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The Development of Molecular Biotechnology
Emergence of Molecular Biotechnology
Commercialization of Molecular Biotechnology
Emergence of Molecular Biotechnology
LONG BEFORE WE KNEW that microorganisms existed or that genes were the units of inheritance, humans looked to the natural world to develop methods to increase food production, preserve food, and heal the sick. Our ancestors discovered that grains could be preserved through fermentation into beer, that storing horse saddles in a warm, damp corner of the stable resulted in the growth of a saddle mold that could heal infected saddle sores, that intentional exposure to a “contagion” could somehow provide protection from an infectious disease on subsequent exposures, and that plants and animals with enhanced production traits could be developed through cross breeding. Following the discovery of the microscopic world in the 17th century, microorganisms have been employed in the development of numerous useful processes and products. Many of these are found in our households and backyards. Lactic acid bacteria are used to prepare yogurts and probiotics, insecticide-producing bacteria are sprayed on many of the plants from which the vegetables in our refrigerator are harvested, nitrogen-fixing bacteria are added in the soil used for cultivation of legumes, the enzymatic stain removers in laundry detergent come from a microorganism, and antibiotics that are derived from common soil microbes are used to treat infectious diseases. These are just a few examples of traditional biotechnologies that have improved our lives. Up to the early 1970s, however, traditional biotechnology was not a well-recognized scientific discipline, and research in this area was centered in departments of chemical engineering and occasionally in specialized microbiology programs.
In a broad sense, biotechnology is concerned with the manipulation of organisms to develop and manufacture useful products. The term “biotechnology” was first used in 1917 by a Hungarian engineer, Karl Ereky, to describe an integrated process for the large-scale production of pigs by using sugar beets as the source of food. According to Ereky, biotechnology was “all lines of work by which products are produced from raw materials with the aid of living things.” This fairly precise definition was more or less ignored. For a number of years, biotechnology was used to describe two very different engineering disciplines. On one hand, it referred to industrial fermentation. On the other, it was used for the study of efficiency in the workplace—what is now called ergonomics. This ambiguity ended in 1961 when the Swedish microbiologist Carl Göran Hedén recommended that the title of a scientific journal dedicated to publishing research in the fields of applied microbiology and industrial fermentation be changed from the Journal of Microbiological and Biochemical Engineering and Technology to Biotechnology and Bioengineering. From that time on, biotechnology has been defined as the application of scientific and engineering principles to the processing of material by biological agents to provide goods and services. It is grounded on expertise in microbiology, genetics, biochemistry, immunology, cell biology, and chemical engineering.
Commodity production by naturally occurring microbial strains on a large scale is often considerably less than optimal. Initial efforts to enhance product yields focused on creating variants (mutants) using chemical mutagens or radiation to induce changes in the genetic constitution of existing strains. However, the level of improvement that could be achieved in this way was usually limited biologically. If a mutated strain, for example, synthesized too much of a compound, other metabolic functions often were impaired, thereby causing the strain’s growth during large-scale fermentation to be less than desired. Despite this constraint, the traditional “induced mutagenesis and selection” strategies of strain improvement were extremely successful for a number of processes, such as the production of antibiotics.
The traditional genetic improvement regimens were tedious, time-consuming, and costly because of the large numbers of microbial cells that had to be screened and tested. Moreover, the best result that could be expected with this approach was the improvement of an existing inherited property of a microorganism rather than the expansion of its genetic capabilities. Despite these limitations, by the late 1970s effective processes for the mass production of a wide range of commercial products had been perfected.
Today we have acquired sufficient knowledge of the biochemistry, genetics, and molecular biology of microbes and other organisms to accelerate the development of useful and improved biological products and processes and to create new products that would not otherwise occur. Distinct from traditional biotechnology, the modern methods require knowledge of and manipulation of genes, the functional units of inheritance, and the discipline that is concerned with the manipulation of genes for the purpose of producing useful goods and services using living organisms is known as molecular biotechnology. The pivotal developments that enabled this technology were the establishment of techniques to isolate genes and to transfer them from one organism to another. This technology is known as recombinant DNA technology, and it began as a lunchtime conversation between two scientists working in different fields who met at a scientific conference in 1972. In his laboratory at Stanford University in California, Stanley Cohen had been developing methods to transfer plasmids, small circular DNA molecules, into bacterial cells. Meanwhile, Herbert Boyer at the University of California at San Francisco was working with enzymes that cut DNA at specific nucleotide sequences. Over lunch at a scientific meeting in Hawaii, they reasoned that Boyer’s enzyme could be used to splice a specific segment of DNA into a plasmid and then the recombinant plasmid could be introduced into a host bacterium using Cohen’s method.
