Adeena ShafiqueSana Javed

Tehreem TanveerUzair Hashmi

Muhammad Nasir Younis

Class: UG-3Submitted to: Sir Zaffar MehboobSubmission date: November 12th, 2012

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TABLE OF CONTENTS:

INTRODUCTION TO rDNA TECHNOLOGY

THE TECHNOLOGY

FOOD AND rDNA TECHNOLOGY USES

RECOMBINANT FOOD CONTROVERSIES

REGULATIONS FOR rDNA-DERIVED FOOD

REGULATIONS FOR rDNA-DERIVED FOOD

APPLICATION OF RECOMBINANT DNA TECHNOLOGY IN AGRICULTURE

DEVELOPMENT OF STRESS TOLERANT PLANTS

DEVELOPMENT OF PLANTS HAVING INCREASE IN QUALITY OF PLANT PRODUCTS

TRANSGENIC PLANTS AS A SOURCE OF BIO PHARMACEUTICALS

RECOMBINANT ENZYMES USED IN FOOD PROCESSING INDUSTRIES

FOOD INGREDIENTS

CONCLUSION

USES OF RECOMBINANT DNA TECHNOLOGY IN WINE INDUSTRY

GENETICALLY MODIFIED GRAPEVINES

CONTROVERSIES AGAINST GM GRAPEVINES

WINE YEAST IMPROVEMENT

WINE BACTERIA IMPROVEMENT

BUSINESS PLAN

INTRODUCTION TO rDNA TECHNOLOGY

The birth of Recombinant DNA technology is accredited to Paul Berg, Herbert Boyer and Stanley Cohen. In his goal to insert new genes into living cells, Berg was the first scientist to splice the DNA segments from bacteriophage Lambda using restriction enzyme ECoR1, and inserting them into the Simian Virus 40 DNA- thus producing the very first recombinant DNA molecule. Later in 1973, Herbert Boyer and Stanley Cohen made further advances in the technology by producing a recombinant plasmid using the same method as Berg, and then introducing this plasmid into E.Coli cells by transformation. This resulted in the production of a recombinant organism for the first time in history.

Further developments which lead to the true advent of rDNA technology included the production of the first human protein, Somatostatin-a growth hormone regulator, using rDNA Technology by Genentech in 1978. The technology has been progressing ever since.

Recombinant DNA Technology modifies the genome of an organism through transformation, by inserting DNA from another organism with desired genes. Also known as genetic engineering, the rDNA technology is now widely used in the production of human proteins and drugs, biotechnological products, research and the food industry to create genetically-modified organisms (GMOs) that produce genetically-modified (GM) products. The first GM food was the Flavr Savr tomato, produced in 1994, which had a longer shelf life and an enhanced flavor. Since then, the number of GMOs has exploded as producers prefer them over traditional crops because they yield more and require less care.

THE TECHNOLOGY

The basic mechanism used in rDNA technology involves selecting the desired gene and the cloning vector, their ligation and formation of the rDNA, the transformation of this rDNA into a suitable host, which would then produce the desired gene product as a result of transformation. Once this gene product is produced, it can result in improved enzymes, crops, vitamins and amino acids, optimized microorganisms for beer and wine industry and several other food uses, depending upon the purpose the rDNA was synthesized. The major steps involved in the technology are described below.

Isolation and Preparation of Gene of Interest: The desired gene which is to be cloned is extracted from the organism which produces it naturally or is obtained from the gene library. This purified donor DNA segment is then treated with restriction endonucleases; these are the enzymes which cleave DNA strands at specific sites and result in the formation of sticky ends.

Preparation of the Cloning Vector: The vector used for cloning is a plasmid in most cases, which is a circular DNA molecule present in bacteria such as E.Coli. The plasmid has independent, self-replicative properties and is manipulated for biotechnological purposes. It also possesses selectable markers and insertion sites, where the foreign donor gene is inserted. Other

than bacterial plasmids, Ti plasmids, bacteriophages, cosmids and yeast artificial chromosomes may also serve as the cloning vector, which require essentially the same procedure as the plasmids for foreign gene expression. The plasmid is also treated with the same endonuclease enzyme as the one used for cleaving the gene of interest. Thus the plasmid DNA is also cleaved at same specific sites, producing sticky ends in it.

rDNA Formation: The use of the same restriction enzyme for cleaving the foreign gene and the vector results in the formation of sticky ends in both, which are complementary to eachother. This complementarity helps the gene of interest and the plasmid vector to ligate together in the presence of ligase enzyme- thus forming the recombinant DNA (rDNA).

