Modern Ag Biotech Applications

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6 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

232 εμφανίσεις

Martina Newell
-
McGloughlin

Director, UC Systemwide Biotech Research

and Education Program



Agronomic Traits


Biotic Stress


Insect Resistance


Bt, cystatin


Disease Resistance


Viral
-

coat protein protection (Papaya ringspot virus)


Bacterial, Fungal, Nematode (Rice blight, rice blast)


Weed
-

herbicide tolerance (Striga, orobanchia)


ABiotic Stress


Drought, Cold, Heat


Poor soil


Salinity


tomatoes with transport protein


Aluminum
-
Citric acid



Yield


Nitrogen Assimilation


nodulation by rhizobia, GDH metab eng


Starch Biosynthesis, O
2
Assimilation, photosynthesis/Rubisco


Quality Traits


Processing


Post harvest loss reduction


Reproduction: sex barriers, male sterility, seedless fruit


Nutrients (Nutraceuticals)


Macro: Protein (Cassava
-
ASP), Carbohydrates, Fats, Fiber


Micro: vitamins, minerals, phytochemicals


Anti
-
nutrients: Phytase, Toxin removal


Novel Crop Products


Proteins: nutraceuticals, therapeutics, vaccines


Renewable resources:
Biomass conversion, feedstocks, biofuels,
phytoremediation



Crop Biotechnology

Source: ISAAA


Biotech Crops 2008: 125 million hectares (310 million acres up 9.4%) X74




25 countries (15 LDC) 12% increase over 2007, 13.3 M farmers (12 M 2007)


90% resource
-
poor LDC farmers (12.3 M
-

11.0 M 2007) most Bt cotton


New: Egypt, Burkina Faso, Paraguay , Uruguay.


India Bt cotton up to 7.4 M Ha.



$44 billion 1996 to 2007, 44% yield gains, 56%
reduction costs (including a 359,000 tonne a.i. in
pesticides); gains of 141 million tons, would have
required 43 million additional hectares


Environmental pesticide footprint down by 15.4
%. GM reduction in 286 million kg of CO2
emissions equivalent to removing 6 M cars from
the roads (Brookes 2008)


HT
-

increase in no
-

till: reduction in erosion,
soils much healthier, organic matter, less soil
compaction, fuel use down by 20 gals/acre
(Fawcett & Towery 2005 )


CP papaya saved Hawaii papaya industry (and
helped organic farmers!) may be the outcome for
plum pox

C5 PTGS insurance against typhoid
Mary in nurseries


Snap shot


No natural resistance anywhere
so could never breed resistance



Removes viral reservoir thus
protects all growers


Field trial of transgenic 'UH
Rainbow' and 'UH SunUp' was
established in Puna in 1995.
Gonsalves, Mansardt, Ferreira







Plums highly resistant to PPV
-

System is totally resistant as
virus is not harbored
unknowingly


Tolerant non
biotech trees can harbor virus


Farmers Choice

Papaya ringspot virus



China BT rice GM used pesticides fewer than 1/season;
conventional rice used pesticides 3.7 times ( Rozell, 2005)
Pesticides cost applied to the conventional rice was 8 to 10
times as high as GM. 80% reduction in pesticide use.
Significant decrease in adverse health effects


Lives saved


India Bt cotton decrease insecticide use 70% and increased
productivity in 58% (737 kg/Ha)



Organisms in “Bt crops” fields fared better in trials than
those with insecticides Monarch butterflies increase



Engineering Modified Bt Toxins to Counter Insect
Resistance
-

pink bollworms cadherin mutation bypass
with modifed Cry protein Science, Nov. 2007



BT corn 90% reduction in mycotoxin fungal fumonisins
-

total US benefit estimated at $23m annually. (Wu, 2006)



Blight
-
resistant potato (BASF
-
Rpi
-
blb1 and Rpi
-
blb2
NBS
-
LRR)
-
UI study concluded for the major potato
-
producing regions of the world would be $4.3 billion.

Snapshot


Number

2

in

GM

production
.



17
%

of

the

global

area

of

GM

plants
.




In

2007
,

98
%

of

soybean

in

Argentina

was

GM
.



Yield

have

reached

over

6

tonns

per

hectare


In

1994
-
95

production

costs

were

182

dolars/Ha
;

in

2007

are

117

dolars/Ha



In

1994
-
95

farmers

spent

78

dolars/Ha

in

herbicides
;

today

they

spend

37

dolars/Ha

and

insecticide

use

has

decrease

90
%
.



Economical

benefits

of

GM

soybean

USD
$

20

billion

+

1

million

jobs



Problems,

yes
.

Due

to

the

economic

success

of

GM

soybean

and

maize,

President

Cristina

Fern
á
dez

de

Kirchne

created

a

new

tax

on

Gm

soybean

exports

that

producers

oppose

The Argentinean Case: GM crops an engine of
economical development


Between

2001
-
05

the

chinese

government

spent

15

billion

US

dólars

on

AgBiotech

projects
;

2006
-
10

a

400
%

increase

has

been

projected


National

Biotechnology

program

work

on

the

development

of

over

130

varieties

of

GM

rice

and

55

varieties

of

GM

cotton



10

GM

products

have

been

aproved

for

human

consumption

(rice,

maize,

soybean

and

potato)
.

