TECHNOLOGY

 
1- HP VICE-PRESIDENT OF THE IPG VISITS PAKISTAN
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APPLICATION OF BIOTECHNOLOGY
 

APPLICATION OF BIOTECHNOLOGY

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Major accomplishments in raising HP leadership in the IT industry has taken

 

By Dr S. M. ALAM
 Apr 18 - 24, 2005
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Due to recent technological revolution in the bio-chemical methodology, the old discipline of fermentation technology has been rejuvenated. Consequently, the new term of "Biotechnology'' has emerged which include process that exploit versatile metabolic machinery or component of living organisms to produce valuable metabolite from renewable resources. Infect biotechnology is a scientific discipline with focus on the exploitation of metabolic properties of living organisms for the production of the valuable products of a very different structural and organizational level for the benefit of men. The product can be the organism themselves (i.e. biomass or the parts of the organismic body), product of cellular or organismic metabolisms (i.e. enzymes, metabolites) or product formed from endogenous or exogenous substrate with the help of single enzymes or complex metabolic routes.

The products of biotechnology are of importance for medicine pharmaceutical sciences, agriculture, food production, chemistry and numerous other disciplines. Biotechnology receives the necessary scientific and technical information from a considerable number of disciplines, cell biology, morphology of the implied organisms, biochemistry, physiology, genetics and various technical field are major sources. In the last two decades, molecular biology and gene technology have substantially contributed to the spectrum of scientific discipline forming biotechnology. As is always true for progress in natural sciences, it is specially true for biotechnology that more rapid development and again of higher standards depend on the improvement of methods.

AGRICULTURE

Genetic engineering of the plants is the next green revolution the technology has already allowed expression of many alien gene into all kinds of plants. This new biology provide tool to augment conventional plant breeding. Plant variety with good agronomy characteristic can be further improved for better processing solid context, flavour, colour, texture, acidity, sweetness size and shape of edible parts. Other characteristic can be modified include suitability for mechanical harvesting, self storage and resistance to disease pests, insects and frost. The two biotechnological techniques most commonly used in agriculture are tissue culture and genetic engineering. The former has proved its potential and is commercially adopted in most countries of the world. In Pakistan, it had made a dent in the economy by the production of disease free potato locally. Other crops are banana, sugarcane and ornamental and horticultural crops.

The recent completion and publication of the first complete genome sequence of a flowering plant the Brassica Arabidopsis thaliana represent a further grant step forward. The full genomic sequence of this plant provide a mean for analyzing gene function that is also important for other plant species, including commercial and agricultural crops. Plant biotechnology greatly benefits from the aradopisis genome project. The large set of identified genes and also the hither to functionally, unknown, predicted genes from the basis for more sophisticated plant genetic analysis and plant improvement by construction of plants better adapted to human needs. Plant biotechnology application, i.e. selection of specific lines, genetic modification or transformation at sites of characteristic plant - specific properties.

A considerable number of the nuclear gene products (approximately 14%) are predicted to be targeted to the chloroplasts as indicated by appropriate signal peptide sequences such as valuable indicates the massive in fluex of nuclear-coded proteins into plastids. Further biotechnology applications of these documented plant transporters are for example, concentration of more facile cellular sequestration processes for biologically active or toxic xenobiotic (i.e. pesticides, parasitic toxins) into vacuoles. A fascinating new technique (DNA micro array technology) allows the determinations RNA expression profile on the genome level with many hundred genes at the same time. The process of specific DNA-mRNA hyberdization can be followed or automatically recorded by various techniques of light emission or colour formation. It is easily to predicted that in the future plant sciences will benefit from DNA micro-array technology to the same extent as already shown for medical and pharmaceutical application.