Transformation: The rDNA is introduced into the host organism, where it would be replicated, transcribed and translated to produce the desired gene product. The host organisms are usually bacteria such as E.coli, which take up the recombinant vector, and help it to replicate and translate, producing copies of the desired recombinant protein. Methods such as electroporation (DNA translocation across the cell membrane using high voltage pulses) and packaging the DNA inside phages may be used for transforming the host organism.

Screening rDNA Clones: The bacteria and other host organisms which are successfully transformed are selected and screened using various methods such as selectable markers present in plasmid vectors, DNA hybridization methods, and antibody probes etc. The selected organisms producing the desired gene products are then utilized for food uses.

FOOD AND rDNA TEECHNOLOGY USES

The recombinant gene products produced through the process above may directly be used for food purposes; for example, these gene products could be vitamins (Vitamin C, riboflavin etc.), which would be taken as supplements. They could also be food additives, artificial sweeteners (aspartate and other amino acids) and flavor enhancers (monosodium glutamate).

Else, these gene products may optimize the bacteria and other hosts to function efficiently; for example, these products could be more efficient enzymes, which are more stable, more specific or have a better productivity. Thus these enzymes (chymosin, rennin etc.) would be used to give better results in industries such as wine, beer and cheese industries. The productivity of these industries may also be enhanced due to the optimization of fermenting bacteria in terms of better ethanol production.

rDNA Technology can also be used to produce genetically modified plants with characteristics such as better productivity, resistance to pesticides, herbicides, insecticides, and diseases, and imparting qualities such as tolerance to environmental stress. The food quality may also be enhanced by improved nutritional density, prolonged shelf life by delayed ripening and better flavors.

RECOMBINANT FOOD CONTROVERSIES

Although the use of rDNA technology in food industry has many benefits of improved food quality and quantity, this area is still under tremendous amount of scrutiny from the public. This is the reason that genetically modified foods have still not revolutionized the food industries particularly in Europe and Asia. Recombinant food safety is a topic of heated debates, and the public is concerned about various issues regarding the safety, ethics and environmental aspects of these foods. Major issues are mentioned below.

Food safety

In rDNA technology, the genetic makeup of an organism is modified by the introduction of genes from another organism. This DNA insertion is an uncontrolled event, which can possibly result in genetic alterations in different ways, giving rise to the potential of creating unintended effects. For example, the inserted DNA may interfere with the regulation and expression of various genes which exert a positive influence on the required GM product. This can produce health hazards in the modified products such as

1. Toxins2. Allergens3. Nutritional loss

Such faulty insertions are unique to each insertional event, and thus it is practically impossible to search for every possible effect that a gene insertion might have.

Environmental Concerns

The genetically modified crops can impact the environment by escaping into the natural process of gene flow. The pollens or seeds of the modified agricultural crops can escape into the environment, and can thus spread the engineered gene into natural strains and species of crops thus threatening the wildtype crop species for competition. Another concern is associated with the insect resistant crops; the resistance mechanism of these crops is non-specific, thus they have

the potential of threatening the beneficial insects too, which could disturb the ecosystem, posing a serious concern for the integrity of biodiversity.

Ethical and Social Issues

There are many concerns over the social and bioethical aspects of the rDNA derived foods. Many people are opposed to the idea of altering the natural genetic makeup of a species in the first place, terming it as an ‘unnatural abomination’. Others are opposed to the idea of transferring genes from animals to plant foods due to their religious beliefs which prohibit the diet of some or all of the animals. Yet others have concerns about the disruption of the ecosystem and thus consider rDNA food technology as unethical.

All these controversies associated with genetically modified food are valid, and are addressed by the regulatory authorities who assess the food safety and quality. These authorities are also responsible for assuring that the environment and biodiversity isn’t affected to a large extent, and are also accountable for labeling the foods which may contain prohibited animal derived products to counter the ethical and social liabilities.

REGULATIONS FOR rDNA-DERIVED FOOD

The safety assessment of the genetically engineered foods is conducted under the ground rules provided by the consensus among WHO (World Health Organization), FAO ( Food and Agricultural Organization) and OECD (Organization for Economic Co-operation and Development) and follows the basic concepts of risk assessment, risk management and risk communication.