Bt

and

disease

resistent

rice

is

commercially

planted

in

China
.


GM

cotton

was

used

by

7
.
1

million

small

farmers

in

3
.
8

million

hectares

in

2007

with

an

economical

benefit

of

USD
$

817

million

in

2006
.

China is awakening


Spain:

farmer Jose Victor Nogues”
After 5 years of GM crops in the area,
most people can appreciate the huge
benefits and lack of negative effects
-
Introducing GM maize was definitely
the way forward









France:

Thierry de l'Escaille,
European Landowners' Organization,
-

“wide
-
scale adoption of these three
biotech crops in Europe could
significantly increase annual
production, improve farmer income
by more than 1 billion Euros
(US$1.18 billion) and reduce spraying
practices. With results like these, it's
easy to understand why farmers want
access to this new technology," said
l'Escaille



Bt corn farmers earn about $85
more per acre, ISAAA (2006), while at
the same time producing a healthier
feed that is better for the environment.

Romania:

Buzdugan farmers reported
price premiums of up to 10 percent for
biotech soybeans due to fewer weed
impurities. Average price gain 2%.
Production gain 29
-
33.5K M tonnes
(16
-
19% )
Earnings increase 35
million and 62.4 million euros (2003).

I can tell you that soybean farmers in
Romania are very interested in biotech
seeds," “Although the seeds are 10 to
15 percent more expensive, the income
gains make the extra cost more than
worthwhile”




Transgenic breeding lines preferred because resistance can be
introduced into commercial lines with greater speed. The
BASF GM potatoes involves the use of two broad spectrum
resistance genes, Rpi
-
blb1 and Rpi
-
blb2. These two genes have
a structure associated with regulatory genes called nucleotide
binding site
-
leucine rich repeat (NBS
-
LRR) class of regulatory
proteins. Many disease resistance genes code for proteins of
that class. Numerous plant NBS
-
LRR genes are present in the
typical plant genome, each protein is specific for a particular
pathogen signaling a defense response, frequently a localized
plant cell death called hypersensitive response. The C
terminus of the protein containing LRR recognizes a ligand
feature of a pathogen activating the NBS signaling module to
initiate the defense response. The blight fungus suppresses the
potato defense genes in sensitive plants but is thwarted by
successful defense genes.


Blight
-
resistant potato (BASF
-
Rpi
-
blb1 and Rpi
-
blb2 NBS
-
LRR)
-
UI study concluded for the major potato
-
producing
regions of the world would be $4.3 billion.

Blight
-
resistant potato

11

“Resistance” Genes natural and otherwise


Rootknot nematodes R in tomato (
Mi
) and (aphids). Alternate to
fumigation (Williamson UC Davis)




Xylella fastidiosa Diffusible signal factor (Dsf) for disrupting Xf
colonization, Inhibition of Xf polygalacturonase (PG). Targeting
other Xf proteins required for virulence. protein/peptide
-
based
inhibitors of pilins and adhesins


alternate to malathion vector
control



Xa21 rice R gene confers resistance to several Xoo. Defense
response triggered by Xo molecule, AvrXa21. Transgenic more
resistant due to copy number (Ronald)



Apple Fireblight (E. amylovora)
-
antibiotic sprays, cecropin lytic
peptide analog worked well in field trials (Norelli)



Apple scab (V. inaequalis) Multiple applications of fungicides used
exclusively to control this disease. 'McIntosh' trees expressing
either the endo
-

or exochitinase gene or both increased resistance
to apple scab



Use of apoptosis inhibition to protect plants from mycotoxin
damage (Gilchrist, UC Davis)



Sclerotinia
-
resist sunflower oxalate oxidase Pioneer wheat



Zinc Fingers Dow/Sangamo


Natural Disease Resistance Genes Have Been Cloned




Xa21
gene has



been cloned from



an African rice



variety and




introduced into



modern cultivars to



confer resistance



to rice leaf blight.




Pam Ronald, Plant




Pathology, UC Davis

Use of apoptosis inhibition to protect plants from mycotoxin damage

David Gilchrist, Plant pathology, UC Davis

David Gilchrist, Plant pathology, UC Davis

.


Rice staple food for 2.4 billion people


Fungal diseases destroy 50 million metric
tons of rice per year; varieties being
developed resistant to fungi
-

proteins with
anti
-
fungal properties.


Insects cause a 26 million tons loss of rice
per year; insecticidal proteins
environmentally friendly control.


Viral diseases devastate 10 million tons of
rice per year; Tungro virus genome
transgenes defense systems. Cassava
Mosaic Virus similar protection system as
papaya working in Kenya


Bacterial diseases cause comparable losses
-

transgenes such as cecropin lytic peptide
basis for inbuilt resistance.


Striga (Scrophulariaceae) is a



genus of obligate root
-
parasitic



flowering plants.



