
menthol
(spearmint)
(peppermint)
P450
Dehydrogenease
P450
menthofuran Isomerase
synthase
Dehydrogenease
Reductase
Reductase
Reductase
Plant Physiol 122, 215
Manipulating Peppermint Oil Synthesis
•
•
Plant
WT
DXR6
MFS1
Introduction of sense DXR increases the flux of MEP pathway
– Increase of up to 50% essential oil
Antisense menthofuran synthase decreases by product of menthol
synthesis
– Decrease of 50% menthanofuran
Oil weight
1.8
2.6
1.7
Menthol(%)
Menthofuran (mg/g FW)
6.9
12.7
23.2
16.8
15.7
2.5
PNAS 98, 8915
Alkaloids
Definition
• Pharmacologically active, nitrogen-containing basic
compounds of plant origin
• Found in about 20 % of plant species
• Many of them serves as a chemical defense of plants
against herbivores
– Many alkaloids are toxic to insects (nicotine, caffeine, etc)
– Herbivory stimulates biosynthesis
• Ingredients of medicinal plants
–
–
–
–
Pharmaceuticals
Narcotics
Stimulants
Poisons
Ref: Alkaloid biosynthesis in plants Ammu. Rev. Plant
Physiol. Plant Mol. Biol. (2001), 52, 29-66
Four classes of alkaloids
Terpenoid-indole alkaloid (Trp)
Benzylisoquinoline alkaloid (Tyr)
Tropane alkaloid (SAM)
Purine alkaloid
Major alkaloid products
Name
Caffeine
Class
Purine
Nicotine
Atropin
Hyoscyamine
Scoporamine
Cocain
Tropane
Anticholinergic
Sedative
Topical anaesthetic
Berberine
Benzylisoquinoline
Morphine
Codein
Heroin (semisynthetic)
Quinine
Vinblastin
Camptothecin
Function
Indole
Analgestic
Antimalarial
Antineoplastic
Anticancer
Purine alkaloids
Ripening beans of Coffea arabica
coffee
tea
coffee
tea
chocolate
Kola nut
metabolite
of caffeine
in animals
Chinese
tea
minor alkaloids in coffee
Ashihara and Crozier (2001)
RNA interference:
Producing decaffeinated
coffee plants
Phenylpropanoids and phenolics
•
Aromatic metabolites that possess one or more
“acidic”-OH groups attachd to the phenyl ring.
•
Major classes
– Lignin/Lignans
• Polymeric (lignins) structure that
reinforce cell wall
• Unique to land plants and not found in
aquatic plants
– Hydrolizable tannins: polyphenols
– Flavonoids:most diverse group, includes
anthocyanin, anthocyanidins, isoflavonoids,
etc.
– Condensed tannins
– Coumarins: defense chemicals, toxic to
mammals.
– Stilbenes: antifungal property, target of
biotechnology.
– Suberin: Structures of polyaromatic,
hydrophobic layer alternated with phenolic
hydrophillic layer
Biosynthesis of phenylpropanoid
Flavonoid
Phenylalanine Cinnamic acid
p-Coumaric acid
Stilbens
Coumarins
Monolignol
Transgenic poplars for better pulp
production
WT
anti-CAD
• 4-year-long field trial is conducted using UK and France sites with COMT and CAD
antisense plants
• UK site was terminated early due to activist‘ vandalism
Lignans
• Lignan is a group of dimeric phenylpropanoid (C6C3). Most of
the lignans are connected by 8-8’ bonds, but other linkages
exists.
8
8’
8’ 8
Lignans with different linkages
Lignans are absorbed by the digestive system and can protect
against breast and prostate cancer
Some plant species uses stylbene synthase to produce defense
compounds against pathogens
3x malonyl CoA
Isoflavonoids
CHS, CHR
Flavones
a-KG
P450
(dihydroflavonol)
OH
|
OH
|
F3H
F3‘H
Dihydroquercetin
DFR
ANS
3GT
Cyanidin
F3‘5’H
Dihydrokaempferol
DFR
ANS
3GT
-Glc
Flavonols
-Glc
Pelargonidin
OH
Dihydromyricetin
DFR
ANS
3GT
Condensed
tannin
-Glc
Delphinidin
Genetic engineering of Blue rose
OH
|
OH
|
F3‘H
Dihydroquercetin
DFR
ANS
3GT
Dihydrokaempferol
OH
Dihydromyricetin
DFR
ANS
3GT
-Glc
Cyanidin
F3‘5’H
-Glc
Delphinidin
Discussion topics
• Controversy about genetically modifying the biochemical
profile of plants for consumption