The safety testing involves nutritional analysis and compositional studies, toxicological studies as well as the allergenicity studies. These analyses follow the ‘substantial equivalence’ concept, according to which the engineered products are compared to the natural products-which are set as standard- and the engineered foods are evaluated according to their natural counterparts. Any health hazards, if suspected under any circumstances in the finished product are labeled so that the consumer is aware of any potential risks of allergies and anaphylaxis, and of the nutritional charecteristics if they vary from the unengineered product.

Environmental concerns which exist in the case of modified crops are controlled by preventing horizontal gene transfer within and among crop species. This can be done by isolating the growth of genetically modified crops from the wild type species. Special fields are allocated to plant rDNA derived crops, which reduces the chances of the fleeing of seeds and pollen of these crops to areas where natural crops are grown.

Dealing with the ethical concerns is tough, and the most accepted regulatory rule is to label the foods which contain animal-derived genes in them so that the believers of certain religions or school of thoughts can differentiate between labeled and unlabeled products.

rDNA technology is a field of tremendous potential for food uses in today’s world of ever-increasing population size and depleting food and nutrients. If managed properly under the

regulatory guidelines, this technology can possibly alleviate food, hunger and malnutrition conditions due to the sufficient availability of rDNA foods such as crops, fermentation industries, enzymes and other nutrients such as amino acids and vitamins.

APPLICATION OF RECOMBINANT DNA TECHNOLOGY IN AGRICULTURE The genetic manipulation of plants has been going on since prehistoric times, when early farmers began carefully selecting and maintaining seeds from their best sow for the next season. Plant breeders have cross fertilized related plants to provide next generation plants with new characteristics such as higher yield, resistance to diseases and better nutrient content long before the science of genetics was developed. Recombinant DNA technology can be used for insertion of genes in plants not only from related plant species, but also from unrelated species such as microorganisms. This process of creation of transgenic plants is far more precise and selective than traditional breeding. Application of recombinant technology is primarily for the production of transgenic plants with higher yield and nutritional values, increased resistance to stress and pests. Several commercially important transgenic crops such as maize, soybean, tomato, cotton, potato, mustard, rice etc. have been genetically modified. During the last couple of decades, considerable progress has been made to understand the function of genes, isolation of novel genes and promoters as well as the utilization of these genes for the development of transgenic crops with improved and new characters. There are many potential applications of plant genetic engineering. In fact, in 2002, more than 5.5 million farmers worldwide cultivated about 58.7 million hectares (about 148 million acres) crops that were genetically manipulated for herbicide tolerance, insect resistance, delayed fruit ripening and improved oil quality. Application of recombinant DNA technology has primarily helped in producing three major types of transgenic plant having improved performances. These are:

1. Development of stress tolerant plants 2. Development of plants having improved yield 3. Transgenic plants as a source of biopharmaceuticals

DEVELOPMENT OF STRESS TOLERANT PLANTS

(a) Plant resistant to environmental stress: Plants need to cope up with abiotic stresses such as drought, cold, heat and soils that are too acidic or salty to support plant growth. While plant breeders have successfully incorporated genetic resistance to biotic stresses into many crop plants through crossbreeding, their success at creating crops resistant to abiotic stresses has been more limited, largely because few crops have close relatives with genes for resistance to these stresses. Therefore rDNA technology is being increasingly used to develop crops that can tolerate difficult growing conditions.

(b) Herbicide Resistant plant: Many effective broad spectrum herbicides do not distinguish between weeds and crops, but crop plants can be modified to make them resistant to herbicides, so as to eliminate weeds more

selectively. For example, the herbicide RoundupTM contains the active ingredient glyphosate,

which kills plants by binding to the active site of enzymes called enolpyruvalshikimate phosphate synthase (EPSP synthase). This enzyme is critical for the synthesis of aromatic amino acids. Roundup is an extremely effective herbicide but it kills almost all species of plants, including most crop plants. On the other hand, it is very safe for humans and animals because they do not have EPSP synthase. By using rDNA technology, modified EPSP synthase gene (that produced enzymes that were still functional but were not inhibited by glyphosate) have been introduced into crop plants such as cotton and soyabean. These genetically modified plants were found to be highly resistant to treatment with Roundup. Genes that provide resistance to other herbicides such as sulfonyl ureas, gluphosinates etc. have also been developed and transferred to produce various transgenic plants.