Secondary metabolites will undoubtedly continue to be a great importance. Detecting isolating and producing biologically or pharmaceutically active secondary plant metabolites are high priority objectives in many laboratories around the world. Another interesting aspect of the search for new secondary products is the fact that most likely all plants have a much greater genetic potential for the fermentation and accumulation of such products than actually expressed during normal condition Under stress (i.e. heat, cold, drought, high salt concentration) and other difficult environment conditions (i.e. UV irradiation, high light intensity) plants tend to form a much wider spectrum of secondary metabolites. Thus, numerous compounds (e.g. alkaloids, quinines, phenolics, lignons) are found as stress related metabolites. Especially in response to pathogen infection, a wide range of anti microbial compounds called phytoalexins are inducibly formed denovo around infection sites. The importance of phytoalexins as efficient anti microbial defense compounds is elegantly demonstrated by the transfer of the genes encoding key biosynthetic enzymes into plants that do normally not produce these compounds. A valuable technique for biotechnological application is connected with cell suspension cultures of the experimental plants in which secondary product accumulation and phytoalexin formation are stimulated or induced by treatment of cultures with microbial elicitors. These signal compounds of very different chemical structure all tend to interfere with cell metabolism via signal transductions cascades to induce stress and defense responses.

Transformation of mitochondria genome is in its infancy. Successful attempt to engineer the chloroplast genome have so for been restricted to very few systems, but routine procedures with other crop plants suitable for biotechnology application are slowly emerging. The new technology can be offered for the introduction of new biosynthesis pathways, resistance management of crop plants and the use of plants as factories for biopharmaceuticals, proteins, enzymes or peptides. Some of the potential gains that can be achieved with the technology include higher yields, developing disease resistance plants, enhancing productivity and improving product quality. All these will have a direct bearing on improve food security and environmental conservation. Pakistan researchers have indigenously evolved genetically modified varieties of various crops which are ready to be launched but absence of biosafety guidelines is a hindrance towards delivery of this new technology to the farmers.

Biotechnology has immense applications in agriculture. Development of agricultural science and technology has a direct bearing on the humans health, the improvement of environment, economic prosperity as well as social stability.

BIOCHEMICAL PRODUCTS

Microbial synthesis of bioactive chemotherapeutics is an important venue of classical fermentation technology. These molecules have a wide application in veterinary medicine, agricultural and human antiviral, antibacterial, antifungal, anticoccidal, antiprotozeal, antihelminthic, antilumor, pesticidal and insecticidal activity. Most of the microbially produced molecules are modified chemically to produce analogs with superior biological activity. Most novel analogues evermectins, levastatin, penciline, cephalosperins, aminoglycosides, chloroamphenicols have been prepared.

Recombinant DNA technology will be used to create substrates for chemical modification and hydride molecule of complex chemical structure. Japanese biotechnologists have already introduced hydrolytic enzymes in Acremonium chrysogenum to synthesize 7-ACA by fermentation. Gene for efficient utilization of soy, carbon and nitrogen sources may also be transplanted into organisms which were originally isolate from soil. Construction of industrial strains with amplified copies of desired genes is now possible. Generation of strains that have enhanced ability to transport metabolites out of the cell and are blocked in acid production will further contribute to yield enhancement of antibiotics. It is believed that worldwide use of enzymes are often products of single genes and modern methods of recombinant DNA technology can be used to enhance their yields and modify their specificities is suitable microbial hosts. To reduce the expense of producing and using enzymes, bacterial strains improvement, enzymes and cell immobilization and stability enhancement are being explored. Protein thermo stabilization design rules may be important guide for engineering of stabilized industrial enzymes. Enzymes complexes are used by cells to perform complex tasks with higher efficiency of fidelity. Artificial enzyme complex may also be constructed to achieve the same objectives. Today scientist are creating modified enzymes that have properties superior to those of natural enzymes are created in three ways:

1. Genetic manipulation of the organisms to select a variant of the natural enzyme.

2. In vitro mutagenesis of the gene and its expression in a suitable host.

3. Production of a completely new enzyme by creating a catalytic antibody. The long-term goal is the rotational design of efficient, biologically active enzymes.

Protein engineering is based on genetic engineering, X-ray crystallography, protein structural modification, synthesis, analysis, interpretation, scale-up and purification. The rotational design of peptide-based HIV proteins inhibitors using the three dimensional structure of the enzyme has already been achieved and approved as a therapy for AIDS.

