Indian Journal of Biotechnology Vol I, January 2002, pp 9-16

Developments in Biotechnology: An Overview

Rintu Banerjee l, Gargi Mukherjee l

, Ashok Pande/* and A Sabu2

I Microbial Biotechnology and Downstream Processing Laboratory,

Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur 721302

2Biotechnology Division, Regional Research Laboratory , CSIR, Thiruvananthapuram 695 019, India

Human quest for improving the natural capabilities of microorganisms, making them capable of novel processes, and discovering microorganisms with new capabilities has led to tremendous development, today referred to as modern biotechnological advances. This has led to the development of recombinant DNA technology, which allows modifying microbes and other organisms to create in them highly valuable, novel and naturally non-existent capabilities. R&D in areas such as stem cell, human genome, transgenic crops, etc. has made untreatable diseases to understand and control. However, research in many areas such as cloning, stem cell, etc. has also posed serious concern on ethical and societal issues, which if not taken proper measures, could be eventually vel'Y dangerous. This paper traces the significant biotechnology developments and their impact on human life with a futuristic approach.

Keywords: biotechnology, bioprocesses, industrial development, DNA technology, gene therapy, human genome, stem cell, biosafety, ethical issues

Introduction Biotechnology is concerned with the exploitation of

biological components for generating useful products. The area covered under biotechnology is vast and the techniques involved are highly divergent. Some standard definitions of biotechnology are, "the controlled use of biological agents such as microorganisms or cellular components for beneficial use" (US National Science Foundation). According to European Federation of Biotechnology, "the integrated use of Biochemistry, microbiology and engineering sciences in order to achieve technological application of the capabilities of microorganisms, cultured tissues/cells and parts thereof'. A British biotechnologist says that "the application of biological organisms, systems or processes" constitutes biotechnology. Thus, in general, biotechnology is the utilization of biological entities (microorganisms, cells of higher organisms, their components or constituents, e.g. enzymes) in such a way that some product/service of human welfare is generated.

History Since 5000 BC, microbes have been employed for

making wine, vinegar, curd, leavened bread, etc. Such processes that are based on the natural capabilities of

* Author for correspondence: Tel : +91-471-515279; Fax: +91-471-491712 E-mail: pandey@csrrltrd.ren.nic.in

microorganisms are commonly referred to as old biotechnology. The term 'Biotechnology' was coined by Karl Ereky in 1919, a Hungarian engineer, who envisioned a biochemical age similar to the stone and iron ages (Bud, 1989).

Biotechnology, in 20th century, brought industry and agriculture together. During World War I, fermentation processes were developed that produced acetone from starch and paint solvents for the rapidly growing automobile industry. During World War II, the manufacture of penicillin shifted the biotechnological focus towards pharmaceuticals. The "cold war" years were dominated by work on microorganisms for the preparation of biological warfare as well as antibiotics and fermentation processes (Goodman, 1987).

After the discovery of double-helix model of DNA, scientists started working on genes. Werner Arber discovered special enzymes in bacteria called restriction enzymes, which cut DNA strands of organism at precise points. In 1973, Stanley Cohen and Herbert Boyer removed a specific gene from one bacterium and inserted it in another using restriction enzymes. This one event marked the beginning of recombinant DNA technology or genetic engineering. In 1977, genes were transferred from one organism to bacteria (Table 1).

10

Year Scientist

Prior to 1750

1797: Edward Jenner 1750-1850: 1859: Charles Darwin 1864: Louis Pasteur

1865: Gregor Mendel 1869: Johann Meischer 1890: 1893: Koch, Pasteur 1902: Walter Sutton

1910: Thomas H Morgan

1927: Herman Mueller 1928: Frederick Griffiths

1928: Alexander Fleming 1920-1930: 1938:

Edward Tatum 1943-1953: Linus Pauling

1944: Oswald A very 1945: Max Delbruck

Mid-1940's: 1950: Erwin Chargaff

1952: Alfred Hershey Margaret Chase

1953: James Watson Francis Crick

1956: Dangr 1957: Francis Crick

George Gamov 1958: Coenberg 1960: 1965: 1966: Marshall Nirenberg

Severo Ochoa 1970: 1971: 1972: Paul Berg

1973: Stanley Cohen Herbert Boyer

1975: 1976:

1977: 1978: Genentech, Inc.