(c) Insect resistant plant: To minimize crop damage by insects, mites and nematodes, farmers use synthetic pesticides extensively which cause severe effects on human health and environment. The transgenic technology provides an alternative and innovative method to improve pest control management which is eco friendly, effective, sustainable and beneficial in term of yield. This involves genetic incorporation of toxic gene (product of which is lethal to insect) into the plant. The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular group of insect pests, and are not harmful to other useful insects like butter flies and silk worms. Transgenic crops (e.g. cotton, rice, maize, potato, brinjal, cauliflower, cabbage etc.) with Bt genes have been developed and such transgenic varieties proved effective in controlling the insect pests and it has been claimed worldwide that it has led to significant increase in yield along with dramatic reduction in pesticides use. The most notable example is Bt cotton (which contains Cry/Ac gene) that is resistant to a notorious insect pest Bollworm (Hellicoperpa armigera) and only last year (2002) Bt cotton was adopted in India. Biotechnology has opened up new avenues for natural protection for plants by providing new biopesticides, such as microorganisms, that are toxic to targeted crop pests but do not harm humans, animals, fish, birds or beneficial insects. As biopesticides act in unique ways, they can even control pest population that has developed resistance to conventional pesticides. Using recombinant DNA technology, the gene that makes these microorganisms lethal to certain insects can be transplanted into the plants on which that insect feeds. The plant that once was a food source for the insect now kills it, lessening the need to spray crops with chemical pesticides to control infestation. One such microorganism is commonly found soil bacterium Bacillus thuringiensis. The spores of Bacillus thuringiensis (Bt) contain a crystalline protein (Cry) which breaks down to release a toxin, known as delta-endotoxin which is highly toxic to lepidopteran larvae. This toxin binds the intestinal lining and creates pores resulting in an ion imbalance, paralysis of the digestive system, and consequent death of the insect. Bt toxin sprays and powders have been in use for many years.Different Cry genes, also known as Bt genes have been identified, cloned and characterized. Effective gene constructs have made it possible to deliver these genes into plant tissues so that they are expressed at levels high enough to kill the insects. The Bt genes are effective against

different orders of insects. Bt cotton and maize which have increased resistance to boll worms have been developed and cultivated since 1996. Farmers get benefited by saving costs by using less of traditional pesticides. However, one of the major concerns about Bt based transgenics is the possibility of development of toxin resistant insects. Efforts are also underway to identify and transfer other genes from Bt, which can impart insecticidal properties to the plants. One example in this is transfer of vip gene i.e. vegetative insecticidal proteins, for which the trials are being conducted in some countries.

(d) Disease resistant plant: Plants are susceptible to viral, bacterial and fungal diseases. Much progress has been made in evolving transgenic plants resistant to viruses. For example, expression of a gene that encodes the coat protein of tobacco mosaic virus (TMV) in transgenic tobacco plants has been shown to cause the plants to resist TMV infection. A number of other viral resistant plants species have been developed including squash and potatoes. Genetic engineering of crop plants for resistance to fungal and bacterial infections has been more difficult. However, by studying the protective genes that are expressed in naturally disease-resistant plants, an encouraging progress has been made. The proteins encoded by these so called pathogenesis related proteins (PR proteins) can, in some cases, provide limited disease protection in transgenic plants. There are several strategies for engineering plants for viral resistance and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. Some viral resistant transgenic plants like papaya resistance to papaya ring spot virus have been commercialized in some countries. Plants respond to pathogens by inducing a variety of defense responses like pathogenesis-related proteins (PR proteins), enzymes that degrade/destroy fungal cell wall (chitinase), antifungal proteins and compounds, phytoalexins, etc. Several transgenic crop plants showing increased resistance to fungal pathogens are being raised with genes coding for the different compounds mentioned above. DEVELOPMENT OF PLANTS HAVING INCREASE IN QUALITY OF PLANT PRODUCTSOne of the most successful research efforts to change the characteristics of a plant produce was carried out with tomatoes. Tomatoes need to be picked while still green so that they are firm enough to withstand mechanical handling and transport. Unfortunately, they do not develop the same flavor and texture of vine-ripened tomatoes. Softening of tomatoes and many other fruits is caused by the enzyme pectinase or polygalacturonase (PGA). This enzyme digests the pectin polysaccharide that cements the plant cells together. Softening of the fruit is caused, in part by this breakdown of pectin. In order to reduce the levels of PGA in ripening tomatoes, researchers placed the PGA gene in reverse orientation relative to the CaMV 35S promoter. This results in transcription of an antisense RNA that is complementary to the normal sense PGA mRNA. Although the exact mechanism is unknown, antisense RNA is able to arrest the translation of the endogenous PGA mRNA in the tomato fruit. Transgenic tomato plants that express an antisense PGA gene only have about 5 to 10% of normal PGA levels. Fruits of these plants have normal

