Martina Newell-McGloughlin
Director, UC Systemwide Biotech Research
and Education Program
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Agronomic Traits
Crop Biotechnology
– 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, O2 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
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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.
Source: ISAAA
Snap shot
– $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
Farmers Choice
Papaya ringspot virus
• 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
Snapshot
– 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 potatoproducing regions of the world would be $4.3 billion.
The Argentinean Case: GM crops an engine of
economical development
 Number 2 in GM production.
 17% of the global area of GM plants.
 In 2007, 98% of soybean in Argentina was GM.
 Yield have reached over 6 tonns per hectare
 In 1994-95 production costs were 182 dolars/Ha; in
2007 are 117 dolars/Ha
 In 1994-95 farmers spent 78 dolars/Ha in
herbicides; today they spend 37 dolars/Ha and
insecticide use has decrease 90%.
 Economical benefits of GM soybean USD$ 20 billion
+ 1 million jobs
 Problems, yes. Due to the economic success of GM
soybean and maize, President Cristina Fernádez de
Kirchne created a new tax on Gm soybean exports
that producers oppose
China is awakening
 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.
 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”
Blight-resistant potato
– 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 NBSLRR) -UI study concluded for the major potato-producing
regions of the world would be $4.3 billion.
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“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
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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
• 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.
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Striga spp. in the savanna
regions alone account for
$7 billion and are
detrimental to the lives of over
100 million African people.
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
Host plants
Parasitic plants
maize
cowpea
Arabidopsis
Striga
Orobanche
Triphysaria
Seed germination
sorgoleone
0h
DMBQ
12 h
24 h
Haustorium development
Abiotic stress limiting factor for crops
reaching genetic potential
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Improved water conservation -Fewer crop
losses -Higher yields on all acres through
improved water utilization -Expand in
drylands - Nuclear Factor Y B subunit
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Crops limited by salinity on 40 % world's
irrigated land (25 % US)
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Cold: Engineering with COR15a Tf, role
in freezing tolerance.
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Plants engineered with Choline oxidase
(codA) soil tolerated saline and cold
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Homeodomain-leucine zipper (HD-Zip)
transcription factors respond to H2O &
osmotic stress, exongenous abscisic acid
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Transport protein. Grow and fruit even in
irrigation water that is > 50X saltier than
normal. > 1/3 salty as seawater.
Blumwald and Zhang)
Abiotic Stress:
Drought, ColdHeat, Salinity
Drought Stressed Rice
Control
+ Gene D
Control + Gene E
Increased Yields
- Improve Nitrogen Assimilation
- Increase Sucrose hydrolysis,
Starch biosynthesis
- Increase O2 availability
- Modify photosynthesis
Yield Gene
Control
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
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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 CO2)
Rapeseed, Botryococcus braunii (Bb) colloidal microalgae
Concerns: Food trade off – Efficiency of production – ecological impact
Seeing the wood for the Trees
• 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 20oxidase (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)
Phytoremediation
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Simultaneously restores soil health and revegetates economical
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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
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Richard Meagher gene from the E. coli soybeans to Arabidopsis pumps
arsenic from the soil and stores it in its leaves,
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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.
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 fruitripening 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.
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Modern Biology / Genomics A new Research-Paradigm in modern Biology
Phenotype
Genotype / Genes / DNA
Appearance and Traits
of an Organism
Inherited Information
Defining 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
Phase 2
Reverse Genetics
Forward Genetics
GeneARNAAProteinsAMetabolitesAOrganism
DNA
Sequence
Map
Transcriptome
Molecular Breeding
New Plant Traits
Proteome
Transgenics
Metabolome
Profiling
Phase 1
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
Beware the flux! High
Lysine maize
• Photo: Fruc-1, 6Bisphosphatase
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)
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
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)
• Mod storage proteins or
2nd metabolic paths >
success than alt 1ry and
intermed metabolism
(ILSI, 2004/ 2008).
• Best: Mod single genes or
series ind enzy. steps.
• Conversion of exist comp
to another rather than
change flux through path
• 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
Improved Nutritional Content
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
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
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• 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 D6
Desaturase: Canola/soybean precursor SDA 3.6X ALA in
generating EPA
• 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
Introduced
enzyme
Micro nutrients Vitamins/ minerals:
(daffodil)
(maize)
• Vit A Golden rice II b-carotene-Rice 25X (CBP)
(bacteria)
(daffodil)
• Biofortified cassva flour- Field trial Nigeria (Sayre)
• Vit B Folate increase in rice (pregnancy deficinicies)
• Vit E a-tocopherol g-TMT; Vit 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 endospermspecific promoters into white maize. Different enzyme
combinations show distinct metabolic phenotypes – resulting
in
• 169X beta carotene (60 mg/g v. 14 by breeding)
• 6X vitamin C, and
• 2X folate (Christou, 2009)
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
Increased b-Carotene in Rice Grains
• Over 120 million children
worldwide are deficient in
vitamin A. Rice engineered
to for b-carotene,
converted to vit A in the
body. This trait in rice
cultivars distributed
worldwide could prevent 1
to 2 M deaths each year.
