Plant metabolic engineering

What is Metabolic Engineering?

Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology

Gene Cloning• Gene cloning is a process of making large quantities

of a desired piece of DNA once it has been isolated• Cloning allows an unlimited number of copies of a

gene to be produced for analysis or for production of a protein product

• Methods have been developed to insert a DNA fragment of interest (e.g. a segment of human DNA) into the DNA of a vector, resulting in a recombinant DNA molecule or molecular clone

Gene Cloning• A vector is a self-replicating DNA molecule (e.g. plasmid or

viral DNA) used to transmit a gene from one organism into another

• All vectors must have the following properties:– Be able to replicate inside their host organism– Have one or more sites at which a restriction enzyme can

cut– Have some kind of genetic marker that allows them to be

easily identified• Organisms such as bacteria, viruses and yeasts have DNA

which behaves in this way• Large quantities of the desired gene can be obtained if the

recombinant DNA is allowed to replicate in an appropriate host

Gene cloning using plasmids• Plasmid vectors, found in bacteria, are prepared for cloning

in the following manner:

1. A gene of interest (DNA fragment) is isolated from any tissue cells2. An appropriate plasmid vector isolated from a bacterial cell3. DNA and plasmid are treated with the same restriction enzyme to produce

identical sticky ends4. DNAs are mixed together and the enzyme DNA ligase used to bond the

sticky ends5. Recombinant plasmid is introduced into a bacterial cell by simply adding the

DNA to a bacterial culture where some bacteria take up the plasmid from the solution

6. The actual gene cloning process (making multiple copies of the gene) occurs when the bacterium with the recombinant plasmid is allowed to reproduce

7. Colonies of bacteria that carry the recombinant plasmid can be identified by a genetic marker such as ampicillin resistance

Gene cloning using plasmids

Using bacteria to make proteins for human use

Gene cloning using viruses• Some bacteriophages are convenient for cloning large fragments of

DNA (15 to 20kbp)

• Main steps in preparing a clone using viral vectors:1. A gene is isolated from tissue cells2. An appropriate bacteriophage vector is selected that is capable

of infecting the target cell3. Tissue and the viral DNA are cut with same restriction enzyme4. DNAs are mixed together and the enzyme DNA ligase used to

bond the sticky ends5. The recombinant DNA is packaged into phage particles by being

mixed with page proteins6. The assembled phages are then used to infect a bacterial host

cell7. The viral genes and enzymes cause the replication of the

recombinant DNA within the bacterial host cell8. The bacterial host cell succumbs to the viral infection. The cell

ruptures (lysis) and thousands of phages, each with recombinant DNA, are released to infect neighbouring bacteria.

Gene cloning using viruses

Transgenesis• Trangenesis, using genetic engineering

techniques, is concerned with the movement of genes from one species to another. An organism that develops from a cell into which foreign DNA has been introduced is called a transgenic organism

• Because of their immense economic importance, plants have been the subject of traditional breeding programmes aimed at developing improved varieties

Transgenesis• Recombinant DNA technology now

allows direct modification of a plant’s genome allowing traits to be introduced that are not even present in the species naturally

• DNA can now be introduced from other plant species, animals or even bacteria

• Micropropagation techniques allow introduced genes to become par of the germ line for plants (the trait is inherited)

Transformation using a plasmid• Ti plasmid isolated from bacteria Agrobacterium tumefaciens.

Agrobacterium tumefaciens causes tumours (galls) in plants. • The Ti plasmid can be succesfully transferred to plant cells

where a segment of its DNA can be integrated into the plant’s chromosome.

• Restriction enzyme and DNA ligase splice the gene of interest into the plasmid as discussed previously for cloning into plasmids

• Introduce plasmid into plant cells• Part of the plasmid containing the gene of interest integrates

into the plant’s chromosomal DNA• Transformed plant cells are grown by tissue culture

Transformation using a plasmid

Transformation by protoplast fusion

• This process requires the cell walls of plant to be removed by digesting enzymes

• The resulting protoplasts (cells that have lost their cell walls) are then treated with polyethylene glycol (PEG) which causes them to fuse

• In the new hybrid cell, the DNA derived from the 2 “parent” cells may undergo natural recombination (they may merge)

