ENGINEERING HERBICIDE
RESISTANCE AND METABOLIC
ENGINEERING
Sana Sikandar
Fasiha Mazhar
Rida Batool
Noor Fatima
METABOLIC ENGINEERING:-
It is a practice of optimizing
genetic and regulatory
processes within cells to
increase the production of
certain substances.”
“
INTRODUCTION:
It is basically the use of genetic engineering to
modify the metabolism of an organism.
 It can involve the chemical networks.
 Series of biochemical reactions and certain
enzymes.
 Allow cells to convert raw materials into
molecules.

GOALS OF METABOLIC ENGINEERING
Metabolic engineering specifically seeks to:
 Mathematical model these chemical networks.
 Calculate a yield of useful products.
 Pinpoint parts of network that constrain the
production of products.
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The ultimate goal of metabolic engineering is to
be able to use the organisms to produce valuable
substances on an industrial scale in a cost
effective manner.
Current examples include the production of:
Beer.
Wine.
Pharmaceuticals.
Cheese.
Biotechnology products.
HOW REGULATORY NETWORK WORKS
EFFICIENTLY:
As cells use the metabolic networks for their
survivals
 There can be some drastic effects on the cell’s
viability.
 In order to decrease its effect; trade-off in
metabolic engineering arises between cells.
 Instead of directly deleting or overexpressing the
genes that encode for metabolic enzymes, the
current focus is to target the regulatory networks
in a cell.

HISTORY:
In the past, to increase the productivity of a
desired metabolite, a micro-organism was
genetically modified by chemically induced
mutation and the mutant strain that overexpressed the desired metabolite was chosen.
 One of the main problem with this technique was
the metabolic pathway for the production of that
metabolite was not analyzed.
 As a result, the constraints to production and
relevant pathway enzymes to be modified were
unknown.

In 1990’s, a new technology called metabolic
engineering was emerged.
 This technology analyzes the metabolic pathway
of a micro-organism, determines the constraints
and their effects on the production of desired
compounds.
 Some examples of successful metabolic
engineering are:
 Identification of constraints to lysine production.
 Engineering of new fatty acid biosynthesis
pathway called reversed beta oxidation pathway.
 Improved production of DAHP.

APPLICATIONS :
Metabolic engineering is a new field with
applications in the production of chemicals, fuels,
material, pharmaceuticals and medicines at the
genetic level. Following are some of the
applications :
 Crop plants with altered nutrient content.
I.
Golden rice.
II. Broccoli.
III. Tomato.
IV. Flavor of strawberry.
 Precise metabolic engineering of carotenoid
biosynthesis in E-coli.

I. GOLDEN RICE:
Metabolically engineered plants.
 Nutrient content of a crop plant can be improved
by metabolic engineering.
 Rice, maize, .wheat, tomatoes and other
vegetables are breaded to enhance the level of
certain nutrients
 Addition of β-carotene pathway to rice to yield
rice having higher levels of vitamin A.

II.
BROCCOLI:
Conventional plant breeding led to the beneforte
strain of broccoli.
 Higher levels of glucosinolate glucoraphanin.
 Early studies have demonstrated that human
consumption of high glucoraphanin broccoli
results in improved metabolism and reduced
level of fatty acids.
 In the lipid compound associated with
inflammation.

III. TOMATO:
o
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By expressing two transcription factors form
snap dragon in tomato, the level of flavonoid
anthocyanin have been increase 3 fold.
A level that was sufficient for this flavonoid to
confer improved chemo-preventive properties in
cancer susceptible mice.
IV. FLAVOR OF STRAWBERRY
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Challenge of improving the taste of a plant can
also be altered by using metabolic engineering
since there are small molecule’s metabolites
which make an important contribution to flavor.
For example in strawberry plant ,the steviol
glycosides and the mixed esters give in
strawberries their distinctive flavor.
Precise metabolic engineering of carotenoid
biosynthesis in E-coli towards a low cost
biosensor.
 Micronutrient deficiencies including zinc
deficiency are responsible for hundreds of
thousands of deaths annually.

Conclusion
At the industrial scale, metabolic engineering is
becoming more convenient and cost effective.
According to Biotechnology Industry Organization
more than 50 bio refinery facilities are being built
across North America to apply metabolic
engineering to produce biofuels and chemicals from
renewable biomass which can help reduce
greenhouse gas emissions.
ENGINEERING OF HERBICIDE
RESISTANCE
in plants
WHAT IS GENETIC ENGINEERING?
Modification of organism’s genetic composition by
artificial means.
 Involing the transfer of specific trait, gene from one
organism into plant or animal of entirely different
species

WHY HERBICIDES ARE USED?
More labour and energy efficient than manual or
mechanical weed control.
 Phytotoxic moleculesare used.
 Most herbicides originate from extensive screening
of large no. of synthetic compounds.

WHAT KIND OF HERBICIDES ARE CHOOSEN
FOR DEVELOPMENT?
1.
2.
Lack of activity towards important crops.
Phytotoxicity towards its major weed
WHY ENGINEERED HERBICIDES ARE
REQUIRED?
Contamination of environment,toxicity to animal
and persistance of residues in soil and water.
 Herbicides effects target protein by inactivating
them.
 For confering resistance to crop against broad
spectrum herbicide.

