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