MICROBIAL BIOTECHNOLOGY: SCOPE, TECHNIQUES CONTD.
Microbial endophytes (bacteria and fungi) are an enormous, highly diverse
component of the microbial world. Plant endophytes live in plant tissues
between living plant cells but generally can be isolated and cultured independent of the host. For some endophytes, there is evidence that genetic
exchange takes place in both directions between the plant and the endophyte. Such exchange raises the possibility that higher plant pathways for
the synthesis of complex organic molecules that have desirable biological activities might be transferred to their endophytes.
The story of the highly effective anticancer drug taxol provides proof of
the validity of this notion. Taxol, a highly substituted diterpenoid with multiple asymmetric centers was isolated in 1965 from the Pacific yew (Taxus brevifolia). In human cells, taxol prevents the depolymerization of microtubules during cell division. It has the same effect in fungi. Consequently, in nature, taxol is a fungicide.
Taxol proved to be an exceptionally effective anticancer drug,and demand far exceeded the amount that could be produced from the Pacific yew. Moreover, the level at which these slow-growing trees were being
utilized for taxol production threatened them with extinction. The development in 1989 of a commercially viable organic synthesis of taxol resolved
the problem. In the early 2000s, a plant cell fermentation process for taxol
production displaced the chemical synthesis. Here, calluses of a specific
Taxus cell line are propagated on a simple defined medium to produce taxol.
Even so, it would be advantageous if taxol could be produced by an inexpensive microbial fermentation. The Pacific yew is not the only tree that
produces taxol. This compound is in fact found in each of the world’s Taxus
species. The possibility was then explored that a taxol-producing endophyte
might be discovered in a Taxus species. In 1993, a taxol-producing endophytic fungus,Taxomyces andreanae, was discovered in T. brevifolia. Subsequently, fungal endophytes in a wide variety of higher plants were found to make taxol. In culture, these endophytes make taxol in sub micro gram per
liter amounts. A great deal of work remains to be done to achieve high levels of microbial taxol production.
Fig: Taxus brevifolia
Methods dependent on microbial biotechnology greatly increases the diversity of genes that can be incorporated into crops plants dramatically shorten the time required for the production of new varieties of plants.It is now possible to transfer foreign genes in the plant cells. Transgenic plants
that are viable and fertile can be regenerated from these transformed cells,
and the genes that have been introduced into these transgenic plants are
as stable as other genes in the plant nuclei and show a normal pattern of
inheritance. Transgenic plants are most commonly generated by exploiting a plasmid vector carried by Agrobacterium tumefaciens, a bacterium. Foreign DNA carrying from one to
50 genes can be introduced into plants in this manner, with the donor
DNA originating from different plant species, animal cells, or microorganisms.
Higher plants have genes whose expression shows precise temporal and
spatial regulation in various parts of plants – for example, leaves, floral
organs, and seeds that appear at specific times during plant development
and/or at specific locations, or whose expression is regulated by light. Other
plant genes respond to different stimuli, such as plant hormones, nutrients,
lack of oxygen (anaerobiosis), heat shock, and wounding. It is therefore possible to insert the control sequence(s) from such genes into transgenic plants
to confine the expression of foreign genes to specific organelles or tissues
and to determine the initiation and duration of such expression. Microorganisms that live on or within plants can be manipulated to control insect pests and fungal disease or to establish new symbioses, such as those between nitrogen-fixing bacteria and plants.
In bacteria and yeast, trehalose-6-phosphate is synthesized from UDP-glucose and glucose-6-phosphate in a reaction catalyzed by trehalose-6-phosphate synthase (OtsA). Trehalose-6-phosphate phosphatase (OtsB) then converts trehalose-6-phosphate to trehalose.
ABILITY TO GROW IN HARSH ENVIRONMENTS
Extending the habitat range for plants may be achieved by imparting traits
such as cold, heat, and drought tolerance; ability to withstand high moisture
or high salt concentrations; and resistance to iron deficiency in very alkaline
soils. Tolerances toward environmental stresses are likely to be polygenic
traits and as a consequence may be difficult to transfer from one kind of
organism to another. However, there are some successes, as illustrated by
the following example.
Trehalose, a disaccharide of glucose, acts as a compatible solute that
stabilizes and protects proteins and biological membranes in bacteria,
fungi, and invertebrates from damage during desiccation. Except for highly
desiccation-tolerant “resurrection plants,” most plants do not accumulate
detectable amounts of trehalose. E. coli genes otsA and otsB for trehalose
biosynthesis were introduced into . An otsA–otsB fusion
gene was generated so that only a single transformation event would be necessary and to achieve a higher catalytic efficiency of trehalose formation. To obtain either tissue-specific or stress-inducible expression, two different constructs were made. In one, the fusion gene, equipped with a transit
peptide, was placed under the control of the promoter ofrbcS, the gene
encoding the small subunit of ribulose bisphosphate carboxylase, to direct
the gene product to the chloroplast. In the second, the gene was placed
under the control of an abscisic acid–inducible promoter. Here, the OtsA–
OtsB enzyme fusion remains in the cytosol. The constructs were introduced
into rice using Agrobacterium-mediated gene transfer.
Compared with non transgenic rice, several independent transgenic lines
showed sustained plant growth under drought,salt,or low temperature stress
conditions. The transgenic rice contained three- to nine fold greater levels
of trehalose than the non transgenic rice. However, the striking finding was
that the trehalose level did not exceed 1 mg/g wet weight of tissue under
any conditions. Consequently, in rice, trehalose must exert its protective
effect indirectly rather than primarily through affecting the bulk properties
of water within the plant cells. A detailed analysis of the transgenic rice with
each of the constructs showed less photo oxidative damage to photosystem II
(allowing maintenance of higher capacity for photosynthesis), higher levels
of soluble carbohydrate, and greater ability to control potassium ion to sodium ion
balance in the roots under the stress conditions, than seen in non transgenic rice controls.
These results indicate that in rice, trehalose acts as a regulatory molecule that
affects the expression of genes associated with carbon metabolism and those
involved with ion uptake and possibly other processes as well. This example offers a valuable lesson. The presence of homologous genes in widely
diverged organisms that catalyze the synthesis of the same product offers
no guarantee of a universal identical role for the product.
Initial field trials on the transgenic rice are promising and offer the
prospect of growing rice in saline soils, or in areas where availability of water
would depend on intermittent rainfall.
Some Useful References Site:
www.cancerresearchuk.org › ... › Treatment › Cancer drugs
www.drugs.com › Drugs by Condition › Breast Cancer
Cited By Kamal Singh Khadka & Shailendra Parajuli
Msc Microbiology, TU.
Assistant Professors In PU, Pokhara Bigyan Thata Prabidhi Campus, PNC, NA, LA.