All of the cultivated food
-
crop


cereals in Africa are parasitized


by one or more Striga spp.



Striga spp. in the savanna


regions alone account for


$7 billion and are


detrimental to the lives of over


100 million African people.

STRIGA

Host plants release factors required by parasitic plants Striga causes
massive losses to crops in Africa:control strategy to inactivate host
recognition factors
-

John Yoder, UC Davis,

Host plants release factors required by parasitic plants:

control strategy to inactivate host recognition factors

DMBQ

sorgoleone

Host plants

Parasitic plants

maize

cowpea

Arabidopsis

Striga

Orobanche

Triphysaria

Seed germination

0 h

12 h

24 h

Haustorium development

+ Gene E

Control

+ Gene D

Control






Abiotic Stress:






Drought, ColdHeat, Salinity

Drought Stressed Rice


Abiotic

stress limiting factor for crops
reaching genetic potential


Improved water conservation
-
Fewer crop
losses
-
Higher yields on all acres through
improved water utilization
-
Expand in
drylands

-

Nuclear Factor Y B subunit


Crops limited by salinity on 40 % world's
irrigated land (25 % US)


Cold: Engineering with COR15a
Tf
, role
in freezing tolerance.


Plants engineered with
Choline

oxidase

(
codA
) soil tolerated saline and cold


Homeodomain
-
leucine

zipper (HD
-
Zip)
transcription factors respond to H
2
O &
osmotic stress,
exongenous

abscisic

acid


Transport protein. Grow and fruit even in
irrigation water that is > 50X saltier than
normal. > 1/3 salty as seawater.
Blumwald

and Zhang)




-

Improve Nitrogen Assimilation


-

Increase Sucrose hydrolysis,



Starch biosynthesis


-

Increase O
2

availability


-

Modify photosynthesis

Yield Gene

Control

Increased Yields

This short
-
day sorghum plant was
used to map the Ma
-
1 gene (genes
which modify photoperiodic
behavior and thus maturity). This
gene which works in other cereals
would offer particular benefits to
biomass and forage crops in which
flowering is undesirable

BioFuels


LDC 30 % global energy. Growth driven by population and economic . Of
the world's 47 poorest countries, 38 are net oil importers, and 25 import all
of their oil, consuming much of their national income to pay for it.


The challenge: 5
-
10 times more efficient
-

2001 $5/gal
-
2005 18c/gal


Biomass Conversion:

Organic polymeric material, lignin, starches celluloses
bioconverted

ethanol;
hemicellulose

hydrolyzed to sugars,
xylose

, glucose .


Modify Plants and algae to improve enzymatic conversion.


Modify enzymes to improve conversion and fermentation


Maize other cereals, Switch grass, elephant grass poplars


Biodiesel
is biodegradable and non
-
toxic
-

alkyl esters made from the
transesterification

of vegetable oils or animal fats., (60% less CO
2
)


Rapeseed,
Botryococcus

braunii

(Bb) colloidal microalgae


Concerns: Food trade off


Efficiency of production


ecological impact





Agronomic: HT, Disease resistance : increased productivity
reduction of plantation establishment , reduced tree losses


Poplar, Aspen, Pine, Walnut, Cottonwood trialed 57% timber


Cell Cycle: LFY gene, PTFL gene from Populus able to induce
early flowering in poplar.


Phytohormones increase
size, biomass, wood quality
. GA 20
-
oxidase (AT) aspen fast growth (D/H) increas biomass (Eriksson)


Paper:

low lignin, faster growing ( Sederoff/Chiang)


Biofuels: low lignin (ArborGen), faster growing


Concerns:

robustness/health, gene flow, longevity, similar trees.
China lost track of Bt poplars.


Male Sterility/Flowering inhibition
: restrict gene flow, grow faster
and produce more wood, since energy not wasted reproducing


Tapetum barnase gene
prevent pollen development
promoters
could lead to damage of non
-
reproductive tissue, (long generation
times of conifers are considered).


RNAi down regulation (full suppression often difficult)


Tissue specific cytotoxic stilbene synthase (STS) competes
chalcone synthase (CHS). Somatic cells STS no competition
resveratrol anti
-
fungal (Höfig et al. 2006)

Seeing the wood for the Trees


Simultaneously restores soil health and revegetates economical



University of Georgia poplar trees with merA, from mercury
-
resistant
bacteria. Soil
-
borne and selected for heavy metals. Absorb from soil,
convert to a relatively inert form, and release as vapor. 10X Mercury removal



Richard Meagher gene from the E. coli soybeans to Arabidopsis pumps
arsenic from the soil and stores it in its leaves,



Bioremediation: Wilfred Chen Riverside developing high
-
affinity microbial
bioadsorbents for heavy metal removal using engineered E. coli with
surface
-
expressed peptide analogues (ECs) of phytochelatin
-

microbial
bioadsorbents
-

removal of heavy metals
-

cadmium, mercury, and lead.