INDIAN J BIOTECHNOL, JANUARY 2002

Table 1- Developments in biotechnology*

Findings

Plants were used for food Plants domesticated; selectively bred for desired characteristics; microbial fermentation used to make cheese, beverages, and bread

Used living microorganisms to protect people from disease Increased cultivation of leguminous crops and crop rotations to increase yield and land use Hypothesized that animal and plant populations adapt over time to best fit the environment Proved existence of microorganisms. Showed that all living things are produced by other living things Investigated how traits are passed from generation to generation - called them factors Isolated DNA from the nuclei of white blood cells Ammonia synthesis Fermentation process patented Coined the term "gene". Proposed that chromosomes carry genes (factors which Mendel said that could be passed from generation to generation) Proved that genes are carried on chromosomes. "Biotechnology" term coined Yeast grown in large quantities for animal and glycerol Made activated sludge for sewage treatment process Increased mutation rate in fruit flies by exposing them to X-rays Noticed that a rough kind of bacterium changed to a smooth type when unknown "transforming principle" from smooth type was present Discovered antibiotic properties of certain molds Plant hybridization Proteins and DNA studied by X-ray crystallography Term 'molecular biology" coined

Described sickle cell anemia calling it a molecular disease. Cortisone made in large amounts DNA is identified as the genetic material Performed transformation experiment with Griffith's bacterium Organized course to study a type of bacterial virus that consists of a protein coat containing DNA Penicillin produced Determined that there is always a ratio of 1: 1 adenine to thiamine in DNA of many different organisms Used radioactive labeling to determine that it is the DNA not protein which carries the instructions for assembling new phages Determined the double helix structure of DNA

Sequenced insulin (protein) from pork Explained how DNA functions to make protein

Discovered DNA polymerase Isolation of m-RNA Classification of the plasmids Determined that a sequence of three nucleotide bases determine each of 20 amino acids Isolation of reverse transcriptase Discovery of restriction enzymes Cut sections of viral DNA and bacterial DNA with same restriction enzyme Spliced viral DNA to the bacterial DNA Produced first recombinant DNA organism Beginning of genetic engineering Moratorium on recombinant DNA techniques National Institute of Health guidelines developed for study of recombinant DNA

First practical application of genetic engineering Genetic engineering techniques used to produce human insulin in E. coli First biotech company on NY stock exchange Contd.-

BANERJEE et at: DEVELOPMENTS IN BIOTECHNOLOGY 11

Year

1979:

1980:

1983:

1985: 1986:

1988: 1993: 1993

1993 1994 1994 1994 1995 1995

1996 1996 1997 1997

1998 1998 1999 1999 2000

2000

Dec18,2000 2001

Scientist

Stanford University

Genentech. Inc.

Genetech, Inc.

Karry Mullis Genentech

Cal gene

Biogene Scottish scientists Scottish scientists

University of Hawaii

Celera Genomics

IBM

Table 1-Developments in biotechnology---Contd.

Findings

First successful transplantation of mammalian gene

Discoverers of restriction enzymes receive Nobel Prize in medicine

Produce human growth hormone and two kinds of interferon DNA from malignant cells transformed a strain of cultured mouse cells - new tool for analyzing cancer genes US. Supreme Court decided that manmade microbes could be patented

Licensed Eli Lily to make insulin First transfer of foreign gene in plants Plants can be patented First field trials of DNA recombinant plants resistant to insects, viruses, bacteria

