Recombinant DNA
Inside recombinant DNA
Recombinant DNA
Recombinant DNA represents a unique potential health hazard. Genetic manipulation has given researchers a powerful tool to define the pathogenesis of many diseases as well as to initiate potential therapies. Recombinant DNA is any foreign or altered DNA sequence that has been spliced into cellular DNA, and which may be replicated. One danger unique to recombinant DNA work is the potential for lab personnel to become directly transfected by foreign DNA. Another potential hazard is the production of microbial agents with enhanced virulence, posing a significant environmental threat.
Traditionally gene transfer was accomplished by the introduction of naked plasmid DNA to the cellular target, relying on unassisted recombination to result in its incorporation into the cellular genome. In an effort to improve the efficiency of gene transfer, a number of viral vectors have been designed by incorporating the desired cDNA into the viral genome. The natural history of all viral propagation begins with introduction of the viral genome into the cell. The type of virus determines whether viral DNA will be incorporated into the host DNA and whether cells will be stably transduced. Most viral vectors have been rendered replication incompetent, allowing the introduction of the cDNA into the cell while not permitting propagation of the virus. These vectors have become widely available and their biosafety status may alter previously established laboratory precautions. A clear understanding of the vector (its biology and virulence) and the gene of interest (its protein product and function) is necessary to define the appropriate safety level.
Introduction
Recombinant DNA has been a transformative technology, providing tools that not only have enabled tremendous understanding of life at the most fundamental levels, but that have also led to a myriad of medical and agricultural applications. Progress in recombinant DNA research continues to revolutionize approaches to life science research and biotechnology and has been possible because scientists taking the lead in developing this technology had the foresight to recognize that the promise of recombinant DNA could only be realized if they assumed responsibility for addressing the safety and ethical concerns that it raised.
The current system of oversight of recombinant DNA research was established almost 40 years ago when the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) were first written [1]. At the federal level, the NIH Guidelines were initially administered by the Office of Recombinant DNA Activities (ORDA) that later became the Office of Biotechnology Activities (OBA) within the Office of Science Policy.
The NIH Guidelines outline the requirements for local oversight, including the establishment of an institutional oversight committee. The first NIH Guidelines articulated the requirements for "Institutional Biohazards Committees" that were later renamed "Institutional Biosafety Committees" (IBCs) to more clearly reflect their role. IBCs must review recombinant and synthetic nucleic acid molecule research for conformity with the NIH Guidelines. In addition, they assess the research for potential risks to health and the environment. This is accomplished by reviewing physical and biological containment for the research and ensuring that researchers are adequately trained to conduct the research they are proposing safely.
The hallmarks of this oversight system from its inception were public participation and transparency. Attention to the concerns of the community and local interests is a major theme that carries forward in the system of biosafety oversight today. This key element has served to preserve public trust in the safety of the life sciences research enterprise. In retrospect, the risks of recombinant DNA technology that were feared early in its evolution did not materialize. That fact notwithstanding, the development of a scientifically based oversight system with the IBCs as the centerpiece permitted the safe development of recombinant DNA as an essential technology in research. Over the years, oversight by IBCs has proven critically important to ensuring safety throughout various research fields – medical, occupational, environmental – as well as in promoting responsible scientific practice. Due to the dynamic nature of the life sciences there remains an ongoing need to assess biosafety dimensions of the research being conducted and to manage any risks associated with work. As life sciences research continues to advance, many lines of research, particularly involving highly pathogenic organisms, continue to generate public concern. Financial support for life sciences research comes primarily from publicly derived tax dollars, and so the life sciences community must demonstrate to the public that it is being a responsible steward of those funds. IBCs today remain critically important in preserving public trust and thus facilitating continued scientific progress. The National Institutes of Health (NIH) and the institutions it funds must continue to ensure that IBCs are equipped to fulfill their responsibilities so that biosafety risks are responsibly managed and public safety and trust are preserved.