color and flavor but they soften more slowly and can be picked and processed after they are ripe. They also have a higher content of soluble solids and are therefore better than normal tomatoes for processed tomato products. Transgenic lines of potato having increased levels of starch also have been developed by introducing a gene construct that expresses a gene from bacteria that produce an enzyme that enhances starch biosynthesis. A promoter from a potato gene that encodes the major protein in potato tubers has been used, so that the expression of the introduced gene is limited to the tuber. Tubers accumulate approximately 3 to 5% more starch than normal potatoes and when they are deep fried absorb less oil and yield chips having fewer calories. Some of the other value added transgenic crops include:

1. Golden rice: containing beta carotene to overcome vitamin A deficiency in regions where rice is the staple food

2. Canola containing high levels of oleic acids and laurate 3. Barley containing feed enzymes 4. Tomatoes which does not rot in room temperature 5. Other vegetables and fruits with delayed ripening as well as modified flavour

characteristics

TRANSGENIC PLANT AS A SOURCE OF BIO PHARMACEUTICALS Plants are among the most efficient bioreactors which produce quantities of material with sunlight and soil based nutrients as inputs. Attempts are being made to replace the traditional fermentation procedure for the production of biopharmaceuticals to plant based production. The benefits of using plants are the ability to increase production at low cost by planting more acres, rather than building fermentation capacity, lower capital and operating cost, simplified downstream processing etc. Therapeutic drugs to treat cancer, infectious diseases, autoimmune diseases, cardiovascular diseases and other conditions and several vaccines can potentially be grown in plants. Plant transgenic technology is being used to produce a plant that will generate a seed that expresses a desired therapeutic protein. This seed can propagate under the right growing conditions to yield plants and seed stock for producing the desired protein. The desired protein can be extracted from the seed to make a biopharmaceutical. Plant based therapeutics are expected to be much more cost effective. For example, Dow Plant Pharmaceuticals is using corn to grow pharmaceuticals by designing and selecting the plant which will contain the active pharmaceutical within the endosperm seed compartment. Benefits of producing the pharmaceuticals in the corn include long term storage advantage, easier purification in view of limited number of soluble seed proteins in a corn seeds, low microbial load, low proteolytic activity and specialized promoters to enable expression of the protein in specific parts of the plants. Edible vaccines:Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The genes encoding antigenic proteins can be isolated from the pathogens and expressed in plants and such transgenic plants or their tissues producing antigens can be eaten for vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops like banana and tomato are useful for immunization of humans since banana and tomato fruits

can be eaten raw. The edible vaccines that are produced in transgenic plants have great advantages like the alleviation of storage problems, easy delivery system by feeding and low cost as compared to recombinant vaccines produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera and hepatitis B by feeding them banana/ tomato, and vaccinating animals against important diseases such as foot and mouth disease by feeding them sugar beets could be a reality in the near future.

RECOMBINANT ENZYMES USED IN FOOD PROCESSING INDUSTRIES

Food industries commonly use enzymes for processing, as they use them in producing food ingredients. As traditional methods of isolation of enzymes, from different organismal sources, often do not adapt to conditions used in modern methods of food production, hence, rDNA technology is used to manufacture such enzymes. These recombinant enzymes are suitable for the conditions that are particular for food processing.

The use of recombinant technology has shown an immense increase in the yield of desired enzymes by introducing gene into production organism thereby influencing the regulator sequences. Among these, the main strategy involves introducing the gene that encodes the enzyme in safe and efficient microorganism. For this purpose, yeasts have been proved to be ideal system for the expression of heterologous proteins.

FDA has received, on food processing enzymes that are derived from recombinant microorganism, number of petitions. Following is the table that includes list of these enzymes along with their source of organisms while the complete list can be found on Enzyme Technical Association website.

FOOD INGREDIENTS

Biotechnological fermentation and biotransformation are the two processes that have been used for producing food products including organic acids, sweeteners, vitamins etc. rDNA technology is applied to improve:

1. The recovery2. Increase the yield 3. Increase in the purification of compounds

Amongst these products flavoring agents and enhancers are the ideal examples of food ingredients, which are best, suited for recombinant DNA techniques’ application for food production.

