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. 2. 3.
4. 5.

6. 7.

A gene of interest (DNA fragment) is isolated from any tissue cells An appropriate plasmid vector isolated from a bacterial cell DNA and plasmid are treated with the same restriction enzyme to produce identical sticky ends DNAs are mixed together and the enzyme DNA ligase used to bond the sticky ends 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 The actual gene cloning process (making multiple copies of the gene) occurs when the bacterium with the recombinant plasmid is allowed to reproduce 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 cells 2. An appropriate bacteriophage vector is selected that is capable of infecting the target cell 3. Tissue and the viral DNA are cut with same restriction enzyme 4. DNAs are mixed together and the enzyme DNA ligase used to bond the sticky ends 5. The recombinant DNA is packaged into phage particles by being mixed with page proteins 6. The assembled phages are then used to infect a bacterial host cell 7. The viral genes and enzymes cause the replication of the recombinant DNA within the bacterial host cell 8. 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 plants 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 plants 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 plants 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 hosts 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 organisms 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

Terpenoid Indole Alkaloid Pathway

Chorismate AS Anthranilate Indole Pathway Tryptophan TDC Tryptamine STR Strictosidine Mevalonate DMAPP

Pyruvate + G3P DXS 1-Deoxy-D-Xylulose-5-Phosphate 2-C-Methyl-D-erythitol-4-phosphate IPP

GPP Geraniol G10H 10-Hydroxygeraniol Loganin Secologanin Terpenoid Pathway

Ajmalicine Serpentine

Tabersonine Lochnericine Hrhammericine Vindoline

Catharanthine
Vinblastine Vincristine

Clone Generation
Plasmid Construction in E. coli Transgene Agrobacterium Adapt to Liquid Media (24 weeks)

Ri

Infection
Sterile Grown Plants (5 weeks) (6 weeks) Selection Media (6 weeks)

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