39.5.1 Rational Protein Design
Revolutionary recombinant DNA technology (RDT) also pioneered protein engineering as RDT made it possible to change specifically any given amino acid in a polypeptide chain due to the precise and specific base replacement in a cloned DNA sequence instead of trial and error of mutations for desired characteristics (Hutchinson et al., 1978; Gillam and Smith, 1979a, 1979b). Thus site-directed mutagenesis was used as an elementary technique of gene manipulation due to simplified DNA manipulation that initiated protein engineering for the design and development of mutant proteins having superior working characteristics or entirely novel proteins. By the application of RDT-based, site-directed protein engineering, at first the formation of designer proteins or enzymes with predictable properties was not achieved (Lutz and Patrick, 2004; Neylon, 2004). For rational designing of proteins/enzymes, a prior knowledge of amino acid sequences and native folding of polypeptide chains, that is, a three-dimensional structure of the target protein and the correlation between structure and function is a prerequisite. These structural and functional understandings facilitate the "rational" prediction for changeable sites on the enzyme, where desired changes would provide desired properties to the targeted enzyme (Bonagura et al., 1999a, 1999b; Cahoon et al., 1997; Carter et al., 1989; Cedrone et al., 2000; Craik et al., 1985; Danielson et al., 1999; Gengenbach et al., 1999; Harris et al., 1998; Mouratou et al., 1999; Wells et al., 1987; Wilcox et al., 1998). After site-directed mutagenesis of flexible amino acids, the required properties containing mutants are selected. Hence for the protein/enzyme engineering by rational designing, the aforementioned information of the "hot spots" on the protein/enzyme is necessary. This advancement has generated considerable data on the role of the primary structure of proteins, that is, the sequence of amino acid residues and their respective role in structural and catalytic mechanisms. This data can be furthered subjugated to produce novel proteins with customized functions as industrially useful enzymes (Estell et al., 1985; Onuffer and Kirsch, 1995; Winter et al., 1982), antibodies (Jacobsen et al., 1997; Neuberger et al., 1984) and transporter proteins (Looker et al., 1992). With the use of the technique, chimeric proteins getting added specific functions of another protein are being formed, for example, the grafting of complementarity-determining regions (CDRs) or cell binding ligands from any other source to construct adapted antibodies (Abderrazek et al., 2011; Jones et al., 1986; Park, 2002; Rybak et al., 1992) and the improvement of protein functions such as hemoglobin and the transporter protein (Komiyama et al., 1995; Natarajan et al., 2011). In this method of protein engineering, gene mutation is carried out either randomly or at a distinct location and then a protein variant with the required property is screened and selected. With the directed evaluation process, the modified mutant can be further used for the next mutation and subsequent selection for more improvements (Myers et al., 1985). "Directed evolution," a widely used term, applies to various methods using a natural evolution strategy of mutagenesis with subsequent selection for varying and improving the functions of different enzymes (Nannemann et al., 2011). Directed evolution (or molecular evolution) does not require prior sequence or three-dimensional structure knowledge, as it usually employs random mutagenesis protocols to engineer enzymes that are subsequently screened for the desired properties (Dalby, 2003; Jaeger and Eggert, 2004; Jestin and Kaminski, 2004; Tao and Cornish, 2002; Williams et al., 2004). Directed evolution-based enzyme engineering is a widely accepted approach for improving the structural enzyme-substrate complex forming as well as the catalytic efficiency of industrially useful enzymes (Lutz and Bornscheuer, 2009). The enzyme subtilisin was used as a paradigm for this approach. This serine protease has every property altered such as reaction rate, specificity for substrate, pH optima, and enzyme stability for different factors (Bryan, 2000). Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research, and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties (Packer and Liu, 2015). Gene shuffling is an alternative approach to directed evolution, where many protein variants already existing in nature with desirable characteristics were used for novel combinations to form variants with more desirable properties (Bommarius, 2015). There are three alternative sources for gene shuffling: (1) the natural existence of polymorphic genes within a single organism or the formation of the gene of interest by random in vitro mutagenesis, (2) isozymic enzymes and their genes in different organisms, and (3) the presence of a protein family with a protein of interest as a member as well as other members with related activities. Ness et al. (1999) used 26 subtilisin family members to form a chimeric proteases library; later, this library was used for gene shuffling. With the screening of the library for four distinct enzyme properties, it was observed that few variants had considerably superior enzymes then any of the parental enzymes. Lehmann et al., initially used phytase sequences of 13 different fungal stains to construct a consensus enzyme (consensus phytase-1) which had increased thermostability than the original parent enzymes and subsequently swapped the active site of Aspergillus niger NRRL 3135 phytase with this synthetic phytase and termed new protein as consensus phytase-7, having limited variation in catalytic reactivity (Lehmann et al., 2000a). Hence "consensus sequence" based gene shuffling can add properties diverse than the parent proteins, an innovative approach in favor of directed evolution (Lehmann et al., 2000b).