Site-directed mutagenesis enables precise amino acid replacement any where in the protein structure. Protein whose three-dimensional X-ray structures have been determined are good candidates for such genetic modification although trying to make meaningful amino acid sequence changes is still very much guesswork. Novo Nordisk has engineered a bleach-resistant detergent protease "Durazym" in which methionine is substituted by other amino acids. This enzymes is active in detergents to remove protein-based strains. This company is designing additional enzymes for various purposes. Use of other enzymes (e.g. heat stable alkaline proteases, amylases and lipases) in automatic dish washing detergents is increasing as a result of demand for safer products.

LIVING ORGANISMS

 

 

Recombinant DNA technology allows the isolation of cDNAs that determine that exist in the human body in minute quantities only. These cDNAs are being expressed in a variety of cells to mass produce their protein products commercially. Human insulin and human growth hormone are already being produced in E.Coli and sold commercially by Genetech. Angen has produced a recombinant granulocyte colony simulating factor (rG-CSF) to fight infections associated with some anticancer drug therapies. Tissue plasminogen activator, produced bacteria is a commercial product worth 300 million. Genentech's gamma interferon is approved for treating chronic granulamatous disease, a genetic deficiency of the immune system that results in life-threatening infections. Erythropoietins is being sold by Amgen to increase red blood cell production and lymphokines produced by inteferons and lymphokines produced by microbial means are in the market and many more are in clinical trails to test their efficiency. Eukaryotic host cell grown in tissue culture. A new generation of serum-free media are now being commercially introduced to facilitate isolate of recombinant DNA protein expressed in Eukaryotic cells.

The development of DNA micro-injection techniques are spawned the emergence of transgenic animals carrying foreign genes and propagating them to their offspring gene expression can be studied in vivo, for example in human growth hormone has be successfully transferred to mice , rabbits , sheep and pigs with the hope of someday, providing a genetic therapy for human dwarfism. Transgenic cows carrying bovine somatotophin have increased milk production by 10-25% although the social benefits of this effect are still debated. DNAX INC. Princeton, NJ has produced genetically-engineered pigs that synthesized human hemoglobin. Researcher at the institute of animal. Physiology and genetic research in Edinburg and Sheatland were the first to produced a transgenic sheep that secreted in their milk low levels of human antithemophilic.

ENGINEERING VACCINE AND ANTIBIOTICS

Unlike traditional polyvalent vaccines, genetically engineered vaccine contain only one or a few major antigen of a pathogenic agent that are capable of producing a neutralizing immune response upon infection. Disease that effect a large proportion of the world's population have received high priority for the production of recombinant vaccine. For example China, more than 85% of population has been exposed to Hepatitis B. The gene for the surface antigen HbsAg of the hepatitis B virus was cloned when expressed in Yeast and used as an immunogen generated a protective immune response. Human disease such as measles polio, tuberculosis, leprosy, and malaria may also someday be controlled or even deradicated by the use of genetically-engineered vaccine.

Genetic engineering approaches have been use to make these antibodies more humanized, that is by replacing the mouse antigen-combining regions (VH AND VL) with human constant heavy and light regions. Such chimeric antibody molecule are not less immunogenic in humans, but also retain the antigen specificity of the original monoclonal antibody. Another approach make use of the antibody fragments that retain antigen-binding properties. Antibody fragments are more effective diagnostic agent for applications , such as radio imaging, where radio isotopes are conjugated.

FLAVORS AND FRAGRANCES

Flavors and fragrance industry may exploit high quality plant derived flavors. Somaclonal variation was used to produce tobacco cultivars was high levels of the musky fragrance, sclareol. Sclareol and abienol are used to create ambrox, a fragrance needed in many products. Haareman and Reiner have described microbial preparation of vanillin from eugenol and isoeugenol. Already available biochemically-produced aroma chemicals are: aldehydes and ketones, (acetaldehyde, diacetyl) acids (acetic, butyric , caproic, caprylic isobutyric, isovelaric, 2-methyl butyric) esters (ethyl and butyl acetate, ethylbutyrate, caproate, isobutyrate, isovalerate, 2-methylbutyrate, methyl acetate) and lactones (gamma-dacalactone).

