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Biology Department, University of New Brunswick,
Fredericton, New Brunswick, Canada

The two of us thank our life partners, Marcia Glick and Patrick Patten, for the enormous support and encouragement that they have provided throughout this endeavor

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Title: Molecular biotechnology : principles and applications of recombinant DNA / Bernard R. Glick, Cheryl L. Patten.

Identifiers: LCCN 2017011321 | ISBN 9781555819361 (hardcover) | ISBN 9781683673101 (ebook)

Potential for Transferring Transgenes from Food to Humans or Intestinal Microorganisms

BASED ON THE DEVELOPMENT OF RECOMBINANT DNA technology, molecular biotechnology emerged as a new research discipline in the late 1970’s. Since those early days, there has been a veritable explosion of knowledge in the biological sciences. With the advent of PCR, chemical DNA synthesis, DNA sequencing, monoclonal antibodies, directed mutagenesis, genomics, proteomics, metabolomics, and more recently, specific genome modification techniques, our understanding of and ability to manipulate the biological world has grown exponentially. When the first edition of Molecular Biotechnology: Principles and Applications of Recombinant DNA was published in 1994, nearly all of the transgenic organisms that were produced included only a single introduced gene. Now, 23 years later it is common for researchers to engineer organisms by both modifying the activity and the regulation of existing genes and also by introducing entire new pathways. In 1994, only a handful of products produced by this new technology had been commercialized. Today, as a consequence of molecular biotechnology hundreds of new therapeutic agents are available in the marketplace with many more in the pipeline as well as dozens of transgenic plants. DNA technologies have become a cornerstone of modern forensics, paternity testing and ancestry determination. A number of new recombinant vaccines have been developed, with many more on the horizon. The list goes on and on. Molecular biotechnology has clearly lived up to its promise and all of the original hype that has existed since the late 1970s. Worldwide there are several thousand biotechnology companies, in virtually every corner of the globe, employing hundreds of thousands of scientists. When the exciting science being done at universities, government labs and research institutes around the world is factored in, the rate of change and of discovery in the biological sciences is absolutely astounding. This fifth edition of Molecular Biotechnology, building upon the fundamentals that were established in the previous four editions, endeavors to provide readers with a window on some of the major developments in this growing field. Given the enormity of the field of molecular biotechnology, we have had to be highly selective in the material we included in this edition. Moreover, the window that we are looking through is moving. This notwithstanding, we both expect and look forward to the commercialization of many of the discoveries that are discussed here, and in the future to the development of many new approaches, insights, and discoveries.

We have throughout endeavored to make the text reader friendly by minimizing the use of technical jargon and unnecessary abbreviations. Moreover, when an important term appears for the first time in the text, it is followed in parentheses with a synonym or brief explanation. The overall size of this edition has been pared down significantly compared to the fourth edition, done, in large measure, by removing some older material that has come to be common knowledge within the past 10–20 years. In addition, to facilitate the book’s flow and ease of understanding, in a number of instances, two or more figures have been combined into a single figure. Endeavoring to be as up-to-date as possible, this edition expands the discussion of interfering RNA and explains CRISPR technology in detail, providing examples of their use in both gene therapy and transgenic plants.

Each chapter opens with an outline of topics and concludes with a summary and list of review questions to sharpen students’ critical thinking skills. All of the key ideas in the book are illustrated by the more than 500 full-color figures and elaborated in more than 80 tables. After introducing molecular biotechnology as a scientific and economic venture in Chapter 1, the next two chapters explain the detailed methodologies of molecular biotechnology. These chapters provide a solid scientific base for the remainder of the book. Chapters 4 to 8 present examples of microbial molecular biotechnology covering such topics as the production of metabolites, new vaccines, both protein and nucleic acid therapeutic agents, diagnostics, bioremediation, and biomass utilization. Chapter 9 describes some of the key components of large-scale fermentation processes using recombinant microorganisms. Chapters 10 to 12 describe the molecular biotechnology of plants and animals. The book concludes in Chapter 13 with a discussion of the interaction of molecular biotechnology with society including controversies that have occurred as a consequence of this technology, coverage of the regulation of molecular biotechnology and patents.

Throughout the text we have relied extensively upon the recent published work of many researchers. In all cases, although not cited directly in the body of a chapter, the original published articles are cited in the references section of the appropriate chapter. In some cases, we have taken “pedagogic license” and either extracted or reformulated data from the original publications. Clearly, we are responsible for any distortions or misrepresentations from these simplifications, although we hope that none has occurred. The references sections also contain other sources that we used in a general way, which might, if consulted, bring the readers closer to a particular subject.

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.

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).

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.

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.

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.

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,

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.