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Genetically engineered bacteria for the degradation of dye and other organic compounds
Arvind Kumar, ... Bhadouria Rahul, in Abatement of Environmental Pollutants, 2020
12 Genetically modified endophytic bacteria and phytoremediation
Application of recombinant DNA technology as a promising tool in the field of contaminant removal has allowed present-day researchers with the possibility of modifying the genetic constitution of endophytes and rhizospheric microbes to facilitate the phytoremediation of soil contaminated with toxic chemical substances (Divya et al., 2011). Selection of a suitable strain for transgenic development and transfer into rhizospheric region is relied on three important points: (1) the generated transgenic microbes should express stability along with enhanced expression of candidate gene, (2) chosen microbe should be able to withstand the harsh environment and able to survive at higher contaminant concentration, and (3) designed microbes should have easy adaptation in plant-specific rhizospheric region (Huang et al., 2004). Numerous bacterial species has been observed to have low degradation abilities for a particular contaminant in rhizosphere. Recent advances in molecular biology techniques have given the fascinating opportunity to develop genetically engineered bacteria equipped with contaminant degrading capability for removal of noxious contaminants present in rhizosphere, i.e., rhizoremediation (Glick, 2010). Experimental investigations pertaining to the molecular details of degradation of contaminants such as trichloroethylene and PCBs have also been conducted. Interestingly, the inoculation of GEMs at the time of sowing into the rhizosphere would be helpful to minimize the competition for resource utilization by microbial consortia. As the transfer of GEMs into natural ecosystem could have a potential threat on the natural microbial communities, detailed risk assessment should be conducted as an inevitable step in this context (Wackett, 2004).
A list of different bacterial species deployed for the remediation of hazardous contaminants is provided in Table 16.1.
Table 16.1. List of some genetically engineered bacterial species used for bioremediation.
S. N.Engineered bacterial speciesContaminantRemarksReferences1.Pseudomonas fluorescens HK44NaphthaleneThe engineered microbe was luminescent; the first engineered bacterium approved for field trials; high survival under natural conditionRipp et al. (2000a,b)2.Pseudomonas fluorescensNaphthaleneLight induction was rapid and was highly responsive to alteration in naphthaleneKing et al. (1990)3.Comamonas testosteroni VP44(pPC3) and VP44(pE43)4-ChlorobenzoateUseful for remediation of polychlorinated biphenyls; complete degradation of contaminants; degradation of high conc. 10 mM; transgenic microbe possessed two different genes "ohb" and "fcb"Hrywna et al. (1999)4.Burkholderia xenovorans strain LB400 (ohb), Rhodococcus sp. strain RHA1(fcb)Aroclor 1242Contaminant favored the growth of transgenic bacteria; degradation was not significantly affected by inoculum density; the population dynamics was monitored by PCR and plate assayRodrigues et al. (2006)5.Rhodococcus sp. strain RHA14-Chlorobenzoates (CBA) and 4-chlorobiphenyl (4-CB)Recombinant strain stored lesser content of chlorinated by-products; recombinant strains were able to grow only in presence of contaminant; fcb operon stable even after 60 daysRodrigues et al. (2001)6.Cupriavidus necator RW112Monochlorobenzoates and 3,5-dichlorobenzoateFirst description of aerobic utilization of Aroclor mixtures; expression of transferred gene was measured by investigation of corresponding enzymatic activitiesWittich and Wolff (2007)7.Sinorhizobium meliloti strain USDA 1936 2′,3,4 PCB congenerEngineered microbe enhanced the degradation of contaminant; gene transfer was confirmed through molecular assays; plants associated with engineered microbes had more than two time degradation capabilities as compared to plants harboring wild strainsChen et al. (2005)8.Sinorhizobium meliloti strain DHK12,4-DinitrotolueneIn situ remediation of 2,4-dinitrotoluene; approximately 95% degradation of tested contaminant (0.55 mM)Dutta et al. (2003)9.Pseudomonas fluorescens RE2,4-DinitrotolueneThe modified organism completely degraded the contaminant; the degradation was also feasible at low temperature (10°C); the inoculation of GEM with plants did not affect the growthMonti et al. (2005)10.Pseudomonas fluorescensHexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)Pollutant degradation was high in presence of α-aminolevulinic acid; application of rhizosphere bacteria for degradation of explosivesLorenz et al. (2013)11.Pseudomonas sp. strain CB153-Chlorobiphenyl (3CB)Emulsification raised the degradation by engineered Pseudomonas; degraded product was accumulated by transgenic bacteriaAdams et al. (1992)12.Pseudomonas hybridsTrichloroethylene and cis-1,2-dichloroethyleneEngineered cells having todC1 gene were able to efficiently degrade a wide range of organic contaminants; possessed the genesSuyama et al. (1996)13.Streptomyces lividansPhenanthrene and 1-methoxynaphthaleneThe rate of biotransformation was high; 200 μM and 2 mM of phenanthrene were converted within 6 and 32 h, respectively; genes responsible for contaminant conversion was recovered from