Plant metabolite engineering
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Transcript of Plant metabolite engineering
Plant Metabolic Engineering
PLANT METABOLIC ENGINEERING BY NEHA P PATEL
M.Sc II
Outline of the TalkIntroduction to Metabolic Engineering
Plants as natural factories
Metabolic Engineering in Plant
Choice of plant system for ME
Stratigies for ME
Applications
Emerging technologies
Future
Introduction to Metabolic Engineering
It is the directed improvement of
product formation or cellular
properties through the modification
of specific biochemical reaction(s) or
the introduction of new one(s) with
the use of recombinant DNA
technology
METABOLIC ENGINEERING
MetabolicNetworks
MODIFICATIONrecombinant
DNA technology
ANALYSIS
Flux Quantification
Analysis of FluxControl
Cell improvement
OTHER TERMS FOR ME
1.MOLECULAR BREEDING
2.PATHWAY ENGINEERING
3.CELLULAR ENGINEERING
Manipulation of
plant metaboli
sm
Food
Feed
industrial
products fine chemical
s
Fuel
Pharm
novel opportun
ities in agricultu
re
Why plants for engineering
What’s so hard about modeling plants?Plant cell metabolism is complex…
Collectively, plants produce over 200,000 (primary and secondary) metabolites
less information is available as compared to bacteria
Proteins Genes Base pairs
1366 (30%+) 4000-5000 4.6M E.coli
3500 (10%+) 30000 135M Arabidopsis
WHY PLANTS AS NATURAL FACTORIES
Cheap availability
easy to grow
Low cost of growing plants
Provide eukaryotic system
Plants cells are highly compartmentalized
PLANT METABOLIC ENGINEERING
Plant metabolic engineering involves the manipulation of existing metabolic pathways by either increasing or diverting flux to desired or from undesired products, respectively, or the generation of chemical entities not normally found in the plant production system (cells or whole plants),through the introduction of genes from other organisms.
Essential elements in the toolbox of the metabolic engineer are
mechanisms to eliminate or
over express gene activity
Introduction of new pathway
Strategies available to eliminate the activity of specific enzymes in a pathway involve one of several possible approaches
Identification of a mutant gene for the corresponding enzyme.
Knocking out gene function by targeted RNA degradation.
Interfering with protein function using specific inhibitors or antibodies.
Process of RNA interference
The expression of genes in plants or plant cells is dependent on several factors that include:
method to introduce genes into the plant
Promoters to direct gene expression in the appropriate spatial and temporal landscape.
source for the gene encoding the enzyme of interest.
CHOICE OF PLANT SYSTEM
FOR METABOLIC
ENGINEERING
Whole Plants
Plant Cells in Culture
STRATEGIES FOR
INCREASING FLUX OF EXISTING
PATHWAYS
Manipulating the Activity of
‘‘Rate Limiting’’
StepsMetabolic Flux Analysis and
ModelingExpression of
Multiple Genes in
Plants:Progress and
LimitationsDiverting Flux Using Loss of
Function ApproachesTargeting
Entire Pathways
withTranscription Factors
PLANTS AS BIOREACTOR
Antibodies
edible vaccines
Polymers
Pharmaceutical drugs etc
ANTIBODIESStable
integration of gene as
compare to microorganis
msProvide
eukaryotic system for production
Low cost production as compare to
fermentation-it costs approx. $5,000 /gm to
produce antibodies from
hybridoma technology cells in culture,$10 to
100 /gm to produce
antibodies from transgenic
plants .
Foreign proteins are mostly produced in
seed
But often not properly
glycosylated
PRODUCTION OF EDIBLE VACCINE
Edible
vaccines
Transgenic Plant Vaccines – Potential for
Edible Vaccines
Pros
No need for equipment such
as needles
No personnel would required
Ability for mass immunization at
low cost
Microorganisms could produce them in foods such as yogurt
and cheeses but would require processing,
adding cost and limiting
accessibility
Plants offer a nutritious vector, especially useful
in third world countries where they would be very important
ConsRestricted to plants whose products are
consumed raw to avoid degradation during
cookingConcerns that ingested proteins
would be broken down in the gut by proteinasesTrying to prove that
antigens being encapsulated by plant
tissue will resist immediate breakdown
Accidental consumption due to
human error in storage and
transportation of crops
Low amounts of highly immunogenic antigen caused no detectable side effects in mice however much more
work must be done in researching different
doses, mixes and antigenicities before
this can be removed as a possible threat
In an experiment with an edible rice vaccination
immunizing against cholera they were
proven effective after being stored at room temperature for 1.5
years
Polymers
polyhydroxyalkanoates (PHA) in
plants .
polyesters of 3-hydroxyacids
have unique biodegradable and
elastomeric properties
used in medical industry, and for making environmental friendly
plastics
PHB granules in Arabidopsis mesophyll cell nucleus
(PHB/PHV block copolymer)Poly(3-hydroxybutyrate- co-3-hydroxyvalerate)
Biopolymer production
GlycerolPropionate
Acetyl-CoAPropionyl-CoA
Acetoacetyl-CoA 3-Ketovaleryl-CoA
3-Hydroxybutyryl-CoA3-Hydroxyvalery-CoA
Acetyl-CoA
HSCoA3-Ketothiolase (PhaA)
NADPH
NADP+
Acetoacetyl-CoAReductase (PhaB)
P(HB-co-HV)HSCoAHSCoA
PHA Synthase (PhaC)
Plants as Pharmaceu
tical FactoriesHave astonishing potential for the
biosynthesis of small (<1000 Da) molecules.
Many Secondary metabolites as drugs.example-
morphines,steroids.
Many phytochemicals like
flavonoids,anthocynins, ,phenolic compounds
etc
Figure showing Drug production in plants
Using Plant Compartmen
ts for Chemical
Sequestration
PROBLEM-Massive accumulation of metabolites if transgenes are
expressed constitutively
throughout the plant
SOLUTION-1.the use of tissue-specific
promoters, allowing for accumulation in
either a specific organ or tissue.
SOLUTION-2.gene expression can be
controlled in such a way that
biosynthetic enzymes or
metabolites are directed to specific cell compartments such as the vacuole
or chloroplast
pathway intermediates or
final products could be sequestered in specific subcellular
compartments
EMERGING
TECHNOLOGIES
Plant Diversity as a Source of New Genes-unique biosynthetic pathways that offer opportunities
to use genomic approaches to capture this biochemical diversity and provide tools for manipulating plants and other organisms that do
not produce the compounds or where accumulation is blocked at a
particular biosynthetic step
Gene Shuffling and Directed Enzyme
Evolution-Gene shuffling technologies can produce a
large number of enzyme variants, by shuffling
fragments from an existing library
The FUTURE
Focus on secondary metabolism
Progress in ‘omics technologies’.
Better use of what we know!Choose model systems we can experimentally validateApply known constraintsDefine appropriate objective functions
Integrate regulatory mechanisms
The FUTURE
Biomass Production
Resistance
Stress Tolerance
Rational Plant Metabolic Engineering
THANK YOU