Recombinant DNA Technology
It was clear to Cohen and Boyer, and others, that recombinant DNA technology had far-reaching possibilities. As Cohen noted at the time, “It may be possible to introduce in E. coli, genes specifying metabolic or synthetic functions such as photosynthesis or antibiotic production indigenous to other biological classes.” The first commercial product produced using recombinant DNA technology was human insulin, which is used in the treatment of diabetes. The DNA sequence that encodes human insulin was synthesized, a remarkable feat in itself at the time, and was inserted into a plasmid that could be maintained in a nonpathogenic strain of the bacterium E. coli. The bacterial host cells acted as biological factories for the production of the two peptide chains of human insulin that, after combining, could be purified and used to treat diabetics who were allergic to the commercially available porcine (pig) insulin. Today, this type of genetic engineering is commonplace.
milestone Construction of Biologically Functional Bacterial Plasmids In Vitro
Cohen SN, Chang ACY, Boyer HW, Helling RB. 1973.
Proc. Natl. Acad. Sci. USA 70:3240–3244.
The landmark study of Cohen et al. established the foundation for recombinant DNA technology by showing how genetic information from different sources could be joined to create a novel, replicable genetic structure. In this instance, the new genetic entities were derived from bacterial autonomously replicating extrachromosomal DNA structures called plasmids. In a previous study, Cohen and Chang (Proc. Natl. Acad. Sci. USA. 70:1293–1297, 1973) produced a small plasmid from a large naturally occurring plasmid by shearing the larger plasmid into smaller random pieces and introducing the mixture of pieces into a host cell, the bacterium E. coli. By chance, one of the fragments that was about 1⁄10 the size of the original plasmid was perpetuated as a functional plasmid. To overcome the randomness of this approach and to make the genetic manipulation of plasmids more manageable, Cohen and his coworkers decided to use an enzyme (restriction endonuclease) that cuts a DNA molecule at a specific site and produces a short extension at each end. The extensions of the cut ends of a restriction endonuclease-treated DNA molecule can combine with the extensions of another DNA molecule that has been cleaved with the same restriction endonuclease. Consequently, when DNA molecules from different sources are treated with the same restriction endonuclease and mixed together, new DNA combinations that never existed before can be formed. In this way, Cohen et al. not only introduced a gene from one plasmid into another plasmid but also demonstrated that the introduced gene was biologically active. To their credit, these authors fully appreciated that their strategy was “potentially useful for insertion of specific sequences from prokaryotic or eukaryotic chromosomes or extrachromosomal DNA into independently replicating bacterial plasmids.” In other words, any gene from any organism could theoretically be cloned into a plasmid which, after introduction into a host cell, would be maintained indefinitely and, perhaps, produce the protein encoded by the cloned gene. By demonstrating the feasibility of gene cloning, Cohen et al. provided the experimental basis for recombinant DNA technology and established that plasmids could act as vehicles (vectors) for maintaining cloned genes. This motivated others to pursue research in this area that rapidly led to the development of more sophisticated vectors and gene cloning strategies. It also engendered concerns about the safety and ethics of this kind of research that, in turn, were responsible for the establishment of official guidelines and governmental agencies for conducting and regulating recombinant DNA research, respectively; and contributed to the formation of the molecular biotechnology industry.
The nature of biotechnology was changed forever by the development of recombinant DNA technology. Genetic engineering provided the means to create, rather than merely isolate, highly productive microbial strains. Not long after the production of the first commercial preparation of recombinant human insulin in 1982, bacteria and then eukaryotic cells were used for the production of insulin, interferon, growth hormone, viral antigens, and a variety of other therapeutic proteins. Recombinant DNA technology also facilitated the biological production of large amounts of useful low-molecular-weight compounds and macromolecules that occur naturally in minuscule quantities. Plants and animals became targets as natural bioreactors for producing new or altered gene products that could never have been created either by mutagenesis and selection or by crossbreeding. Molecular biotechnology has become the standard method for developing living systems with novel functions and capabilities for the synthesis of important commercial products.
