ENGINEERING HERBICIDE RESISTANCE AND METABOLIC ENGINEERING

Sana Sikandar

Fasiha Mazhar

Rida Batool

Noor Fatima

“It is a practice of optimizing genetic and regulatory processes within cells to increase the production of certain substances.”

METABOLIC ENGINEERING:-

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.

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 over-expressed 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 By expressing two transcription factors form snap dragon in tomato, the level of flavonoid anthocyanin have been increase 3 fold.

o A level that was sufficient for this flavonoid to confer improved chemo-preventive properties in cancer susceptible mice.

IV. FLAVOR OF STRAWBERRY

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.

ConclusionAt 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

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

WHAT IS GENETIC ENGINEERING?

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.

WHY HERBICIDES ARE USED?

1. Lack of activity towards important crops.2. Phytotoxicity towards its major weed

WHAT KIND OF HERBICIDES ARE CHOOSEN FOR DEVELOPMENT?

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.

WHY ENGINEERED HERBICIDES ARE REQUIRED?

There are two approaches:1. Modification of the target of herbicide action.2. Detoxification or degradation of the herbicide.

MECHANISMS FOR THE ENGINEERING OF HERBICIDE RESISTANCE IN PLANTS:

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.

MODIFICATION OF THE TARGET OF HERBICIDE ACTION:

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.

METHOD:

HERBICIDES THAT RESIST BIOSYNTHESIS PATHWAYS.

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

TRIAZINE HERBICIDES: (PHOTOSYNTHESIS INHIBITING HERBICIDE)

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.

ENGINEERED TRIAZINE

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 non-

phytotoxic derivatives.

• Differential metabolism is the most important factor.

• Genes encoding for enzymes might be herbicide resistant determinants.

HERBICIDE-RESISTANT PLANT ENZYMESMixed-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. The detoxifying enzyme should be encoded by a single gene or few genes.

2. Enzyme should not require complex co-factors for its activity.

3. The residual metabolite(s) should not be phytotoxic

4. 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 CB. Vitamin AC. Vitamin A and DD. Vitamin B

2. Metabolic Engineering emerged in ?A. 1989B. 1990C. 1991D. 1993

3.Metabolic Engineering involves :A. Biochemical reactionsB. Certain enzymesC. Biochemical path waysD. All of the above

4.Steriol glycosides and a mixed esters :A. Give flavor to strawberriesB. Improve metabolismC. Provide nutrientsD. Act as a biosensor

5.Metabolic engineering can :A. Improve the flavor of any substanceB. Improve nutrient content C. Both D. None

Thank You