First living mammal was patented Fllivr savr tomatoes sold to public FDA declares that genetically engineered foods are not "inherently dangerous" and thus require no special regulation Gets Nobel Prize for PCR Discovery Genentech's Nutropin approved for growth hormone deficiency treatment Breast cancer gene discovered Calgene's Flavr savr tomato resistant to rotting, approved for sale First baboon to human bone marrow transplant performed for an AIDS patient First full gene sequence of a living organism completed for bacterium Haemophilus injluenzae (i .e., other than virus) Biogene's Avonex approved for multiple sclerosis treatment Cloning of identical lambs from early embryonic sheep Sheep cloned using DNA from adult sheep cells New technique combining PCR, DNA chips and a computer program providing new tool for disease causing genes Three generations of mice from nuclei of adult ovarian cumulus cells Rough draft of human genome map produced showing locations of more than 30,000 genes Europe rejects genetically modified products 90% of human genome sequenced Rough draft of human genome completed by Celera Genomics and the Human Genome Project "Golden Rice," modified to make vitamin A, promises to help third-world countries alleviate blindness. Super computer unveiled by IBM to study the human genome The sequence of the human genome published in Science and Nature

*Bullock, 1987; Crespi, 1991 ; DaSilva, 1991 ; Joshi & Pandey, 1999; Pandey, 1998,2000; Old & Primrose, 1990, Swamy, 1995.

Present and Future Biotechnology is leading a,sudden new biological

revolution. It has brought us to the brink of a world of "engineered" products that are based in the natural world rather than on chemical and industrial processes. A common misconception among people is that biotechnology only includes DNA and genetic engineering. However, in reality, genetic engineering is a tool/technique, which can be applied to all disciplines of biotechnology like microbes, plants, animals, and even for the remediation of environmental pollution.

Biotechnology is currently being used in many areas including agriculture, bioremediation, food processing, and energy production. DNA fingerprinting is becoming a common practice in forensics. Production of insulin and other medicines is accomplished through cloning of vectors that now carry the chosen gene. Immunoassays are used not only in medicine for drug level and pregnancy testing, but also by farmers to aid in detection of unsafe levels of pesticides, herbicides and toxins on crops and in animal products. These assays also provide rapid field tests for industrial chemicals in ground water, sediment, and soil. In agriculture, genetic engineering

12 INDIAN J BIOTECHNOL, JANUARY 2002

is being used to produce plants that are resistant to insects, weeds and plant diseases. "Flavr-savr" tomato was brought about through modern techniques and was the first food created by the lise of recombinant DNA ever to go on sale. New biotechnological techniques have permitted scientists to manipulate desired traits. Today's biotechnology has its "roots" in chemistry, physics, and biology.

Biotechnology as Interdisciplinary Area Biotechnology is multidisciplinary and includes

disciplines of basic sciences and engineering. Main science disciplines are microbiology, biochemistry , genetics, molecular biology, immunology, cell and tissue culture , and physiology. On the engineering side, it includes chemical and biochemical engineering processes such as the large-scale cultivation of microbes and cells, their downstream processing etc., e.g. the production of a compound by microorganisms, animal/plant cells. Such processes can be separated into five major operations: i, strain selection and improvement; ii, mass culture; iii, optimization of cell responses; iv, process operation; and v, product recovery or downstream processing (Singh, 1998).

Areas of Biotechnology Major branches of biotechnology are

microbial/industrial , plant/agricultural, animal, medical and environmental biotechnology . Biotechnology has potential impact on virtually all domains of human welfare, ranging from food processing, environmental protection, to human health. As a result, it now plays an important role in employment, productivity, trade, economy, and the quality of human health. This is clearly reflected by the emergence of numerous biotechnological companies throughout the world, including India, and the joining of noted scientists, including Nobel Laureates, to some of these companies. In 1885, Roux demonstrated embryonic chick cells could be kept alive outside an animal's body. For the next one hundred years, advances in cell tissue culture have contributed to many different areas such as biological clocks and cancer therapy.

Cloning has generated large quantities of pure human proteins, which are used to treat diseases such as diabetes. In future, a resource bank for rare human proteins or other molecules is a possibility. For instance, DNA sequences, which are modified to correct a mutation to increase the production of a

specific protein or to produce a new type of protein, can be stored. This technique will probably playa key role in gene therapy.