PL and PG from Recombinant Penicillium

Pectinase is an enzyme that is used for clarification of juices, their filtration and extraction of fruit juices and wines.

Polygalacturonase is the enzyme that is used frequently as a sweetener. Experiments have done to obtain a recombinant organism that will be having the ability to obtain pectin lyase (PL) and polygalacturonase (PG) and for that penicillium griseoroseum that produced both PL &PG simultaneously. Major steps of the experiment are as follows

1. Firstly a strain that was reported to produce high concentration of PL was taken.2. It was then transformed using pAN52pgg2 plasmid which was having a foreign gene of

PG of P. grieoroseum and it was having a promoter from Aspergillus nidulans3. The newly transformed P. grieoroseum T20 when checked was producing higher

concentrations of both PG and PL, around 143 folds higher PL, and 15 folds greater PG.4. This recombinant strain uses carbon sources of low costs that is very economical5. The enzyme preparation commercially available is free of cellulolytic and proteolytic

activities.6. This is an efficient system that uses P. griseoroseum to express and secrete proteins.

Chymosin:

Chymosin is an example of one of pioneer enzymes that are obtained by modifying microorganism genetically. Chymosin is a milk clotting protease that is produced via recombinant DNA techniques and has been approved to be use in food.

Chymosin or rennin is an enzyme found in rennet. It is produced by ruminant animals in the lining of the abomasum. Chymosin is produced by gastric chief cells in infants to curdle the milk they ingest, allowing a longer residence in the bowels and better absorption. Bovine Chymosin is now made in E. coli, Aspergillus Niger recombinantly. Its gene is also found in humans (on chromosome 1), but it is not expressed.

Commercial preparation of 100 % pure Chymosin has been done through genetic technology. And the produced Chymosin is frequently referred to as fermentation produced Chymosin. The microorganisms are usually non pathogenic that are used to produce it, including E.coli K-12, A. Niger etc. prochymosin genes are isolated from young calves and then they are transferred to plasmid and this plasmid is then introduced into microorganisms. On expression prochymosin is activated into Chymosin. Producing 100 % pure Chymosin.

The first step is the milk clotting process in cheese making in which k-caeinolytic enzymes contribute to micelle precipitation and because of its specificity toward k –casein it the best enzyme for this purpose.RBC (recombinant bovine Chymosin ) is commonly used but other sources are now available that includes goat ,camel ,or buffalo. The studies have shown that recombinant goat Chymosin exhibits

1. Best catalytic activity when compared with all the above mentioned Chymosin sources as well as

2. The best proteolytic activity 3. Lower glycosylation degree4. Wider pH range of action5. Proposed as an alternative to recombinant bovine Chymosin

The whole process is shown by the following figure

CONCLUSION

So overall the recombinant enzymes in food processing have improvements as microbial strains are developed to increase the enzyme yield which is done by deletion of genes for proteases. Fungal strains have been modified by reducing or possibly eliminating their potential for toxic secondary metabolites. Scientists are still finding alternatives to make the production cost efficient, with low cost in the preparation, highly specific activity, working efficiently at wider conditions range, to overcome all the loses that industries are still facing. Somehow scientists are also aiming to develop ways leading to better and safer development procedures for enzyme used in food processing industries.

USES OF RECOMBINANT DNA TECHNOLOGY IN WINE INDUSTRY

If we look back in time during the early days of winemaking, different techniques were used to produce wine of different quality, taste and styles. But during the last 150 years, scientific basis of winemaking has become clearer to us, and many practices which were once impossible to be done, are now done in routine.

One of the latest technologies used in the winemaking process is recombinant DNA technology. During the last decade, it has been identified as a most promising technology and it has widened the possibilities to introduce new properties in wine Recombinant DNA technology is used in the wine fermentation i.e primary and secondary--also sometimes described as aerobic and anaerobic fermentations.

In the process of winemaking, rDNA technology is used to produce genetically modified grapevines with improved qualities, genetically modified yeast which is used in the primary fermentation of wine, genetically modified bacteria that works during secondary fermentation of wine.

GENETICALLY MODIFIED GRAPEVINES

Recombinant DNA technology is used to introduce new and improved variety of grapevine which are used in winemaking process New initiatives are being seized from recombinant DNA technology, extensive efforts are underway to characterize the genomes of agriculturally important species. Target for the grapevine improvement is to produce high quality fruit with reduced susceptibility to disease and pests and other stress conditions, as well as the enhanced nutritional value. They also look at the optimal ripening in the grapevine. Transgenic approaches have also speeded up the development of plant lines able to adapt to adverse climatic conditions, among them drought and salt stress, photo-damage and freezing tolerance.