MECHANISMS FOR THE ENGINEERING OF
HERBICIDE RESISTANCE IN PLANTS:
There are two approaches:
1.
Modification of the target of herbicide action.
2.
Detoxification or degradation of the herbicide.
MODIFICATION OF THE TARGET OF
HERBICIDE ACTION:
The modification of the enzyme or other target for
herbicidal action in the plant to render it
insensitive to the herbicide or by inducing the over
production of the unmodified target protein , thus
permitting normal metabolism to occur in spite of
the presence of the herbicide.
METHOD:
This approach depends on the identification in
molecular terms of the biochemical site of herbicide
action in plant cell
 This is done by physiological , biochemical and
genetic studies.
 Some herbicides have been shown to disrupt amino
acid biosynthesis pathways and other or interfere
with photosynthesis.

HERBICIDES THAT RESIST BIOSYNTHESIS
PATHWAYS.
TRIAZINE HERBICIDES: (PHOTOSYNTHESIS INHIBITING
HERBICIDE)

Triazine herbicides: atrazine and simazine
inhibit photosynthesis by inhibiting
electron transport by binding to protein in
photosystem 2.
 These are taken up by roots.
 Many weeds such as amaranthis and
compestris have developed triazine resistance ,
which can be traced to a mutation in PsbA gene
that codes for D1 protein. The change of single
amino acid from serine to glycine lower the
affinity of this for triazine 100 folds
ENGINEERED TRIAZINE
The triazine tolerant trait was transferred from a
resistant weed biotype to cultivated rapeseed
cultivars.
A great advantage of genetic engineering approach is
that the gene can be used to transform different
plant species.
The wild type gene cannot easily be deleted from the
plant genome and consequently , am mixture of
wild type and mutant gene products in transgenic
plants yeild an inconsistent resistance phenotype.
DETOXIFICATION OR DEGRADATION
OF HERBICIDE
Gene
Transfer involving enzymes that
inactivate the herbicide before
inhibition of plant cell target site.
Herbicide
Detoxification and
degradation occur in many plant
species and micro-organisms.
PLANT DETOXIFYING ENZYMES
Many
crop species found tolerant to
specific herbicides – encouragement
for the use of these herbicides for
selective weed killing.
FACTORS INVOLVED IN HERBICIDE
SELECTION
Rate
of absorption in plants.
Translocation.
Subcellular localization.
Variation in target site sensitivity.
Metabolic detoxification to nonphytotoxic derivatives.
• Differential metabolism is the most
important factor.
• Genes encoding for enzymes might be
herbicide resistant determinants.
HERBICIDE-RESISTANT PLANT
ENZYMES
Mixed-function
oxidases- involved in
detoxification of 2,4-D in pea and
dicamba in tolerant barley.
Decarboxylases.
Thiol-sugar conjugative enzymes.
Amino-acid conjugative enzyme
systems.
Mechanism by which plant species
transform foreign compounds involve:
• Hydroxylation of aryl groups.
• Oxidation of alkyl substituents.
• Hydrolysis of carboxylic acid ester
groups.
For example:
• Detoxification of atrazine and alachor
in tolerant maize line.
• Involves conjugation with tripeptide
glutathione – glutathione-S transferase.
• Conjugate is non-toxic to plant cells.
• Pyridinium Herbicide paraquat
generates active oxygen.
• Lipoxidizes plant membranes, causing
cell death.
• Enhanced levels of enzymes can
detoxify oxygen radicals.
• Three enzymes: superoxide dismutase.
catalase.
peroxidase.
BACTERIAL DETOXIFYING ENZYMES
Micro-organisms
are the potential
source for herbicide resistance genes.
Pseudomonas, Arthrobacter,
Alcaligenes metabolize 2,4-D.
The 2,4-D metabolism pathway is
plasmid encoded.
CONCLUSION
Detoxification
of herbicides has
advantage over target modification.
The process can be done when:
i.
biochemical site of action of
herbicide is not known.
ii.
target is difficult to engineer
because of the existence of dual
pathway.
BUT FEW CRITERIA ARE REQUIRED:
1.
2.
3.
4.
The detoxifying enzyme should be
encoded by a single gene or few
genes.
Enzyme should not require
complex co-factors for its activity.
The residual metabolite(s) should
not be phytotoxic
And they should eventually be
further metabolized.
• The process of herbicide degradation
and detoxification is little known.
• Alternative of plant enzymes- micro
organismic enzymes.
• Elucidation of metabolic pathway is
much simpler, bacterial genes express
well in plants.
MULTIPLE CHOICE QUESTIONS
1.Addition of beta carotene to rice yields rice
having:
A. Vitamin C
B. Vitamin A
C. Vitamin A and D
D. Vitamin B
2. Metabolic Engineering emerged in ?
A. 1989
B. 1990
C. 1991
D. 1993
3.Metabolic Engineering involves :
A. Biochemical reactions
B. Certain enzymes
C. Biochemical path ways
D. All of the above
4.Steriol glycosides and a mixed esters :
A. Give flavor to strawberries
B. Improve metabolism
C. Provide nutrients
D. Act as a biosensor
5.Metabolic engineering can :
A. Improve the flavor of any substance
B. Improve nutrient content
C. Both
D. None
Thank You