1. Dr. Hisashi Kiowa Texas A&M University
Biosynthesis
Dr. Hisashi Kiowa Texas A&M University

2. 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

3. Natural Products are often called as 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

4. Nature may not be the best source of Natural Products
Accumulation of natural products can take years

5. In vitro system to produce Natural Products
Copis plants
Selected alkaloid-
producing cells
Biosynthesis in roots
Products are transferred
to leaves to be stored
Berberine crystals
5-6 years
5% Dry Weight
3 weeks
8-10% Dry Weight

6. 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

7. Topics for natural products
Terpenoid (isoprenoid)
Alkaloids (N-containing basic compound)
Phenylpropanoids and Phenolics
Lignins
Flavonoids

8. 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

9. 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

10. 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).

11. 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

12. Acyclic and Cyclic Natural Terpenes:

13. IPP synthesised by 2 different pathways
MVA pathway MEP pathway
Archaebacteria, Most eubacteria
fungi and animals
(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

14. Non-mevalonate pathway functions in plastids
chlorophyll
DXS
thiamine
pyridoxol
DXR
fosmidomycin
CDP-ME synthase
carotenoid
CDP-ME kinase
ME-cPP synthase
HMBPP synthase
IDP/DMAPP synthase

15. Formation of parent carbon skeltons
Sidechains of
PQ & UQ
Polyterpenes
(C30,000)
Rubber
C45-50 PT
Phytol
Gibberellins
Abscisic acid
Carotenoids
Sterols
Triterpenes
Monoterpenes
Cyclase
Sesquiterpenes
Diterpenes
Isopentenyl pyrophosphate (IPP)
Geranyl PP
(C10; GPP)
Farnesyl PP
(C15; FPP)
Geranylgeranyl PP
(C20; GGPP)
Squalene
C30
Phytoene
C40
Coupling
Coupl-
ing
Cytokinins
Substituted tRNA
bases
Prenyln

16. Terpene synthase/cyclase
Monoterpenes
Cyclase
Sesquiterpenes
Diterpenes
Isopentenyl pyrophosphate (IPP)
Geranyl PP
(C10; GPP)
Farnesyl PP
(C15; FPP)
Geranylgeranyl PP
(C20; GGPP)

17. 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

18. Modification of limonene to menthol
(peppermint)
Modification of limonene to menthol
Dehydrogenease
P450
(spearmint)
P450
Reductase
menthofuran
synthase
Isomerase
Dehydrogenease
Reductase
Reductase
Plant Physiol 122, 215

19. Manipulating Peppermint Oil Synthesis
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
Plant Oil weight Menthol(%) Menthofuran (mg/g FW) WT
DXR
MFS
PNAS 98, 8915

20. 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

21. Four classes of alkaloids
Terpenoid-indole alkaloid (Trp)
Tropane alkaloid (SAM)
Benzylisoquinoline alkaloid (Tyr)
Purine alkaloid

22. Major alkaloid products
Name Class Function
Caffeine Purine
Nicotine -
Atropin Tropane Anticholinergic
Hyoscyamine
Scoporamine Sedative
Cocain Topical anaesthetic
Berberine Benzylisoquinoline
Morphine Analgestic
Codein
Heroin (semisynthetic)
Quinine Indole Antimalarial
Vinblastin Antineoplastic
Camptothecin Anticancer

23. Purine alkaloids Ripening beans of Coffea arabica coffee
tea
chocolate
Kola nut
metabolite of caffeine in animals
Chinese tea
coffee
tea
minor alkaloids in coffee
Ashihara and Crozier (2001)

24. RNA interference:
Producing decaffeinated coffee plants

25. 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

26. Biosynthesis of phenylpropanoid
Flavonoid
Phenylalanine
Cinnamic acid
p-Coumaric acid
Monolignol
Stilbens
Coumarins

27. Transgenic poplars for better pulp productionWT
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

28. 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’

29. Lignans with different linkages

30. Lignans are absorbed by the digestive system and can protect against breast and prostate cancer

31. Some plant species uses stylbene synthase to produce defense compounds against pathogens

32. a-KG P450 Cyanidin Pelargonidin Delphinidin 3x malonyl CoA
Isoflavonoids
CHS, CHR
Flavones
a-KG
P450
(dihydroflavonol)
Flavonols OH | OH |
F3H
F3‘H
F3‘5’H OH
Dihydroquercetin
Dihydrokaempferol
Dihydromyricetin
DFR
ANS
3GT
DFR
ANS
3GT
DFR
ANS
3GT
Condensed tannin
-Glc
-Glc
-Glc
Cyanidin
Pelargonidin
Delphinidin

33. Genetic engineering of Blue roseOH | OH |
F3‘H
F3‘5’H OH
Dihydroquercetin
Dihydrokaempferol
Dihydromyricetin
DFR
ANS
3GT
DFR
ANS
3GT
-Glc
-Glc
Cyanidin
Delphinidin

34. Discussion topics
Controversy about genetically modifying the biochemical profile of plants for consumption