• Golden Rice 23X rice 1.
Ferritin, an iron-rich bean
storage protein,
Introduced
enzyme
(source)
Normal rice
(daffodil)
(maize)
(bacteria)
Seeds
Phytoene
Synthase
Canola
Carotenoids
are Elevated
40 Times Oil
Phytase, an enzyme that breaks
down phytate making Fe
available, reabsorption of iron, a
UCDAVIS
gene
for a cystein-rich
metallothionein-like protein has
been engineered into rice
“Golden” rice
(daffodil)
Ye et al. (2000) Science 287: 303-305.
A lack of dietary iron, zinc and
calcium results in unhealthy
increases in cadmium uptake into
the kidney and liver
“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-cell dysfunction. In African-Americans the
opposite.
• 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. NAcetyltransferase 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.
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?
• "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)
How to Produce a Bio-therapeutic
Glycosylation Folding
Complexity
Volume
required
HIGH Volume> 100
KGs
Protein
Complexity
YES
Volume< 100
KGs
LOW
Glycosylation
Required
NO
Volume> 100
KGs
HIGH
Protein
Complexity
LOW
System
Comments
• System well
understood
Transgenic unproven
Animals • Appropriate for
Mabs, fusion
proteins
• Well characterized
Cell
System
Culture
(Mammalian)
(mammalian,
Insect, plant) • Limited capacity
Opt Manufacturing
strategy
Transgenic • System early
unproven
Plants • Appropriate for
Mabs, new
direction
Volume<100
KGs
• Well characterized
Microbes System
• Excess capacity
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
Why Make
Pharmaceuticals in
Plants?
• Supply the increasing
demand for new biotech
drugs (esp. antibodies)
– 50 Mabs by 2010
• Significantly decrease unit
costs
• Improve patients’ access to
biotech medicines
• 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
Why Plants?
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.
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
Grow rice crop
in the field,
harvest grain at
maturity.
Imbibe seeds to
Extract and
induce expression of purify proteins
a-amylase promoter from
& production of
germinating
desired protein.
seeds.
Rice Lactoferin Lysozyme
Peru 30% Less Diarrhea,
Quicker recovery 3/6 days,
1/3 less recurrence
Ray Rodriguez, Molecular and Cellular Biology, UC Davis
Plant Based Expression Systems for
Efficient Production of Human Therapeutics
KAREN A. McDONALD/ BRYCE FALK Fellow: MICHAEL A. PLESHA
Human deficiency in alpha-1-antitrypsin (AAT)
results in non-smoking related emphysema
AAT expression kinetics via agroinfiltration of
Nicotiana benthamiana plants will be optimized
AAT is used as a
model human
protein; other
proteins will
also be
expressed and
optimized
Alternate
scalable
induction
methods
will be
evaluated
The optimized human AAT gene has been cloned into
Agrobacterium and is expressed using a chemically
inducible plant viral (CMV) replicon expression system
Inducer
Inducible Promoter CMV 1a
Promoter
CMV 2a
Promoter
CMV 3a
RNA 3a
1a
2a H
Replicase
CMV 2b
AAT
750
Functional AAT (mg / kg FW tissue)
500
250
RNA AAT
AAT
AAT
AAT
AAT
0
Negative Control
(Healthy Plant)
pCMV-SPAAT +
Inducer; Cotton
pCMV-SPAAT +
Inducer; Syringe
Genetic Engineering Technology
Allows Production of Novel Products
Passive Vaccines
Ab enteric bacteria
E.coli O157:H7 meat
foodborne path
Polyhydroxybuterate
biodegradable plastic
Acetylenic &Vernolic
Acid Containing
Chemical feedstocks
Metabolic
Pathways
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
Active Vaccines
Transmissible
gastroenteritis virus
Antigen
Cholera/Hep B/banana
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-CellProduced System is totally biocontained 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, nonreplicating 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
Tomato , Potato
Banana (someday!)
Wheat
Potato
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
Edible vaccines: Enterotoxigenic E. coli, Norwalk virus,
Hepatitis B, Vibrio cholera, Rabies virus-intact Glycoprotein
Antimicrobe peptides,
Carcinoembryonic antigen - Murine IgG signal peptide
Polyhydroxybuterate biodegradable plastic
Concerns?
• potential gene flow to food
crops of the same species
• co-mingling of food and nonfood 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!
Principles of Confinement
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!).
Concerns/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
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.
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