Nocardioides sp. strain KP7Chun et al. (2001)14.Ralstonia sp. KN1-10ATrichloroethylene (TCE)Most of the chlorine present in TCE was released as chloride ion; supply of external donors did not affect the degradation; contaminant did not show toxicity on engineered bacteriumIshida and Nakamura (2000)15.Escherichia coli and Pseudomonas putida strainsTCENearly absolute degradation was recorded for the contaminants; recombinant E. coli bacteria did not require the presence of isopropyl-β-d-thiogalactopyranosideFujita et al. (1995)16.Escherichia coli JM109C.I. Direct Blue 71The bioaugmented system was not functional at pH 5; at pH 9 there was dye content removal from 150 to 27.4 ppm within 12hJin et al. (2009)17.Escherichia coli JM109Acid red GR The dye degradation was affected by inoculums density and best was recorded at 10%; immobilization on foam carrier enhanced their degradation; the engineered cells maintained higher metabolic rate in batch reactorsJin et al. (2008)18.Rhodococcus erythropolis strainsPhenolThe transformed cells represented enhanced phenol hydroxylase activity; engineered cells were 50% much effective in phenol degradation; the plasmids harboring the genes responsible for contaminant degradation were recovered even after 288 h indicating stabilityZÝdkovß et al. (2013)19.Pseudomonas putida BH (pS10-45)PhenolEngineered bacteria enhanced the degradation; higher sludge settling was observed for GEM inoculated systems; presence of engineered Pseudomonas cells affected the native microbial community participating in phenol degradationSoda et al. (1998)20.Sphingomonas paucimobilis 551 (pS10-45)PhenolThe designed floc forming phenol degrading bacteria was effective in phenol removal; engineered cells maintained population density up to four timesSoda et al. (1999)21.Cupriavidus necator JMP134 -ONPNitrophenolThe transgenic bacteria was able to degrade different nitrophenols simultaneously; engineered organism was unable to utilize the metanitrophenol as the sole nitrogen sourceHu et al. (2014)22.Escherichia coli BL21AI-GOSOrganophosphatesThe bacteria could survive only in the presence of contaminant and commended suicide in the absence of contaminant; the bacteria was able to emit fluorescent light; the engineered microbe is safe for environmental applicationsLi and Wu (2009)23.Pseudomonas putida KTUeOrganophosphates, pyrethroids, and carbamatesThe engineered bacteria was able to simultaneously degrade three different pesticides; strain was able to survive at low oxygen availability; can be applied for in situ remediation of pesticide polluted soil; the modified bacteria was able to degrade 50 ppm of selected pesticides within 30 h in minimal medium containing glucoseGong et al. (2018)
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Future Prospectives for Enzyme Technologies in the Food Industry
Hita Rastogi, Sugandha Bhatia, in Enzymes in Food Biotechnology, 2019
49.2.1 Recombinant DNA Technology
The utilization of recombinant DNA technology or genetic engineering has complemented the improvement in the microbial strains as well as the enzymes associated with food processing and fermentation techniques. Since the advent of this tool in the 1970s, it has completely revolutionized the food industry. Novel as well as known enzymes can be specifically tailored using this technology to produce enzymes with the desired specificity and sensitivity along with reducing the production cost of the enzymes. The considerable success in the development of improved microbial strains has led to many applications in the food industry, such as engineering microbial host strains to enhance enzyme yields by deleting native genes encoding for extracellular proteases. Certain fungal strains have been manipulated to reduce or eliminate their potential for releasing metabolites that are toxic to the final product formation (Olempska-Beer et al., 2006). Genetically engineered strains can be effectively implemented for various batch and continuous operations. This reduces the requirement for the enzyme extraction and purification and can be easily cultured using cheaper raw materials. The genetically improved strains in the production of high fructose syrup from starch is also made suitable for several variable parameters, such as compatibility with addition of high concentrated substrates in the intermediate steps of a multistep biotransformation, metal ions removal/addition step, processing temperatures, and pH adjustments. Therefore, this technique has increased productivity and reduced the cost connected with upstream product processing (Liu and Xu, 2008; Panesar, 2010). Other enzymes (such as α-amylases and pullulanases) used in starch hydrolysis are actually produced by utilizing improved strains. Genetically modified (GM) microbial cultures, along with the production of enzymes, are also utilized for the production of monosodium glutamate (MSG), polyunsaturated fatty acids, and amino acids. However, the use of genetically improved strains is also restricted in some food technologies, owing to incongruity with production and purification procedures, lack of being certified as safe for consumption in the United States and the European Union, and the rising public consciousness regarding the nonconsumption of GMO or GMO-processed products (Agarwal and Sahu, 2014; Olempska-Beer et al., 2006; Spök, 2006).