New Developments Recombinant DNA technology has aroused public

interest and concern and has influenced medicine, industry, agriculture and environment. The recent emphasis on environmental awareness has challenged scientists to find solutions for better and safer living conditions. The added threat of deadly diseases such as AIDS and resistant strains of tuberculosis have forced scientists to look for new therapies within the fie ld of biotechnology.

In medicine, faster and more efficient diagnosis and treatment of diseases such as cystic fibros is, cancer, sickle cell anemia, and diabetes are soon to be developed. Recombinant organisms will be used in industry to produce new vaccines, solvents, and chemicals of all kinds. Scientists are developing disease and herbicide resistant crops, disease resistant animals, seedless fruits and rapidly growing chicken. Microbes are being engineered to digest compounds that are currently polluting our environment.

An exciting frontier of biotechnology includes protein based "biochips" which would be faster and more energy efficient and may replace silicon chips. Biochip implants in the body could deliver precise amounts of drugs to affect heart rate, hormone secretion, or to control artificial li mbs. Biosensors are monitors that use enzymes, monoclonal antibodies, or other proteins to test air and water quality , to detect hazardous substances, and to monitor blood components in vivo .

Gene therapy. It involves correction of defects in genetic material. In this process, a normal gene is in troduced to replace a malfunctioning one. "Gene therapy" will be dealt with by health care biotechnology as an individual industry or join the traditional pharmaceutical industry . New delivery systems, called liposomes, are being developed to get cytotoxic drugs to tumour sites with minimal damage to surrounding healthy tissues. New monoclonal antibodies would be isolated for use in cancer treatment, diagnostic testing, bone marrow transplantation and other applications.

Stem cell research. It is an extremely complex biomedical science, which have raised the hopes to treat otherwise untreated diseases such as Parkinson's and Alzheimer diseases. Stem cells are Master cells, which can develop into any of the 216 types of cells

BANERJEE et al: DEVELOPMENTS IN BIOTECHNOLOGY 13

that make the human body. Stem cells present in embryonic tissue grow vigorously in culture and could form any cell in human body. However, adult stem cells can make only a few types of cells with limited life in culture. R&D in stem cell could provide heart and blood muscles, nerve tissue and brain cells, which could eventually ensure availability of 100% matching cells and tissues for replacement in human body with minimal risks associated with the rejection of foreign tissues by the body.

However, R&D in stem cell could be misused or abused such as for the human reproductive cloning, which is banned in many countries. If a total ban could be put on human reproductive cloning, there would be great hopes that stem cell research could be used for the benefit of human society to study the problems of infertility, miscarriage and congenital diseases.

Human genome research . The Human Genome Project (HGP) is an international research programme with the aim to make a detailed map of human DNA. In this project, which is estimated to take 15 years, chromosome maps are being developed in various laboratories worldwide through a coordinated effort guided by the National Institute of Health, USA. The genetic markers for over 4000 diseases caused by single mutant genes have been mapped. HGP has following main objectives:

i) To construct detailed genetic and physical maps of the human genome, ii) To determine the complete nucleotide sequence of human DNA, iii) Storing information in databases, iv) Improvement of tools for data analysis, v) Localizing the estimated 50,000-100,000 genes within the human genome, vi) Transfer of related technologies to the private sector, vii) To address the ethical, legal, and social issues (ELSI) that may arise from the project, viii) For performing similar analyses on the genomes of several other organisms used extensively in research laboratories as model systems.

The ultimate goal of the HGP is to obtain the sequence of the 3 billion DNA subunits present in human DNA. The scientific products of the HGP would comprise a detailed information resource about the structure, organization and function of human DNA. An important outcome of the HGP would, thus be the generation of technology for biomedical research. From the inception of the HGP, it has been recognized that acquisition and use of such genetic knowledge would have enormous implications for individuals and society and pose a number of policy

choices for public and professional deliberation. Analysis of the ethical, legal, and social implications of genetic knowledge, and the development of policy options for public consideration are therefore, another major component of the human genome research effort.