Phytoremediation

Modify Fruit Ripening

Fruit ripening modified
to Improve Quality and
Reduce Postharvest
Losses by altering the
activity of cell wall
enzymes, such as
polygalacturonase,
involved in softening
and deterioration.


The biosynthesis of
ethylene, the fruit
-
ripening hormone, has
also been blocked in
several ways to delay
fruit ripening. Ethylene
can then be applied to
induce ripening when
desired, as is currently
done with tomatoes and
bananas.

Caffeine
-
free coffee plants
and controlled ripening
coffee plants.

Coffee and tea plants with
improved disease resistance
and tolerance of
environmental stresses such
as cold and drought.

Controlling the ripening
process will allow the farmer
to both control and lengthen
the harvest season.


25

Modern Biology / Genomics
-


A new Research
-
Paradigm in modern Biology

Genotype

/
Genes / DNA
Inherited Information

Defining an Organism

Phenotype


Appearance and Traits

of an Organism

Improved

Crop Plants


Genomics


= the Totality of the Information

of all Genes and their Functions

From Genomics to Improved Crops

The 2 Phases of Biology

Gene
A
剎R
A
偲潴敩es
A
䵥瑡扯汩l敳
A
佲条O楳i




DNA



Sequence

Map

Transcriptome




Proteome




Metabolome







Profiling





Reverse Genetics


Forward Genetics

Phase

1

New Plant Traits

Phase 2

Molecular Breeding


Transgenics


Metabolic Pathways are the target for complex modifications

Genome

Transcriptome

Proteome

Metabolome

Unraveling protein collaborations could change the way
pathways are manipulated for improvement of valuable traits
in plants.

Attempts to modify storage proteins or secondary metabolic
pathways more successful than have alterations of primary
and intermediary metabolism

Plant Metabolic Engineering
Challenges

Transcription Factors! Maize
C1 and R, reg flavonoids
aleurone , accumul rate
anthocyanins activating the
entire path (Bruce al 2000)

Wheat Rescue ancient TFs
NAC (Uauy 2006)


ID rate
-
limiting steps in
synthesis


Target to channel metab
flow into new paths,


Gene
-
silencing reduce or
eliminate undesirable
comps, traits, or switch off
genes to inc desirable


Direct DNA cassette Paul
Christou 2009


Mod storage proteins or
2nd metabolic paths >
success than alt 1
ry

and
intermed metabolism
(ILSI, 2004/ 2008).

Other Considerations


Site of synthesis


Site of activity


Charge and size


Redundancy



Enzymes and Intermediaries
known
-

Little known on
controls and integration

Metabolic

Pathway

Engineering

Beware the flux! High
Lysine maize


Photo: Fruc
-
1, 6
-
Bisphosphatase
Phosphoribulokinase

3X

10X, minor
photosynth rate


Plastid aldolase,
catalyzes reverse
reaction not subject
to allosteric regulate,
signif
↓↓

in
photosynth rate & C
partitioning (Haake
et al., 1998)


Best:

Mod single genes or
series ind enzy. steps.


Conversion of exist comp
to another rather than
change flux through path

Many common food crops not perfect for nutritional requirements of
humans or animals.


Proteins: Maize, wheat, Sweet potato and cassava


WHO: 800 million people suffer from malnutrition, Protein
-
energy
malnutrition (PEM), the most lethal form, affects 1 in 4 children:
150 M (26.7%) underweight; 182 M stunted.

70% live in Asia, 26% Africa, 4% Latin America, Caribbean



Grains low in Lysine


LDCs food
-

Feed Rations/pollution




High Lysine maize: Use non feedback
-

enzyme (5X ppm)


N assimilation modified pathway GDH 12% increase protein



SRP Nonallergenic Amaranthus Albumin for potato



High Protein: Cytokinin rescue flower pair kernels fused single
kernel two embryos
-

high protein/oil low CHO



NAC Tfs (NAM) senescence and nutrient remobilization leaves to
grains, RNAi delay senes 30% protein, Zn, Fe



Artificial Proteins:



ASP
-
1
-
sweet potato 67% increase protein (EAA 80%)



Improved Nutritional Content

Improved Nutritional Content

Carbohydrates




Starch High Amylose (resistant starch) inhibit 2 SBE


Sorbitol role in fruit carbon metabolism and affects quality
attributes sugar
-
acid balance and starch accum



Wheat puroindoline genes in rice better starch/flour

Fibre


Humans increase



Polymers, Inulins, Fructo
-
oligosaccharides (FOS)



SC Fructans sucrose taste: GI Tract health
-

fermented colonic


bifidobacteria (compete pathogenic bacteria)


SC Fatty acid


anticancer/ inhibit HMG
-
CoAR less LDL



SC fructans 1
-
SST Jerusalem artichoke. 90% sucrose
converted "fructan beets“ (Koops, 2000)



Potato synthesize the full spectrum of inulins from globe
artichoke roots



Lignans: enterodiol/lactone estrogen
-
dependent cancer


Fibre


Animals Decrease



Brown midrib (COMT)

Decreased lignin increase
digestibility better feed conversion, livestock prefer
(Sorghum)

