Science of Foods for Health

Biosynthesis

Dr. Hisashi KiowaTexas 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

• Not essential for plants • Complicated regulation of the biosynthesis

– Can be both constitutive and inducible– Tissue specificity

• Bioengineering– Complex chemical structure (difficult though not imp

ossible to chemically synthesize)– Beneficial for human use

Primary metabolitesSecondary metabolites

Nature may not be the best source of Natural Products

Accumulation of natural products can take years

Biosynthesis in rootsProducts are transferredto leaves to be stored

Berberine crystalsSelected alkaloid-producing cells

5-6 years5% Dry Weight

3 weeks8-10% Dry Weight

Copis plants

In vitro system to produce Natural Products

Tissue culture system has been used to study secondary metabolite synthesis

Induction of shikonin biosynthesisPigments 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 PhenolicsLigninsFlavonoids

• 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

Terpenoids

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?

Diversity of terpenoids first arises from diverse way of joining multiple isopentanes (C5 unit).

isopentenyl PP (IPP, C5)

OPP

Architecture of terpenoids

Hemiterpenes: isoprene

Monoterpenes: volatile essences of flowersessential oil

Sesqiterpenes: essential oilphytoalexins

Diterpenes: phytol (chlorophyll side chain)gibberellin, resin, taxol

Triterpenes: brassinosteroidsmembrane sterols

Tetraterpenes: carotenoid pigments

Sesterterpenes:

Acyclic and Cyclic Natural Terpenes:

• 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

IPP synthesised by 2 different pathways

MVA pathway MEP pathway Archaebacteria, Most eubacteria fungi and animals(cytosol and ER) (plastid)

MonoterpenesDiterpenesCytokininsAbscisic acidGibberellinsCarotenoidsPhytol PlastoquinoneTocopherols

BrassinosteroidsPhytosterolsPhytoalexinsPrenyl groupsUbiquinone (mitos)

Non-mevalonate pathway functions in plastids

fosmidomycin

carotenoid

chlorophyll DXS

DXR

CDP-ME synthase

CDP-ME kinase

ME-cPP synthase

HMBPP synthase

IDP/DMAPP synthase

thiaminepyridoxol

Formation of parent carbon skeltons

Sidechains ofPQ & UQ

Polyterpenes(C30,000)

Rubber

C45-50

PT

PT PT

PhytolGibberellins

Abscisic acid

Carotenoids

SterolsTriterpenes

Monoterpenes

Cyclase

SesquiterpenesCyclase

DiterpenesCyclase

Isopentenyl pyrophosphate (IPP)

Geranyl PP (C10; GPP)

Farnesyl PP(C15; FPP)

Geranylgeranyl PP(C20; GGPP)

SqualeneC30

PhytoeneC40

Coupling

Coupl-ing

CytokininsSubstituted tRNAbases

Prenyln

Terpene synthase/cyclase

Monoterpenes

Cyclase

SesquiterpenesCyclase

DiterpenesCyclase

Isopentenyl pyrophosphate (IPP)

Geranyl PP (C10; GPP)

Farnesyl PP(C15; FPP)

Geranylgeranyl PP(C20; GGPP)

Limonene synthase as a prototypical monoterpene synthase

Oil grand EST project yielded identification of several biosynthetic enzymes

Plant Physiology, 120, 879

next slide for mechanism

Plant Physiol 122, 215

Modification of limonene to menthol P450

(spearmint)

Dehydrogenease

ReductaseIsomerase

Reductase

Reductase

menthofuran

synthase

P450

Dehydrogenease

(peppermint)

• 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 Plant Oil weight Menthol(%) Menthofuran (mg/g FW) Oil weight Menthol(%) Menthofuran (mg/g FW)

WTWT 1.8 1.8 6.9 6.9 16.816.8DXR6DXR6 2.62.6 12.7 12.7 15.715.7MFS1MFS1 1.7 1.7 23.223.2 2.5 2.5

PNAS 98, 8915

Manipulating Peppermint Oil Synthesis

Ref: Alkaloid biosynthesis in plants Ammu. Rev. Plant Physiol. Plant Mol. Biol. (2001), 52, 29-66

AlkaloidsDefinition• Pharmacologically active, nitrogen-containing basic c

ompounds 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

Four classes of alkaloidsTerpenoid-indole alkaloid (Trp)

Benzylisoquinoline alkaloid (Tyr)

Tropane alkaloid (SAM)

Purine alkaloid

Major alkaloid productsName Class FunctionCaffeine Purine

Nicotine -Atropin Tropane AnticholinergicHyoscyamineScoporamine SedativeCocain Topical anaesthetic

Berberine BenzylisoquinolineMorphine AnalgesticCodeinHeroin (semisynthetic)

Quinine Indole AntimalarialVinblastin AntineoplasticCamptothecin Anticancer

Purine alkaloids

Ripening beans of Coffea arabica

Ashihara and Crozier (2001)

coffeetea

coffeetea

chocolateKola nut

metabolite of caffeine in animals

Chinese tea minor alkaloids in coffee

RNA interference:Producing decaffeinated coffee plants

Phenylpropanoids and phenolics• Aromatic metabolites that possess one or more “ac

idic”-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 anth

ocyanin, anthocyanidins, isoflavonoids, etc.– Condensed tannins– Coumarins: defense chemicals, toxic to mamm

als.– Stilbenes: antifungal property, target of biotec

hnology.– Suberin: Structures of polyaromatic, hydroph

obic layer alternated with phenolic hydrophillic layer

Biosynthesis of phenylpropanoid

Phenylalanine Cinnamic acidp-Coumaric acid

Flavonoid

Coumarins

Stilbens

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 t

he lignans are connected by 8-8’ bonds, but other linkages exists.

88’

88’

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

Isoflavonoids3x malonyl CoA

CHS, CHR

DFRANS3GT

DFRANS3GT

DFRANS3GT

-Glc -Glc -Glc

Condensed tannin

Flavones

FlavonolsOH |

OH |

OH

Dihydroquercetin Dihydrokaempferol Dihydromyricetin

F3‘H F3‘5’H

Cyanidin DelphinidinPelargonidin

F3H

a-KGP450

(dihydroflavonol)

DFRANS3GT

DFRANS3GT

Dihydroquercetin Dihydrokaempferol Dihydromyricetin

F3‘H F3‘5’H

Delphinidin

OH |

OH |

OH

Cyanidin

Genetic engineering of Blue rose

-Glc-Glc

Discussion topics

• Controversy about genetically modifying the biochemical

profile of plants for consumption