The roll of lectococcal peptidases in the production of flavor peptides during cheese manufacturing is known (Mulholland 1991). Corynebacterium glutamicum produces 3g/l of tetramethylpyrazine in glutamic acids fermentation. Pyrazines are powerful aroma compounds that occur naturally in food as a result of browning and heating. Biotechnology could expend the opportunities for producing flavors and fragrances that are difficult to synthesize chemically (Moyler 1991). Chocolate flavors, thaumatin and monellin can be produced in yeast (kondo et al, 1997 ).

AQUACULTURE

The oceans offer abundant resources for research and development, yet the potential of this domain as the basis of new biotechnologies remains largely unexplored. Indeed, the vast majority of marine micro-organisms have yet to be identified. Even for known organisms, there is insufficient knowledge to permit exploitation. Oceanic organism constitute a major share of the earth's biological resources and often possess unique structure, metabolic pathway, reproductive systems, and sensory, and defense mechanisms. Aquaculture will involve farming of aquatic organisms, including fishes, mollusks, crustaceans, and plants. The genetic nutritional and environmental factor that control the production of primary and secondary metabolites in marine organisms need to studied Unicellular and multicellular microorganisms that are unique to marine world are emerging as a significant chemical resources. The method of transferring genes of interest into non-marine microorganisms also should be developed. For example the capability to produced a marine polysaccharide, a complex molecule that could be useful as a food additive or a water-resistant adhesive, could be transferred to an easily grown plant or bacterium.

Marine natural product may provide a major new class of drugs. The compound monoalide from a specific sponges, for example, has spawned more than 300 chemical analogs as anti inflammatory agents. Rapidly developing assay technology can facilitate exploration of the bioactivity of newly discovered compounds. Enzymes produced by marine bacteria are important in biotechnology due to range of unusual properties. Some are salt-resistant, a characteristic that is often advantageous in industrial processes, such as in cleaning reverse-osmosis membranes. An unusual group of marine microorganism from which enzymes have been isolated are the hyper thermophilic archaea, which can grow at temperature over 1000C and produce that are stable at high temperatures. Thermo stable DNA-modifying enzymes, such as polymerase, ligasase, and restriction endonuclease, already have important research and industrial applications.

Marine biochemical process can be exploit to produce new biomaterials. One type of biosensor employ the enzymes responsible for bioluminescence. The lux, gene, which encode these enzymes, have been cloned from marine bacteria Vibrio fisheri and transferred successfully to a variety of plants and other bacteria. When lux genes are `inserted into a toluene operon, the genetically-engineered system "reports" that biodegradation of a specific chemical is proceeding.

OPU AND IVM/IVF

OPU in mammals allows the repeated pick-up of immature ova directly from the ovary without any major impact on the donor female and the use of these ova in IVM/IVF programmes. Making much greater use of genetically valuable females at a very early age may substantially increase genetic progress. What potential uses of these technologies are feasible in developing countries? What are the required technical and/or policy elements that will enable developing countries to make practical use of these technologies?

Sexing: Technologies for rapid and reliable sexing of embryos allow the generation of only the desired sex at specific points in a genetic improvement programme, markedly reducing the number of animals required and enabling increased genetic progress. Sexing of semen using flow-cytometric sorting has decisively progressed in recent years but still with limited sorting rates, even for IVF. Sexed semen could markedly increase genetic improvement rates and have major implications for end-product commercial production. What is the scope for the use of these technologies in developing countries?

Cloning: IVM/IVF are a source of large numbers of low cost embryos required for biotechnologies such as cloning and transgenesis. Three different types of clones are distinguished, as a result of: (1) limited splitting of an embryo (clones are genetically identical); (2) introducing an embryonic cell into an enucleated Zona (clones may differ in their cytoplasmic inheritance); (3) introducing the nucleus of a somatic cell (milk, blood, dermal cells), after having reversed the DNA quiescence, into an enucleated Zona (clones may differ in their cytoplasmic inheritance and there is likely to already exist substantial knowledge of the phenotype of the parent providing the somatic cell). Cloning will be used to multiply transgenic founder animals. Cloning technologies offer potential as research tools and in areas of very high potential return. The sampling of somatic tissue may assist collection and transfer of breed samples from remote areas for conservation purposes.