Improved Nutritional Content

Oils and Fatty acids




Altering chain length and saturation level





Novel genes to produce unusual fatty acids in oilseed



MUFA: High Oleic Acid: more stable than PUFA heat/
oxidation resistant, little or no postrefining
(hydrogenation): AS oleate desaturase soybean gave >80%
oleic acid (23%), Less SF milk/meat of animals





MCT: medical foods, ergogenic aids. Acyl
-
ACPT canola,
increase in capric (C10) and caprylic (C8)



High
-
CLA: Antiox
-

free radicals heart disease/cancer



Omega
-
3 DHA
-
EPA “Fish Oil” CV/thrombosis/ Cancer/
Arthritis/ Cognitive/Mental/ premies
-

D

䑥獡瑵牡獥㨠䍡C潬愯獯s扥慮b灲散畲獯u 卄A ㌮㙘 䅌A 楮i
来湥g慴楮朠䕐A



GLA safflower oil (C18:3n
-
6) anti
-
inflammatory effect,
improved skin health and maintaining weight loss



Sitostanol: phytosterol phospholipid Block cholesterol

Slide courtesy of Bruce Chassy

Improved Nutritional Content

Micro nutrients Vitamins/ minerals
:



Vit A

Golden rice II

b
-
捡c潴o湥
-
剩捥 ㈵2 ⡃䉐(


Biofortified cassva flour
-

Field trial Nigeria (Sayre)


Vit B Folate increase in rice (pregnancy deficinicies)


Vit E

a
-
瑯t潰o敲潬o
g
-
呍吻TV楴i
C

increase corn DHAR

Minerals:
Ferritin (bean S protein), Metallothionein (Rice,
wheat). Ca/proton antiporter (sCAX) Ca transport into
vacuoles. Ca
-
fortified carrots enhanced absorption.

Multi vitamin Corn


Combinatorial direct DNA transformation rapid production of
multi
-
complex metabolic pathways


transferred 5 constructs controlled by different endosperm
-
specific promoters into white maize. Different enzyme
combinations show distinct metabolic phenotypes


resulting
in


169X beta carotene (60
m
术朠g. ㄴ1批 扲敥摩d朩


6X vitamin C, and


2X folate (Christou, 2009)



(bacteria)

(daffodil)

(daffodil)


Introduced

enzyme


(maize)

Improved Nutritional Content


Functional components
-

greater than nutrient value alone


Phytochemicals
:



Carotenoids
: Golden Rice, Sweet Potato, Cassava
-

(sight,
development)


Lycopene
: polyamine Tomato


(reduce LDL, cancer
)


Isoflavones
:
genistein

and
daidzein
;
Isothiocyanates


Phenolics
:
resveratrol

antioxidant
Sirtuins

(anti
-
aging)


polyphenol

oxidase

: help sequester protein during ensiling,


Gallic acid
hydrolyzable

tannins, sequester protein in the
rumen, more efficiently absorption


Flavanols
:
Catechins
, Flavones:
quercetin

(less adjuncts)


Anti
-
nutrients
:

Trypsin

Inhibitors; oxalic acid; furans;
Phytate
, Bioavailability Phosphate, divalent ions:
Phytase

(Rice, alfalfa)



Allergens:

soy P34 removal;

peanut; gluten digestion


Toxins:

Glycoalkaloid

(potato) AS
solanine


Cyanogenic

glucoside

(cassava)
hydroxynitrile

lyase



Ye et al. (2000) Science 287: 303
-
305.

“Golden” rice



Over 120 million children
worldwide are deficient in
vitamin A. Rice engineered
to for
b
-
捡牯瑥湥Ⱐ
捯cv敲瑥搠瑯 v楴⁁ 楮i瑨攠
body. This trait in rice
cultivars distributed
worldwide could prevent 1
to 2 M deaths each year.




Golden Rice 23X rice 1.

Increased
b
-
䍡牯瑥湥n楮⁒楣i⁇牡楮i

(bacteria)

(daffodil)

Normal rice

(daffodil)

Introduced

enzyme

(source)

UC
DAVIS

Ferritin,

an iron
-
rich bean
storage protein,

Phytase,

an enzyme that breaks
down phytate making Fe
available, reabsorption of iron, a
gene for a cystein
-
rich
metallothionein
-
like protein has
been engineered into rice

A lack of dietary iron, zinc and
calcium results in unhealthy
increases in cadmium uptake into
the kidney and liver

Seeds
Phytoene
Synthase
Canola
Carotenoids
are Elevated
40 Times Oil

(maize)

“Golden” rice 2

38

Nutrigenomics Nutritional Genomics

"Leave your drugs in the chemist's pot if you can heal the patient
with food." (Hippocrates)



Nutrigenomics

refers to the prospective analysis of differences
among nutrients in the regulation of gene expression


Nutrigenomics/Nutritional Genomics/ Nutrigenetics?:

Genetically based, nutrition intervention that maximizes the
health and effectiveness of each individual.