The first commercial recombinant enzyme approved by the US Food and Drug Administration for used in food processing was bovine chymosin, involved in cheese manufacturing (Flamm, 1991). Another example of enzymes manipulated using genetic engineering (extensively reviewed in Olempska-Beer et al., 2006) and used in food processing includes alpha amylases engineered for increased heat stability for use in the production of high-fructose corn syrups. The Phospholipase A1 gene from Fusarium venenatum was expressed in GM Aspergillus oryzae to over-produce the phospholipase A1 enzyme. It is used in the dairy industry for cheese manufacture to improve process efficiencies and yields.
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Principles of Salmonid Culture
Edward M. Donaldson, Robert H. Devlin, in Developments in Aquaculture and Fisheries Science, 1996
Introduction
The development of recombinant DNA technology has resulted in improved knowledge of the structure and function of many genes from agricultural animals, including fish. A large number of genes have now been isolated and characterized from salmonids, and many of these are associated with commercially important traits. For example, genes involved directly in growth regulation include growth hormone (GH), insulin-like growth factors (IGF-I and IGF-II), growth hormone-releasing factor (GRF), and Pit-I. From this emerging molecular genetic information, it has become possible to understand the expression of important genes and to modify their structure accordingly to alter regulatory properties. An overall strategy for producing transgenic fish is shown in Figure 11, which indicates the multigeneration protocol required to produce strains useful for commercial aquaculture.

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Figure 11. Flow diagram showing the multigenerational procedure required to produce pure breeding lines of transgenic salmon.
Early gene transfer work in mice produced very dramatic effects, for example, doubling the size of mice containing mammalian GH (Palmiter et al. 1982,1983) or GRF (Hammer et al. 1985) gene constructs. Indeed, it is this series of reports on growth acceleration technologies in higher vertebrates that motivated much of the existing work on transgenic fish during the past decade. The ability to grow fish more rapidly was immediately recognized as having great potential for improving the efficiency of production in aquaculture. The first gene transfer study in fish was carried out using goldfish (Carassius auratus) (Zhu et al. 1985), and was soon followed by reports in loach (Misgurnus anguillacaudatus) (Zhu et al. 1986), salmonids (Chourrout et al. 1986b; Maclean et al. 1987; Fletcher et al. 1988), and catfish (Ictalurus punctatus) (Dunham et al. 1987). Since that time, a large number of studies have been performed in more than 15 fish species (see reviews by Maclean and Penman 1990; Chen and Powers 1990; Houdebine and Chourrout 1991; Fletcher and Davies 1991; Hackett 1993). The generation of transgenic fish for aquacultural purposes has also been reported for loach (Enikolopov et al. 1989), carp (Chen et al. 1993), catfish (Dunham et al. 1992), and pike (Esox lucius) (Gross et al. 1992), as well as five salmonid species including rainbow and cutthroat trout (Oncorhynchus clarki), and Atlantic, coho and chinook salmon (Chourrout et al. 1986b; Maclean et al. 1987; Fletcher et al. 1988; Guyomard et al. 1989; Penman et al. 1990; Du et al. 1992; Devlin et al. 1994c, 1994d, 1995a). Initial studies with salmonids focused on gene transfer methodologies, but it was not until the early 1990s that modification of any phenotypic character was achieved in transgenic salmonids (Du et al. 1992; Devlin et al. 1994c, 1994d, 1995a, 1995b).