Following are the potential targets which are adapted to include aspects that become important to the industry in future.

1. The improvement of disease and pest resistance 2. The improvement of virus resistance and diagnosis 3. The improvement of stress tolerance, including drought resistance

4. The improvement of plant/fruit metabolism and quality characteristics 5. The improvement of nutritional value

Grapevine health is threatened by different viral, fungal and bacterial diseases. To cope with this, resistant genetically modified grapevine lines have been developed. They have increased tolerance to pathogens, so it has a positive impact on the conservation of environment and production cost as it has resulted in the reduced use of agrochemicals and fungicides.

CONTROVERSIES AGAINST GM GRAPEVINES:There are several controversies about the use of GM grapevines, one of them is that the DNA from a GM grape persists one year after wine fermentation, thus contradicting claims that wine fermentation eliminates DNA. This GM DNA is the carrier of all the risks of recombination and horizontal gene transfer which results in the creation of new viruses and bacteria that cause diseases and cancer in case of human cells. Another hazard includes the toxins and allergens from transgenic products or from unexpected metabolic disturbances to the host plant.

These are the uncertainties that act as a hurdle in the wine industry evaluation of transgenic grapevines. There is a consequent fear because of its strong identities and deep cultural roots, that this technology will accelerate the tendency to standardize wines, leading to loss of local identity, variety and uniqueness.

Wine yeast improvement: In 1863, Louis Pasteur, proved that yeast is the primary catalyst in wine fermentation. The knowledge that yeast is responsible for the biotransformation of grape sugars into alcohol and carbondioxide has helped the winemakers to control the process from vineyard to bottling plant.

A lot of progress has been made in the development of genetically modified yeast strains from rDNA technology which are used in wine, brewing and baking industries. These genetically modified yeast are used in the primary fermentation during wine production.

Commercially available yeast strains used in winemaking are;

MLO1: This GM yeast strain is commercialized and authorized by USA. It has been made by Springer Oenologie. This yeast strain is the recipient of two transgenes. First is malate transporter gene, this gene has been taken from another yeast named Schizosaccharomyces pombe. Second is a malolactic enzyme gene from Oenococcus oeni, a bacteria which is responsible for secondary fermentation i.e. malolactic fermentation after alcoholic fermentation. This yeast is therefore able to carry out malolactic fermentation that is normally done by bacteria at the same time as alcoholic fermentation. So this yeast strain is designed to allow malolactic fermentation to proceed more efficiently, thereby producing fewer biogenic amines, such as histamines, which cause headaches and asthmatic-type reactions in some people.

ECMo01: It is a strain of Saccharomyces cerevisiae, it has been genetically manipulated to better degrade urea during the wine making process. The benefit of such a characteristic is that

the wine contains less ethyl carbamate, a chemical considered by some regulatory bodies to be a human health risk.

In general, five major targets for the genetic improvement of wine yeast strains have been identified which could add benefit to the wine industry:

There are five major targets for the genetic improvement of wine yeast that has been identified to add benefit to the wine industry.

1. Efficiency of the fermentation process can be improved by better sugar utilization, increase tolerance to ethanol, resistance to heavy metals and reduced foam and floculance

2. Processing of wine: Yeast secreting pectinase, glucanases, xylaneses and proteases has ability to clarify and filter efficiently.

3. Wholesomeness

4. Sensorial quality: Secretion of glucanases and glucosidases may also enhance wine flavor by hydrolysis of flavor precursor glycosides

5. Control of microbial spoilage.

Wine bacteria improvement:

Lactic acid bacteria is used in fermentation processes of many food and beverages. In winemaking process it is a must component and performs secondary fermentation called as malolactic fermentation. Considerable progress has been made in the last decade for the development of tools for the genetic modification of lactic acid bacteria. Oenococcus oeni is the lactic acid bacteria most widely used in the wine industry.

Oenococcus oeni: It is a lactic acid bacteria used commercially in the production of wine. Their function is to do the malolactic fermentation of wine. It also has the quality to create a characteristic aroma profile and deaccidification to the wine. Lactic acid bacteria have been found to produce antimicrobial agents that can inhibit the growth of spoilage lactic acid bacteria, and thus might decrease the levels of sulphur dioxide used in wine.