Monogenic Diseases
: 97% of the “disease
-
associated” genes


Phenylketonuria, phenylalanine hydroxylase PHE
-
> TYR. Leads
to neurological damage and mental retardation. PHE restricted
TYR
-
supplemented diets
-
no Aspartame!


Lactose Intolerance


Juvenile enzyme active into adulthood


Polygenic diseases

obesity, cancer, diabetes, and
cardiovascular diseases,


SNP in haemochromatosis linked gene (HFE) risk,


MTHFR PM higher intakes of folic acid serum homocysteine


Most dietary effect specific interactions on molecular level,
regulation of gene expression directly and indirectly activity of
transcription factors

Nancy Fogg
-
Johnson:

Nutrigenomics will revolutionize health and
nutrition


It will inform how we prevent and treat disease and how
food is grown, processed, and made. Eventually nutrigenomics will
be able to discover diets that prevent or retard the onset of the most
serious and widespread of today's killer diseases, like cancer, as well
as degenerative diseases like Alzheimer's. When/if?



Sitosterolemia (hyperabsorption of sterols hypercholesterolemia risk for
atherosclerosis). Regulation of sterol uptake



Mice was treated with a lipid metabolism
-
altering drug


DNA microarray used for expression profiling of various tissues.


Differential display with a control led to the discovery of an unknown gene.


Computer simulation found that two proteins gene regulated reverse
transport of dietary sterols out of the apical surface of intestinal cells.


Exploring human gene databases, found a human homologue


This explained why dietary sterols, which are structurally similar to
cholesterol, are not absorbed in normal individuals.


By scanning sitosterolemic individuals for this gene, it was found that all of
them had a mutation in this gene responsible for their uncontrolled
hyperabsorption of dietary sterols.

Nutrigenomics Nutritional Genomics


Number of genes regulate lipid metabolism/insulin
sensitivity, affect susceptibility to T2 diabetes mellitus.


SREBP
-
1c (sterol response element binding protein)
mutations led to fatty livers, hypertriglyceridemia, severe
insulin resistance, type 2 diabetes mellitus. One
polymorph expression highly induced in mice on high
fructose diets. Two missense mutations in exons
domain of SREBP
-
1c were found in individuals
displaying severe insulin resistance. Another
association was found between an intronic single
nucleotide polymorphism (C/T) between exons 18c and
19c and the onset of diabetes in men, but not in women.


T2DM Asian/Hispanic populations insulin resistance
rather than
b
-
捥汬 摹獦畮捴楯渮†u渠䅦物捡r
-
䅭敲A捡湳c瑨攠
潰灯獩瑥⸠


Hyperlipidemia: E4 allele in the apolipoprotein E higher
LDL compared with the other (E1, E2, E3) for same fat
intake levels


Nutrigenomics Nutritional Genomics


One single nucleotide polymorphism (
-
75 G/A) in the
apolipoprotein A1 gene in women is associated with an
increase in HDL . ApoA1 women showed increase in HDL
with increase in PUFA compared to G variant taking
similar amounts of PUFA.


Haplotype (HapK) in leukotriene A4 hydrolase (LTA4H)
risk of myocardial infarction (MI) European and African
Americans. MI significantly greater in African
-
Americans
HapK. (n6/n3)


Barbecue Heterocyclic aromatic amines acetylated to
reactive metabolites bind DNA
-

colon cancers. N
-
Acetyltransferase NAT is a phase II metabolism enzyme
that exists in two forms: NAT1 and NAT2. HAA can be
activated through acetylation to reactive metabolites
which bind DNA and cause cancers. Only NAT2 fast
acetylators can perform this acetylation. Studies have
shown that the NAT2 fast acetylator genotype had a
higher risk of developing colon cancer in people who
consumed relatively large quantities of red meat.


"
Nutrigenetic Testing: Tests Purchased from Web Sites Mislead Consumers.“


Government Accountability Office (GAO) commercial "nutrigenetic" testing
dubious clinical validity of commercial genetic tests, and unethical practices


Investigators posed as 14 clients used the DNA from just 2
-

man (48)
-
girl
(9mnth). Despite this, the test 'results' were contradictory and warned of risks
for various conditions. osteoporosis, heart disease, diabetes and more. Affiliated
companies then offer nutritional supplements to stave off these predicted
sicknesses


but the pills turn out to be little more than multivitamins, offered
with a hefty dose of misleading medical advice. Cost to you a mere $89 to $395!
(Nature, 2006)

Caveat Emptor!

Will there be implications for
your insurance if you have a
susceptibility to heart disease?
Will there be implications if
you fail to follow a diet to
retard the onset of symptoms?

Glycosylation

Folding

Volume

System

Comments



Complexity


required

Glycosylation

Required

Protein

Complexity


Protein

Complexity



Volume< 100

KGs



Volume<100

KGs


Volume> 100

KGs


Volume> 100

KGs


Cell

Culture

(mammalian,

Insect, plant)

Microbes

Transgenic

Animals


Transgenic

Plants


YES

NO

HIGH

HIGH

LOW

LOW


System well
understood
unproven


Appropriate for
Mabs, fusion
proteins


Well characterized
System
(Mammalian)


Limited capacity
Opt Manufacturing
strategy


System early
unproven


Appropriate for
Mabs, new
direction


Well characterized
System


Excess capacity

How to Produce a Bio
-
therapeutic


The Cow Pock or the Wonderful Effects of the
New Inoculation!