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Transforming the Healthcare System Through Therapeutic Enzymes
Archana Vimal, Awanish Kumar, in Enzymes in Food Biotechnology, 2019
35.7.1 Microbial Strains Used for Production
The use of genetic engineering recombinant DNA technology enables the safer, economical, and desired amount of protein/enzymes production at controlled conditions. The production therapeutics on a large scale in the industry require a host. There are about 151 recombinant pharmaceuticals that are approved by the USFDA and/or by the European Medicines Agency (EMEA). Their production is mediated through the use of microbial cells, either bacteria or yeast, as hosts and they are often termed microbial factories. The most common choice of bacteria is E. coli. Other than this, Bacillus subtilis is also used. However, its use is restricted when a posttranslation modification (PTM) is desirable in the protein produced. To meet the requirement of PTM, yeast cells (Saccharomyces cerevisiae) are used. Apart from these, insect cell lines, hybridoma cell lines, hamster cell lines, and human cell lines are also preferred (Ferrer-Miralles et al., 2009).
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Nutrition of Entomophagous Insects and Other Arthropods
S.N. THOMPSON, K.S. HAGEN, in Handbook of Biological Control, 1999
Genetic Engineering of Entomophagous Species
Advances in molecular biology (recombinant DNA technology) further suggest the possibility for genetic manipulation of the nutrition of entomophagous insects (Thompson, 1990; Chapter 4). Genetic engineering, that is, incorporation of foreign or in vitro altered genes for expression of desirable traits by an organism, has advanced considerably (Beckendorf & Hoy, 1985; Whitten, 1989; Crampton, 1992). Heilmann et al. (1993) discussed the potential of genetic engineering for improving the performance of entomophagous insects. The technology, however, is still in its infancy, and methods for genetic transformation of eukaryotic organisms are limited at the present time (Schmidt, 1990; Finnegan, 1992). Nevertheless, stable genetic transformation of the phytoseiid mite predators Metaseiulus occidentalis (Nesbitt) (Presnail & Hoy, 1992; Presnail et al., 1997) and Amblyseius finlandicus (Oudemans) (Presnail & Hoy, 1994) and the braconid parasitoid Cardiochiles diaphaniae (Marsh) (Oudenmans) (Presnail & Hoy, 1996) has been achieved by microinjection, and Hoy (1993, 1995) and McDermott and Hoy (1997) have discussed the use of transgenic beneficial arthropods in pest management.
The future holds considerable promise for dramatic advances in our understanding the nutritional and dietary requirements of entomophagous insects. That knowledge will play an important role in the development of efficient rearing programs for parasitoids and predators.
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Biomass for Biorefining
Stephen R. Hughes, Nasib Qureshi, in Biorefineries, 2014
2.5.3 Regulation of Genetic Engineering
Genetic engineering is a process in which recombinant DNA technology is used to introduce desirable traits into organisms. It provides powerful tools to enhance animals, plants, and microbes for the benefit of society. Crops have been genetically engineered to decrease pesticide and herbicide usage, protect against stressors, improve yields, and extend shelf life. Consumers stand to benefit from the development of food crops with increased nutritional value, medicinal properties, enhanced taste, and esthetic appeal. Biomass can be genetically altered to improve its use for biofuel production. In the area of genetically engineered microbes, one important application is the development of microbes such as yeast that are engineered to ferment the constituent sugars in lignocellulosic biomass that holds promise as a source of cheap, plentiful, renewable energy (billion-ton report update). Microbes are being custom-engineered not only to ferment the sugars in such feedstocks but also to express high-value coproducts [45,46]. However, biotechnology poses unique challenges to the regulatory process because of the wide variety of changes that can conceivably be created in almost any organism [41,47].