A draft sequence of human genome comprised of 10,000 base pair-sized fragments whose approximate chromosomal locations are known (Anonymous 2001a & 2001b). 47% (1,660,078,000 bases) of the High-Quality Sequence has been finished and the goal of finishi ng 100% may be achieved by 2003. For high-quality sequence generation, additional sequencing is necessary to close gaps, and reduce ambiguities. A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5 .lI -fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies- a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera genornics group and the publicly funded genome effort. The agreed-upon standard for HGP finished sequence is to allow for only a single error every 10,000 bases. Investigators believe that a high-quality sequence is critical for recognizi ng regulatory components of genes that are very important in understanding human biology and disorders like heart disease, cancer, and diabetes. So far, finished sequences have been generated for only two human chromosomes - 21 and 22 (Hazbun & Fields, 2001; Yeh et ai, 2001 , Green & Chakravarti,2001)

Crop production/protection and transgenic research. In 1983, first transgenic plant, in which a bacterium gene was inserted, was produced. In 1996, the first commercial transgenic crop variety (Flavr­savr tomato) was produced. This crop \'/as modified by r-DNA techniques by Calgene Co. to delay post­harvest maturation. Since then, more than 15 products have come in the market. They include a cotton variety lngard, released in 1996 in Australia, which contains Bacillus thuringienses (Bt) genes to provide resistance to Lepidoptera insects. Another important product is Roundup Ready soybean, resistant to the herbicide glyphosate. Some other products include corn hybrids with Bt, virus resistant potato, improved canol a variety, etc.

14 INDIAN J BIOTECHNOL, JANUARY 2002

There has been tremendous growth in transgenic research all over the world, although USA has remained the major player which exports 80% genetically engineered products (Tables 2 & 3). The global area of transgenic crops in 1999 for Industrial and Developing countries was 32.8 and 7.1 million hectare accounting 82 and 18% share, which in 2000 was 78 and 22%, respectively, showing a remarkable increase of 34% for developing nations (9.5 million hectares in 2000 against 7.1 in 1999).

Transgenic research has posed several questions in the society. For example, will large-scale cultivation of transgenic plants bring high risks or only benefits? There is a fear that the transformation of different crop species with the same resistance gene, i.e. clones from the same Bt strain may result in an endemic risk; if the insect overcomes the resistance provided by the Bt gene, a phenomenon called Inter-specific Biotechnological Vulnerability. Obviously, genetic manipulation could pose risks with ecological and socio-economic dimensions, which indeed are of major concern and importance to middle and low income developing nations in the world.

Bioinformatics. It is the study of information content and information flow in biological systems/ processes. Its evolution serves as a bridge between observations (data) in diverse biologically related disciplines and the derivations of understanding (information) about how the systems/processes function, and subsequently its application (knowledge). According to NIH Biomedical Information Science and Technology Initiative Consortium, "Bioinformatics is the research, development, or application of computational tools and approaches for expanding the use of biological, medical, behavioural or health data, including those to acquire, store, organise, archive, analyze, or visualize such data". It is an interface of biology, computer sciences, mathematics, and physical sciences, and has a lot of applications in pharmaceutical, food and agricultural industries. The data generated from various genome sequencing projects is stored and analyzed using bioinformatics to identify disease causing genes, for crop yield improvement, production of disease resistant crops, and designing new drug targets.

Biotechnology and Intellectual Property Rights The intellectual property rights include patents,

trade secrets, trademarks and copyrights, which can be protected through a number of laws in different countries. The patentable examples of biotechnology

Table 2--Global area of transgenic crops

Year 1996 1997 1998 1999 2000

Country-wise share (1999)

US Argentina Canada China

Area (m ha) 1.7

11.0 27.8 39.9 43 .0

28.7 6.7 4.0 0.3

72% 17% 10% -1 %

Other countries: Australia, South Africa, Mexico, Spain, France, Portugal, Romania and Ukraine with very minor shares. RIS, 2000.