James Gillray (1757
-
1815)
Vide
--
The Publications of ye Anti
-
Vaccine
Society, June 12, 1802, Library of Medicine

Pasteurization


Vaccines create antibodies to neutralize the causative virus,
bacteria or toxin.Antigens may be introduced by:Killed
Vaccines


Killed vaccines designed to create antibodies without the
negative effects of infection so are generally considered to be
safe. However, during the inactivation process, some of the
surface antigens needed to create the desired antibodies may
be destroyed thus reducing their effectiveness.


Modified live vaccines contain an attenuated or weakened
version of a disease agent. Modified live vaccines are
effective but can negatively impact the health of the animal.
Subunit vaccines use only the necessary parts of the virus to
stimulate immunity. Unlike modified live vaccines, subunit
vaccines stimulate the immune system to prevent disease
without stressing the animal. And unlike killed vaccines,
subunit vaccines do a better job of disease prevention as they
only contain concentrated amounts of the target antigen. These
qualities make subunit vaccines both safe and effective.
Plant
-
made vaccines are a new type of subunit vaccines.

Rinder Pest

Devastating Disease

100% Fatal

Existing Vaccine: Problems
transport, lack of refrigeration, and
lack of a simple system for
administration. Recombinant
product, freeze
-
dried, abating
problems with transportation and
handling, administered to scarified
skin to regenerate the serum.


The vaccinia virus is attenuated,
The recombinant vaccine
-

two of
the viral surface antigens H and F
eliminates risk of contracting the
disease it is easy to determine if the
animal has been vaccinated and is
not just a survivor.


Vaccination of cattle results in a
high level of immunity, affording
protection against test inoculations
of 1000 times the lethal dose of
rinderpest virus.

Tilahun Yilma, UC Davis


Supply the increasing
demand for new biotech
drugs (esp. antibodies)


50 Mabs by 2010


Significantly decrease unit
costs


Improve patients’ access to
biotech medicines

Why Make
Pharmaceuticals in
Plants?


Plants are an efficient producer of proteins


Plants are scalable bioreactors


Plants provide cost advantages to mammalian cell culture systems


3
-
5 times faster than mammalian systems


Plant cells are similar to human cells


Similar protein synthesis machinery


Read the same genetic code


Assemble, fold and secrete complex proteins

Produce protein ( primarily Mabs)


Correct post
-
translational modification mostly (unlike Microbes)


No propagation of human pathogens or other mammalian contaminants;
no other mammalian contaminants de novo;


Asepsis can begin at purification, not inoculation (less go wrong)


Scale
-
up utilizes the same technology used in agriculture to
-
day.


Faster, cheaper, more convent, more efficient than CHO cells




Why Seeds?


Protein at the highest levels in the harvestable seed.


Seeds are easier, more economical than whole plants to transport to a
processing factory


Proteins can be extracted and purified in prep. for packaging.


Seeds can be stored for prolonged periods with protein intact.


Hundreds of acres of protein
-
containing seeds could inexpensively double
the production CHO bioreactor factory.


Why Plants?

Imbibe seeds to
induce expression of
a
-
amylase promoter
& production of
desired protein.

Plants as Chemical Factories

Malting grain model (Ventria, Inc., CA and defunct attempt in MO, now Kansas)

Transform rice with

desired protein
controlled

by
a
-
amylase promoter
in the aleurone layer.
proteins lactoferrin and
lysozyme

Extract and
purify proteins
from
germinating
seeds.

Grow rice crop
in the field,
harvest grain at
maturity.

Ray Rodriguez, Molecular and Cellular Biology, UC Davis

Rice Lactoferin Lysozyme


Peru 30% Less Diarrhea,
Quicker recovery 3/6 days,
1/3 less recurrence

KAREN A. McDONALD/ BRYCE FALK


Fellow: MICHAEL A. PLESHA

Plant Based Expression Systems for

Efficient Production of Human Therapeutics

Human deficiency in alpha
-
1
-
antitrypsin (AAT)
results in non
-
smoking related emphysema

The optimized human AAT gene has been cloned into
Agrobacterium

and is expressed using a chemically
inducible plant viral (CMV) replicon expression system

1a

2a

H

AAT

AAT

AAT

AAT

RNA 3a

RNA AAT

CMV 2a

CMV 2b

Promoter

CMV 3a

AAT

Promoter

Inducible Promoter

CMV 1a

Replicase

Inducer

AAT expression kinetics via agroinfiltration of
Nicotiana benthamiana

plants will be optimized

Alternate
scalable
induction
methods
will be
evaluated

0
250
500
750
Negative Control
(Healthy Plant)
pCMV-SPAAT +
Inducer; Cotton
pCMV-SPAAT +
Inducer; Syringe
Functional AAT (mg / kg FW tissue)
AAT is used as a
model human
protein; other
proteins will
also be
expressed and
optimized