Most new scientific disciplines do not arise solely on their own. They are often formed by the synthesis of knowledge from different areas of research. For molecular biotechnology, the biotechnology component was perfected by industrial microbiologists and chemical engineers, whereas the recombinant DNA technology portion owes much to discoveries in molecular biology, bacterial genetics, and nucleic acid enzymology (Table 1.1). In a broad sense, molecular biotechnology draws on knowledge from a diverse set of fundamental scientific disciplines to create products that are useful in a wide range of applications (Fig. 1.1).
Table 1.1 Selected developments in the history of molecular biotechnology
Figure 1.1 Many scientific disciplines contribute to molecular biotechnology, which generates a wide range of commercial products.
The Cohen and Boyer strategy for gene cloning was an experiment “heard round the world.” Once their concept was made public, many other researchers immediately appreciated its potential. Consequently, scientists created a large variety of experimental protocols that made identifying, isolating, characterizing, and utilizing genes more efficient and relatively easy. These technological developments have had an enormous impact on generating new knowledge in practically all biological disciplines, including animal behavior, developmental biology, molecular evolution, cell biology, and human genetics. Indeed, the emergence of the field of genomics was dependent on the ability to clone large fragments of DNA into plasmids in preparation for sequence determination.
Commercialization of Molecular Biotechnology
The potential of recombinant DNA technology reached the public with a frenzy of excitement and many people became rich on its promise. Indeed, within 20 minutes of the start of trading on the New York Stock Exchange on 14 October 1980, the price of shares in Genentech, the company founded by Boyer with chemist and entrepreneur Robert Swanson that produced recombinant human insulin, went from $35 to $89. This was the fastest increase in the value of any initial public offering in the history of the market up to that time. It was predicted that some genetically engineered microorganisms would replace chemical fertilizers and others would eat up oil spills; plants with inherited resistance to a variety of pests and exceptional nutritional content would be created; and livestock would have faster growing times, more efficient feed utilization, and meat with low fat content. Many were convinced that as long as a biological characteristic was genetically determined by one or a few genes, organisms with novel genetic constitutions could be readily created. Today, in many cases, the promise of recombinant DNA technology has become a reality.
In the 35 years since the commercial production of recombinant human insulin, more than 300 new drugs produced by recombinant DNA technology have been used to treat over 300 million people for diseases such as cancer, multiple sclerosis, rheumatoid arthritis, cystic fibrosis and strokes, and to provide protection against numerous infectious diseases. The majority of these are therapeutic monoclonal antibodies, hormones, and growth factors, many of which are more effective and have fewer side effects than other therapies. Moreover, hundreds of new biological drugs are in the process of being tested in human clinical trials to treat various cancers, autoimmune diseases, and infectious diseases. Similarly, many new molecular biotechnology products for enhancing crop and livestock yields, decreasing pesticide use, and improving industrial processes such as the manufacture of pulp and paper, food, energy, and textiles have been created and are being marketed.
The impact on agriculture has been tremendous. While the global population is expanding rapidly, yield increases of all major crops have decreased due to poor agricultural management practices, decreased acreage of arable land, and increased reliance on fertilizers and pesticides that diminish soil quality. To produce more food on less land, 18 million farmers in 28 countries are now planting genetically engineered crops on 450 million acres of land. These crops are predominantly soybeans, corn, cotton, and canola that are resistant to herbicides and insects. The global market value of genetically modified crops is currently $15.3 billion. Small resource-poor farmers are among the beneficiaries of agricultural biotechnology. In a comparative study of small cotton farms in South Africa, it was found, over three seasons, that the yield of cotton from plants that were genetically engineered to produce a bacterial insecticide was on average about 70% greater than those from nongenetically modified plants. Higher yields and reduced pesticide and labor costs translated into doubled revenues despite the slightly higher costs of the transgenic seeds. In India, which is the largest cotton producer in the world, revenues from insect-resistant cotton increased by $1.6 billion in 2014 compared to the previous year.