The importation, interstate movement, or environmental release of genetically engineered organisms that may pose a plant pest risk, including plants, insects, or microbes, is under the regulatory control of the USDA, APHIS. In August 2002, the USDA created a new unit within APHIS called BRS to focus on USDA's key role in regulating and facilitating biotechnology [48]. BRS derives its authority to write regulations from provisions of the Plant Protection Act (PPA), which is a part of the larger Agriculture Risk Protection Act of 2000 [49]. According to the Federal PPA, any transgenic crop containing DNA of a known plant pest is viewed as a potential plant pest. All regulated introductions of genetically engineered organisms must be authorized by APHIS under either its permitting procedure or its notification procedure. APHIS issues permits for the introduction of genetically engineered organisms that pose a plant pest risk, including plants, insects, or microbes. All regulated genetically engineered organisms are eligible for the permitting procedure [48]. Applicants submit scientific information for APHIS to review before APHIS issues the permit. Notification is an administratively streamlined alternative to a permit. Developers must meet several criteria for APHIS to accept the notification [19]. After the developer has collected sufficient data, APHIS will accept a petition to deregulate the crop. APHIS grants nonregulated status if the genetically engineered organism poses no more of a plant pest risk than an equivalent nonengineered organism or if a regulated article is very similar to a genetically engineered organism that has already been granted nonregulated status. Nonregulated status means that permits and notifications are no longer required for introductions of this organism [48]. The regulations governing biotechnology as overseen by APHIS-BRS are found in the Federal Register and in the Code of Federal Regulations, Title 7, Section 340 (7 CFR 340) [50].
Genetically engineered microbes that are engineered to ferment the constituent sugars in lignocellulosic biomass or to express high-value coproducts that are used as food or feed must be approved by the Food and Drug Administration (FDA) for specific uses or meet FDA requirements for "generally recognized as safe" (GRAS) substances. A substance may be GRAS only if its general recognition of safety is based on the views of experts qualified to evaluate the safety of the substance. GRAS status may be based either on a history of safe use in food prior to 1958 or on scientific procedures, which require the same quantity and quality of evidence as would be required to obtain a food additive regulation. The food additive regulations for those substances with GRAS status has been affirmed by the FDA and substances that the FDA considers GRAS based on a history of safe use in food are found in the Code of Federal Regulations Title 21 (21 CFR) [51]. Any substance intentionally added to food is a food additive and is subject to review and approval by the FDA before the food is marketed, unless the substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use. Microorganisms and microbe-derived ingredients may be the subject of food additive and GRAS regulations [51].
Because these microorganisms and the substances derived from them are affirmed as safe for use in food products, from a regulatory standpoint, the microorganisms could serve as acceptable host strains for genetic engineering to express desired enzymes and valuable coproducts for cellulosic ethanol biofuel production. Numerous recombinant microorganisms have been the subject of notices submitted to the FDA Center for Food Safety and Applied Nutrition in accordance with the FDA's proposed regulation, 21 CFR 170.36 in the Federal Register of April 17, 1997 [52]. The FDA's Response Letters to these notices indicate that the FDA has no questions regarding the conclusions in the notices that the subjects of the notices are GRAS, but it has not yet made its own determination regarding their GRAS status. GRAS status would be needed if the coproducts of ethanol biofuel production were intended for food use. For organisms or inserts that are not well characterized, the applying party must establish GRAS status by providing sufficient scientific data on the host and production strains, the introduced genetic material, the manufacturing process, and the toxicity and genotoxicity of the recombinant organism. If the recombinant yeast were used for animal feed, similar information would need to be provided to the FDA Center for Veterinary Medicine [53].
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Bacillus thuringiensis in Biological Control
B.A. FEDERICI, in Handbook of Biological Control, 1999
Recombinant Bacteria
During this decade, several bacterial strains constructed using recombinant DNA technology have been registered in the United States. The first products registered were developed by Mycogen (San Diego, CA), and used Pseudomonas fluorescens as the host strain. The bacterium was engineered to produce large amounts of wild-type or recombinant Cry proteins, after which the cell wall was chemically fixed around the crystal to provide protection from ultraviolet light. Examples of products and the protein(s) they contain include MVP®, which contains a Cry1Ac-Cry1Ab chimera toxic to lepidopteran insects; MTRAK®, which contains Cry3A toxic to coleopteran insects; and MATTCH®, which contains Cry1Ac and Cry1C, toxic to lepidopteran insects (this product is a physical mixture of two P. flourscens strains, each producing one of the toxins).