Viruses PlantAb

protected mice

against genital herpes

similar physical props to MCC

remained stable in human

exhibited no diff in affinity

for binding, neutralizing HSV

Genetic Engineering Technology
Allows Production of Novel Products



Metabolic

Pathways




Active Vaccines


Transmissible
gastroenteritis virus

Antigen

Cholera/Hep B/banana

Acetylenic &Vernolic
Acid Containing


Chemical feedstocks


Passive Vaccines

Ab enteric bacteria

E.coli O157:H7

meat

foodborne path

Polyhydroxybuterate
biodegradable plastic

The Dow

AgroSciences unique
Concert™ Plant
-
Cell
-
Produced System
uses plant cells, rather than whole
plants, in a culture medium comprised
primarily of water, sugar and salt. This
eliminates the possibility of
contamination and environment
concerns. The Concert™ Plant
-
Cell
-
Produced System is totally bio
-
contained as all production is done in a
sealed and sterile production process.
This means only the required inputs get
into the vaccine and only a safe, non
-
replicating vaccine comes out.


Alfalfa

Plasma Proteins, Foot
-
and
-
mouth disease vaccine


Maize

Anti
-

HIV and Anti herpes Simplex Antibodies
Microbiocides

for pulmonary infection
Mabs

for cancer, autoimmune disease RA, Vaccines hepatitis B,
Norwalk virus (Travelers disease), Vaccines &
Mabs

for
animal,
Aprotinin

for blood loss, Gastric lipase cystic fibrosis,


Lettuce

Vaccines for Hepatitis B


Moss

Factor IX for hemophilia B


Rice

Lactoferrin Lysozyme for GI health, Alternatives to abs in
poultry diets, Topical infections, inflammations, B
-
cell
lymphoma vaccine


Safflower

Therapeutics and oil
-
based products for oral/dermal delivery


Spinach

Protective antigen for vaccine against Bacillus anthracis


Soybean

Tobacco
extensin

signal peptide
-

Anti
-
HSV
-
2 (
IgG
)


Tobacco

Non
-
Hodgkins

B
-
cell lymphoma, TGF
-
b
glucocerebrosidase

for
Gauchers

Syndrome , Alpha
galactosidase

for enzyme
replacement therapy,
IgGs

for prevention of dental decay,
common cold, GAD 7 cytokines for type 1 Diabetes, Colon
cancer surface antigen


Fabrazyme

fat
-
storage disorder

Tomato , Potato
Banana (someday!)

Edible vaccines: Enterotoxigenic E. coli, Norwalk virus,
Hepatitis B, Vibrio cholera, Rabies virus
-
intact Glycoprotein
Antimicrobe peptides,

Wheat

Carcinoembryonic antigen
-

Murine IgG signal peptide

Potato

Polyhydroxybuterate

biodegradable plastic


potential gene flow to food
crops of the same species


co
-
mingling of food and non
-
food crops


worker exposure to plant
material containing active
pharmaceutical ingredients
(APIs).


ProdiGene paid $3 million bond
for violations in Nebraska and
Iowa reimburse the USDA for
the costs, involved in disposing
of the contaminated crops.


Now making brazzein ( a sweet
protein) in maize!




Concerns?

Physical and biological

Confinement essentially means keeping the crop and its products on the
land where it was grown until removed for processing, with no
inadvertent exposure to the public and minimal exposure of products to
workers and the environment.


Identity Preservation: A Closed Loop System

Preventing co
-
mingling or gene flow prime directive for industry as well as
regulatory agencies. (No StarLink®, repetition!).




Principles of Confinement


Potential gene flow to food crops of the same species


Vectors: Birds, insects, viruses


Co
-
mingling of food and non
-
food crops


Worker exposure active pharmaceutical ingredients (APIs).

Identity Preservation: A Closed Loop System


Preventing co
-
mingling or gene flow prime directive for industry
as well as regulatory agencies.

Physical and biological


Confinement: physical, temporal and biological isolation, as well
as appropriate spatial isolation, acreage limitations, dedicated
equipment, facilities


“Trap” Plant borders, Color coded kernels, Male sterile plants,
work well
-

limited to a few species.


Transformation plastid (not all pollen is plastid
-
free, illegit recomb
none of the transgenes functional in nuclear genome)


Genetic Use Restriction Technologies, or GURTs

Concerns/Confinement

Depending on the timing, expression of the lethal gene leads either to seeds that are
incapable of germinating or to death of the seedling.

During seed maturation stage lea promoters ( “late embryogenesis abundant”) activate the
genes whose products control and accompany the drying process. Shortly after the seeds
have swollen, germination
-
specific promoters activate the genes whose products break
down substances from the storage tissue and transport them to the young seedling.

T
-

GURT seed will germinate only when treated with this inductor chemical.



V
-
GURT seed treated with an inductor that activates the lethal gene. Produce seeds that
mature but that are no longer able to germinate and cannot therefore be used for re
-
sowing.

61