There are several controversies about the application of recombinant DNA technology in wine industry, there are certain questions which are not answered satisfactorily yet. In order to assure any future possibility of the use of recombinant technology in wine industry, there are things about which we have to be assure about, including that the existing desirable characteristics will not be damaged, engineered culture will be stable in practice, requirements of beverage legislation should be met and there will be no adverse effects to the human health. So in order to have a useful approach, two traditional wine sciences, viticulture and oenology, need to be combined, in this way we will be able to have a histolic approach.

BUSINESS PLAN: EXECUTIVE SUMMARY: This report is to make a business plan to commercialize products produced by help of recombinant DNA technology. The purpose of this report is to provide a possible solution to enhance the quality of cheese available in market, to bridge gap between supply and demand gap between consumers while providing economic stability to poor people of the villages. Business plan is made using Chymosin as a product to be commercialized. This Business plan is made for a presentation to students of Applied Biosciences. Our aim is to make students of applied biosciences to think about the economic aspects and communal impact of their research. Online data was searched on the business planning. Various Business models were searched and applied on our product and best one was applied.A member of the group participated in Discover NUST business plan competition/Workshop held on 10-11-Nov-2012 in order to get the task of making Business Plan accomplished. This report is presented under the guidance and experiences of participant of Discover.Our aim is to make the cheese of high quality readily available to all categories of customers in a affordable price while providing maximum benefit to the poor farmers and producers of the villages.We have focused on the market size, needs, quality of cheese and also the supply chain of the cheese from the producer to the consumer at market. We have planned our organizational structure, Business Model and our financial strategy in accordance with the market size, target group and market needs. Interest of biology students towards the economical impact of their research requires the inculcation of entrepreneurial trends, business planning and team building strategies among the students of ASAB.1. Background:

An estimate of livestock population and milk production in Pakistan is given in following tables. Following data for year 2011-2012 is generated according to the 2005-2006 livestock consensus

Province wise distribution of live stock (http://www.pakdairyinfo.com/introduction.htm)

Total milk consumption of Pakistani Population (source http://www.pakdairyinfo.com/introduction.htm)

According to FAO the prices of dairy products are on the rise throughout the year 2012 especially the cheese.

(Source http://www.fao.org/index_en.htm)

OUR RECOMMENDATIONS: we have devised a business model in order as solution Go to market strategy

1. Installment of mini Industrial units in villages: According to our proposed business model we will establish mini industries in villages where the supply of raw materials is very easy and also install mini industrial units for the production of cheese in small villages. For every 25 houses there will be 1 industrial unit. Research department will make sure that the all the different enzymes are available to these industrial units.

2. Goods export to market:Cheese produced there will be exported nearby markets via vehicles and then from these cheese collection centers it will be sent to the mega cities like Lahore, Islamabad, Karachi etc.

3. Sales/profit: Dairy shops, Pizza shops, 5 star hotels and Restaurants will be our intended target places for cheese to be sold. At least 10 of profit will be used for R and D purposes.

4. Product export: In cities additional measures will be taken to convert cheese into export quality product. Also the enzymes produced in Research wing will be exported to the foreign trade market.

5. Villagers: It is planned to ensure that the standard of living and working conditions of villagers living in villages is enhanced through revenue generated in local or foreign markets. 5 % will be used for the economic development of villagers who will work in our cheese industry.

6. Exit Strategy: Initial Public Offering will be the exit strategy for our business.

ORGANIZATIONAL STRUCTURE: The organization will work under the supervision of central executive office who will make sure that all the heads of different sections are working in a collaboration with each other. There will wings or post which include HR section for human resource, Finance will look over all the financial matters, sales/marketing will be ensured by people of marketing department. Pr and communication will keep updating about market variables and market trends, all the legal recommendations will be made by the legal sections and taxes sections.

FINANCE:

Finance Year 1 Year 2 Year 3

Total Revenue No profit loss 10 lacks 15 lacks

Gross Profit 0 5 lacks 10 lack

Net Income 0 2 lacks 5 lacks

Investment Needed 5 lacks 0 0

There will be no profit in the first year with a total investment of 5 lacks. Then in 2nd year gross profit after paying all the cost of process, Vages and marketing cost will be 2 5 lacks and net income after paying off taxes will be 2 lacks. No investment needed for 2nd and 3rd year. Gross profit will increase to 10 lacks and net profit will be 5 lacks according to proposed plan.

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