Rumen biology: Rumen biotechnology has the potential to improve the nutritive value of ruminant feedstuffs that are fibrous, low in nitrogen and of limited nutritional value for other animal species. Biotechnology can alter the amount and availability of carbohydrate and protein in plants as well as the rate and extent of fermentation and metabolism of these nutrients in the rumen. The potential applications of biotechnology to rumen microorganisms are many but technical difficulties limit its progress. Current limitations include: isolation and taxonomic identification of strains for inoculation and DNA recombination; isolation and characterization of candidate enzymes; level of production, localization and efficiency of secretion of the recombinant enzyme; stability of the introduced gene; fitness, survival and functional contribution of introduced new strains.

Methods for improving rumen digestion in ruminants include the use of probiotics, supplementation with chelated minerals, and the transfer of rumen microorganisms from other species.

INCREASING THE SPEED OF GENETIC IMPROVEMENT OF LOCALLY ADAPTED BREEDS: There are many links in the chain to realizing rapid genetic progress in the desired goals, with the objective being to rapidly transmit from selected breeding parents to offspring those alleles which contribute to enhanced expression of the traits of interest. In developing countries, generation intervals are generally longer for all animal species of interest than in developing countries. How can DNA technologies be used to reliably realize intense and accurate selection and short generation intervals and to enable genetic improvement of these many locally adapted breeds to contribute to the required livestock development?

There is rapid progress in the preparation of sufficiently dense microsatellite linkage maps to assist in the search for genetic traits of economic importance. Can these linkage maps be used to develop strategies of marker-assisted selection (MAS) and marker assisted introgression (MAI) to meet developing country breeding goals? How should this be approached? Given the limited financial resources, how might work for the developing country breeding programmes strategically utilise the rapidly accumulating functional genomic information of humans, mice and drosophila?

Transgenic animals have one or more copies of one or various foreign gene(s) incorporated in their genome or, alternatively, selected genes have been 'knocked out'. The fact that it is possible to introduce or to delete genes offers considerable opportunities in the areas of increasing productivity, product quality and perhaps even adaptive fitness. In initial experiments, genes responsible for growth have been inserted. The technology is currently very costly and inefficient and applications in the near future seem to be limited to the production of transgenic animals as bio-reactors. What is the potential significance of these advanced technologies for developing countries and what are the technical, societal, political and ethical determinants of their application?

CONSERVING GENETIC DIVERSITY: Global surveys indicate that some 30% of all remaining livestock breeds are at risk of loss, with little conservation effort currently invested. The majority of domestic animal breeds are in developing countries. Whilst animals cannot be re-formed from DNA alone, the conservation of genomic DNA may be useful. Under what circumstances should DNA genomic material be conserved and how should this be done by developing countries? What other information should be retained and what policy issues need to be taken into account?

PRODUCT CATEGORY

EXAMPLE

END USE

Amino acids

Cysteine, lysine,glutamate, metionine, proline, threonine, phenylalanine, tryptophan

Food enrichments, flavorants.

Biopolymers

Polyamylose, poly-B-hydroxybuterate, alginate, cellulose, curdlan, levan, dextran, xenthan.

Biodegradable wrapping. Food thinking

Microbial cells

Yeast, lactobacilli, streptococci.

Brewing baking, wine and heese making

Steroids

Cortisone

Treatment of arthritis

Ergot alkaloid

Ergotamine, agroclavine

Therapeutics

Microbial proteins

Methylophuius methylotropus, yeast, algae

Single cell proteins

Vitamins

B-2, B-12D, C, nicotinic acid

Food supplement

Peptides,proteins

Aspartame, thaumatin

Sweeter

Carotenoid

B-Carotene

Colorant

Organicacids

Geraniol, isobutylene, linalool, nerol, nucleotides, aceticacids, benzoicacids, citric, acids, fumaric acids.

Flavorants

Organicacids

Gibberellins

Plant growth promoters

Organic feed stocks

Ethanol, butanol, aceticacids, propylene, glycerol, 2, 3-butanol, methyl ethyl ketone.

Solvents, fuels

Secondary metabolites

Cyclosporin, FK-506

Immunomodulators

Enzymes inhibitors

Antipain, chymostatin, leupeptin, pepstatin L-phenylacetylcarbinol.

Protease inhibitors. Precursor of L-ephedrine