The ultimate objective of all biotechnology research is the development of commercial products. Consequently, molecular biotechnology is driven to a great extent by the prospect of financial gain. By nightfall on 14 October 1980, the principal shareholders of Genentech stock were worth millions of dollars. The unprecedented enthusiastic public response to Genentech encouraged others to follow. Between 1980 and 1983, about 200 small biotechnology companies were founded in the United States with the help of tax incentives and funding from both stock market speculation and private investment. Like Herbert Boyer, who was first a research scientist at the University of California at San Francisco and then a vice president of Genentech, university professors started many of the early companies.
Today, there are about 2,500 biotechnology companies in the United States and 2,100 in Europe, with annual earnings of $132 billion in 2015. The biotechnology industry in these regions employs more than 200,000 people. Large multinational chemical and pharmaceutical companies, such as Monsanto, Bayer, Du Pont, Pfizer, GlaxoSmithKline, Merck, Novartis, Hoffmann-LaRoche, Gilead Sciences, and Amgen, to name but a few, have made significant research commitments to molecular biotechnology. During the rapid proliferation of the biotechnology business in the 1980s, small companies that tended to specialize in one particular type of recombinant DNA product were often absorbed by larger ones, strategic mergers took place, and joint ventures were undertaken. For example, in 1991, 60% of Genentech was sold to Hoffmann-LaRoche for $2.1 billion. Inevitably, and for various reasons, there were a number of bankruptcies of biotechnology companies. This state of flux is a characteristic feature of the biotechnology industry. Currently, the roster of biotechnology companies is extensive and includes those focused on vaccines, protein and nucleic acid therapeutics, drug delivery, molecular diagnostics, genomics, industrial processing, and agricultural biotechnology.
Concerns and Consequences
While many people appreciate the potential of molecular biotechnology to solve important problems in agriculture, medicine, and industry, they recognize the need to be cautious about its widespread application. Indeed, one of the first scientific responses to this new technology was a voluntary moratorium on certain experiments that were thought to be potentially hazardous. This research ban was self-imposed by a group of molecular biologists, including Cohen and Boyer. They were concerned that combining genes from two different organisms might accidentally create a novel organism with undesirable and dangerous properties. Within a few years, however, these apprehensions were allayed as scientists gained laboratory experience with this technology and safety guidelines were formulated for recombinant DNA research. The temporary cessation of some recombinant DNA research projects did not dampen the enthusiasm for genetic engineering. In fact, the new technology continued to receive unprecedented attention from both the public and the scientific community.
Molecular biotechnology can benefit humanity by
providing opportunities to accurately diagnose, prevent, or cure a wide range of infectious and genetic diseases
increasing crop yields by creating plants that are resistant to insect predation, fungal and viral diseases, and environmental stresses such as short-term drought and excessive heat, and at the same time reduce applications of hazardous agrichemicals
creating microorganisms that will produce chemicals, antibiotics, polymers, amino acids, enzymes, and various food additives that are important for food production and other industries
developing livestock and other animals that have genetically enhanced attributes
facilitating the removal of pollutants and waste materials from the environment.
Although it is exciting and important to emphasize the positive aspects of new advances, there are also social concerns and consequences that must be addressed. For example,
Will some genetically engineered organisms, or their products, be harmful to humans or other organisms, or to the environment?
Will the development and use of genetically engineered organisms reduce natural biological diversity?
Should humans be genetically manipulated?
Will new diagnostic procedures, especially those based on genome sequencing, undermine individual privacy?
Will financial support for molecular biotechnology constrain the development of other important technologies?
Will the emphasis on commercial success mean that the benefits of molecular biotechnology will be available only to wealthy individuals or nations?
Will agricultural molecular biotechnology undermine traditional farming practices?
Will medical therapies based on molecular biotechnology supersede equally effective traditional treatments?
Will the quest for patents inhibit the free exchange of ideas among research scientists?