Transformation by protoplast fusion

Transformation using a gene gun

• This method of introducing foreign DNA into plant cells, literally shoots it directly through cell walls using a “gene gun”

• Microscopic particles of gold or tungsten are coated with DNA and propelled by a burst of helium through the cell wall and membrane

• Some of the cells express the introduced DNA as if it were their own

Transformation using a gene gun

Transformation using liposomes

• Liposomes are small spherical vesicles made of a single membrane. They can be made commercially to precise specifications

• When coated with appropriate surface molecules, they are attracted to specific cell types in the body

• DNA carried by the liposome can enter the cell by endocytosis or fusion

• They can be used to deliver genes to these cells to correct defective or missing genes

Transformation using liposomes

Transformation using viral vectors

• Some viruses are well suited for gene therapy – they can accommodate up to 7.5kbp of inserted DNA in their protein capsule

• When viruses infect and reproduce inside the target cells, they are also spreading the recombinant DNA gene

• A problem with this method involves the host’s immune system reacting to and killing the virus

• Common viruses used for viral transformation of target cells are retroviruses, lentiviruses and adenoviruses

Transformation using viral vectors

Transformation using microinjection

• DNA can be introduced directly into an animal cell (usually an egg cell) by microinjection

• This technique requires the use a glass micropipette with a diameter that is much smaller than the cell itself – the sharp tip can then be used to puncture the cell membrane

• The DNA is then injected through it and into the nucleus

Transformation using microinjection

Making an artificial gene• Biologists get genes for cloning from two main sources

– DNA isolated directly from an organism– complementary DNA (cDNA) made in the laboratory from mRNA

templates• One problem with cloning DNA directly from an organism’s cell is that it

often contains long non-coding regions called introns• These introns can be enormous in length and cause problems when the

gene as a whole is inserted into plasmids or viral DNA vectors for cloning:– Plasmids tend to lose large inserts of foreign DNA– Viruses cannot fit the extra long DNA into their protein coats

• To avoid this problem, it is possible to make an artificial gene that lacks introns

• This is possible by using the enzyme reverse transcriptase which is able to reverse the process of transcription

• The important feature of this process is that mRNA has already had the introns removed, so by using them as the template to recreate the gene, the cDNA will also lack the intron region

Metabolic Engineering of the Terpenoid and Indole Pathways in Catharanthus roseus hairy roots

Catharanthus roseus (Madagascar Periwinkle) Produce a wide range of secondary metabolites Ajmalicine and Serpentine – hypertension Vinblastine and Vincristine – anticancer drugs used to

treat lymphomas and leukemia

vinblastine vincristine

Chorismate

Anthranilate

Tryptophan

Tryptamine

Pyruvate + G3P

1-Deoxy-D-Xylulose-5-Phosphate

2-C-Methyl-D-erythitol-4-phosphate

IPPDMAPP

GPP

Geraniol

10-Hydroxygeraniol

Loganin

SecologaninStrictosidine

Ajmalicine

Serpentine

Tabersonine

Lochnericine

Hörhammericine

Vindoline

Catharanthine

VinblastineVincristine

Terpenoid Indole Alkaloid Pathway

Indole Pathway

Terpenoid Pathway

MevalonateAS

DXS

STR

G10HTDC

Clone Generation

Plasmid Construction

in E. coli

Agrobacterium

Ri

Sterile Grown Plants

(5 weeks)

Infection

(6 weeks) Selection Media

(6 weeks)

Adapt to Liquid Media

(24 weeks)Transgene

Monoterpenoid Biosynthesis in Mint

•Metabolic Engineering of Terpenoid Biosynthesis

Why? Metabolic Engineering of Terpenoids in Plants

• In addition, plants altered in the profile of terpenoids (and pool of precursors) make an important contribution to fundamental studies on their biosynthesis and regulation

• REFERENCES• Monoterpene Metabolism. Cloning,

Expression,and Characterization of Menthone Reductasesfrom Peppermint

• Genetic Engineering on Shikonin biosynthesis: Expression of the Bacterial ubiA Gene in Lithospermum erythrorhizon

• Legume Natural Products: Understanding and Manipulating Complex Pathways for Human

and Animal Health

FaNES1, a Dual Linalool / Nerolidol Synthase

• SynthaseUsing FaNES1 allows evaluation of both mono and sesquiterpene