Table 3--Global area of transgenic crops (2000)

Country Hectares % +1- 1999/2000 (millions)

USA 30.2 70 + 1.5 Argentina 8.8 21 + 2.1 Canada 3.0 7 - 1.0 China 0.5 + 0.2 South Africa 0.2 <I +0.1 Australia 0.2 <I +<0.1 Romania <0.1 <I +<0.1 Mexico <0.1 <I +<0.1 Bulgaria <0. 1 <I +<0.1 Spain <0. 1 <I -<0.1 Germany <0. 1 <I -<0.1 France <0. 1 <I -<0.1 Total 43.0 100 +3 .1

RIS, 2000

are modified antibiotics, hormones/enzymes, organ specific drug delivery, heart valves, artificial teeth, plastic bags for storage of blood, etc. including some products in agriculture and animal husbandry. However, biotechnology developments like techniques used in medical sciences (bypass heart surgery, organ transplant, artificial limbs, use of drugs, antibiotics and vaccines), a number of culture methods, and biocontrol of pests and weeds are not patentable.

Biosafety and Ethical Issues Biosafety and ethical practice are required to be

addressed to ensure biotechnology application in right perspective. The issues have been raised on various forums, much serious attention is being paid since last 10 years or so at international level. Among several agreements signed, the European Council Directives

BANERJEE et al: DEVELOPMENTS IN BIOTECHNOLOGY 15

on use and deliberate release of genetically modified organisms (GMO) into the environment, the Food and Agricultural Organization (FAO) International code of conduct on distribution and use of pesticides, the UNIDO code of conduct, UNEP regulations and Convention on biological diversity held from time to time since 1990, are important ones. A more significant development in this regard was constitution of an ad-hoc group of experts in Biosafety (BSWG) in 1994 under UN umbrella. BSWG held its first convention of parties (COP) at Bahmas in 1994 and second at Jakarta in 1995. In the COP meeting at Montreal in 2000, 68 countries signed a treaty, which, however, has no legal binding and no internationallega: ~eeth .

Biosafety and ethicaL issues-Indian scenario. Since the enacting of the Environmental Protection Act (EPA) in 1986, there has been continuous vigil and monitoring of the biosafety issues and ethical practices on the biotechnology developments in the country. On these issues, the Department of Biotechnology (DBT), New Delhi, acts as the nodal agency and coordinates a number of sub-committees like Review Committee on Genetic Manipulation (RCGM), the Recombinant DNA Advisory Committee (RDAC), Genetic Engineering Approval Committee (GEAC), State Biotechnology Co­ordination Committee (SBCC; Chief Secretary of the State being its chairman), District Level Committee (DLC; District Collector as its Chairman), and Institutional Biosafety Committee (IBSC). Interestingly, in these committees, NGOs have no role. But significantly enough, the breach of code provides stringent punitive measure against the offenders.

Conclusion Biotechnology is the oldest activity of mankind in

terms of wines and alcoholic beverages and the present modern fermentation industries. The advent of recombinant DNA technology led it to be claimed as the 'New Biotechnology' , assuming that molecular biology and genetic engineering would be the major tools to solve most of the problems of human society. particularly human health-care and crop protection. Obviously, the present era belongs to biotechnology and the future of humanity would largely depend on biotechnology for sustaining life and its comforts. To feed the world population, which is expected to stabilize around 8.5 billions in 2050 with less per capita water and land availability, the mankind would

look up on the tools of biotechnology to meet such demands.

However, along with the growth of biotechnology, a number of ethical and societal issues would be needed to be tackled to ensure that the developments are made for the benefit of the society and not misused or abused. Needless to say that it has created a socio-ethical debate all over the world on the extent of its application in humans. Also, one cannot rule out the consequential impact of biotechnological developments on society . For example, as we have mentioned previously, will large-scale cultivation of transgenic plants bring high risks or only benefits. Surely risk is there, but its occurrence and potency will depend on how the genetic resources made available through genetic transformation, will be used in farming. The scenario on other fronts also such as cloning, genome research, stem cell research, etc. would be the same.

In Indian scenario, there is a strong need to continue the R&D projects exploring the biotechnological potential of flora and fauna. Transgenic research and biosafety are important issues and these must be dealt with utmost care, keeping regional richness in biodiversity in mind so as to fully safeguard the national interests at international platforms and agreements.

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