These and many other issues have been considered by government commissions, discussed extensively at conferences, and thoughtfully debated and analyzed by individuals in both popular and academic publications. On this basis, regulations have been formulated, guidelines have been established, and policies have been created. There has been active and extensive participation by both scientists and the general public in deciding how molecular biotechnology should proceed, although some controversies still remain.
Molecular biotechnology, with much fuss and fanfare, became a comprehensive scientific and commercial venture in a remarkably short period of time. Many scientific and business publications are now devoted to molecular biotechnology, and graduate and undergraduate programs and courses are available at universities throughout the world to teach molecular biotechnology. It could be debated whether the early promise of biotechnology has been fulfilled as it was predicted to in a 1987 document published by the U.S. Office of Technology Assessment which declared that molecular biotechnology is “a new scientific revolution that could change the lives and futures of … citizens as dramatically as did the Industrial Revolution two centuries ago and the computer revolution today. The ability to manipulate genetic material to achieve specified outcomes in living organisms … promises major changes in many aspects of modern life.” It does, however, offer solutions to some serious global problems including the spread of infectious diseases, the burden of waste accumulation, and food shortages that may become increasingly dire as the climate changes. The potential of molecular biotechnology to solve some of these imminent problems is the subject of this book.
summary
In 1973, Stanley Cohen, Herbert Boyer, and their coworkers devised a method for transferring genetic information (genes) from one organism to another. This procedure, which became known as recombinant DNA technology, enabled researchers to isolate specific genes and perpetuate them in host organisms. Recombinant DNA technology has been beneficial to many different areas of study. However, its impact on biotechnology has been extraordinary.
Biotechnology uses organisms, often on a large scale, for the production of commercially important products. Before the advent of recombinant DNA technology, the most effective way of increasing the productivity of an organism was to induce mutations and then use selection procedures to identify organisms with superior traits. This process was not foolproof; it was time-consuming, labor-intensive, and costly, and only a small set of traits could be enhanced in this way. Recombinant DNA technology, however, provided a rapid, efficient, and powerful means for creating organisms with specific and expanded genetic attributes. The tools of recombinant DNA technology enable microorganisms, plants, and animals to be genetically engineered. Combining recombinant DNA technology with biotechnology created a dynamic and exciting discipline called molecular biotechnology.
A large number of molecular biotechnology products are currently available. Many vaccines and protein and nucleic acid therapeutics are produced in genetically engineered microorganisms, mammalian cells, or transgenic animals and used to treat a variety of diseases in humans and animals. Molecular diagnostic tests produced through recombinant DNA technology detect specific proteins or nucleic acid sequences that indicate susceptibility to, or progress of, a disease, or response to a treatment. Proteins or small molecules of industrial importance are often produced on a large scale by genetically engineered microorganisms. Increased agricultural output has been possible due to the development of transgenic plants that are resistant to pests, pathogens, and abiotic stresses such as drought and salt. A recent milestone was the approval of the first genetically engineered animal (a salmon) for human consumption by the U.S. Food and Drug Administration in 2015. The salmon, which has an enhanced growth rate, is expected to be in markets before 2018. Many more exciting solutions to medical, environmental, industrial, and agricultural problems are under development.
Because of its broad impact, molecular biotechnology has been scrutinized carefully for its potential effects on society. Some of the concerns that have been raised are its safety, possible negative effects on the environment, and the private or public ownership of genetically engineered organisms.
REFERENCES
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Cohen SN, Chang AC. 1973. Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc. Natl. Acad. Sci. USA. 70:1293–1297.
Cohen SN, Chang ACY, Boyer HW, Helling RB. 1973. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA. 70:3240–3244.
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review questions
1. What is biotechnology?
2. Distinguish between traditional biotechnology and molecular biotechnology.
3. What are the shortcomings of the “mutation and selection” method for developing enhanced organisms for commercial purposes?
4. Why was the work reported by Cohen and Boyer and their coworkers in 1973 considered important?
5. How did recombinant DNA technology enable the production of human insulin?
6. What are some of the problems that molecular biotechnology has the potential to solve?
7. Discuss the statement “Molecular biotechnology is a diverse science.”
8. Discuss some of the social concerns that have been raised about molecular biotechnology.
9. Go to your favorite news website and conduct a search with the word “biotechnology.” Describe and discuss three recent biotechnology news stories.