Ecogen (Langhorne, PA) pioneered a strategy in which strains of B. thuringiensis were engineered to produce more complex mixtures of toxins using recombinant plasmids. Current products on the market are CRYMAX®, which contains Cry1Ac, and Cry2A, and is toxic to lepidopteran insects; Lepinox®, which contains Cry1Aa, Cry1Ac, a Cry1Ac-Cry1F chimera, and Cry2A, toxic to lepidopteran insects and designed especially for the Spodoptera complex; and Raven®, which contains Cry1Aa, Cry3A and Cry3Ba, and is toxic to both lepidopteran and coleopteran insects (Baum et al., 1998).
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A Perspective on Pathogens as Biological Control Agents for Insect Pests
B.A. FEDERICI, in Handbook of Biological Control, 1999
Use of Recombinant DNA Technology to Improve Nuclear Polyhedrosis Viruses
The two most significant limitations of conventional viral insecticides are the relatively slow speed of kill and the narrow host range. Recombinant DNA technology is being used to overcome both of these limitations (Miller, 1988; Maeda, 1989; Hawtin & Possee, 1992; Black et al., 1997; Treacy, 1998). When a conventional virus is used against an insect population (e.g., against noctuid larvae), the population typically consists of a mixture of instars and may contain more than one economically important species. A virulent NPV sprayed at an appropriate rate will kill the first and second, and in some cases the third instars, within 2 to 4 days. However, the more advanced instars may live for a week or more, causing further damage to the crop. Thus, current approaches to improving the efficacy of viral insecticides are aimed at developing broad-spectrum viruses that will cause a cessation of larval feeding within 24 to 48 h of infection, either by death or by paralysis. This is being done by deleting genes that delay death from viruses, and by engineering viruses to express genes encoding enzymes or peptide hormones that disrupt larval metabolism, or peptide neurotoxins that paralyze or kill the insect directly. Because it already has a broader host range than most occluded baculoviruses and can be genetically manipulated with ease in several cell lines, the Autographa Californica MNPV (AcMNPV) is the virus that has been the subject of most of the engineering studies to date.
The virus is engineered by deleting the gene encoding EGT (ecdysteroid UDP-glucosyltransferase) and/or by adding one or two genes encoding toxins under the control of strong viral promoters. By using this strategy, genes for juvenile hormone esterase (Hammock et al., 1990), B.t. endotoxins (Martin et al., 1990; Merryweather et al., 1990; Pang et al., 1992), insecticidal neurotoxins from the straw itch mite (Pyemotes tritici), scorpions (Androctonus australis, Buthus epeus, and Leirus quinquestriatus lebraeus), and spiders (Agelenopsis aperta, Diguetia canites, Tegenaria agnestis) have been engineered into the AcMNPV (McCutchen et al., 1991; Tomalski & Miller, 1991; Black et al., 1997; Treacy, 1998). Of these, the most promising results have been obtained with viruses producing neurotoxins, where the time between feeding and paralysis has been reduced by as much as 40% in comparison to wild-type AcMNPV (Treacy, 1998).
Engineering viruses to express insecticidal proteins in many cases could also result in an expanded host range. This is because in many lepidopteran host species that do not develop a patent disease when infected by conventional viruses, there can be limited viral replication, with these less susceptible hosts developing a mild disease and surviving infection. However, the same hosts infected by an engineered virus that expresses a potent insecticidal protein will likely succumb because the virus does not need to replicate extensively to paralyze or kill the larva.
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Hybridomas, Genetic Engineering of
Michael Butler, in Encyclopedia of Physical Science and Technology (Third Edition), 2003
XIV Recombinant Antibodies
A further possibility is the humanization of monoclonal antibodies originally produced from mice. This process involves antibody engineering which relies on the techniques of recombinant DNA technology to rearrange some of the molecular domains of an immunoglobulin. Examples of these are shown in Fig. 10. In a chimeric antibody the mouse variable regions are linked to human constant regions. Thus in such a construct the antigen-binding site of the murine antibody is retained but the human constant region contributes the immunogenicity through the effector functions. A further step to humanizing the antibody by replacing portions of the V region that are not required for the antigen-binding site. The framework regions (FR residues) which were originally murine are replaced by human regions. Thus only the complementarity-determining regions (CDR) are retained as of murine origin. Hybrid antibodies of this type have now been used as human therapeutic agents.

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FIGURE 10. Ways to humanize an antibody.
The elimination of the murine constant regions reduces the previously experienced HAMA response. It is not always certain that complete humanization has an advantage over a chimeric antibody because humanization of the V region may result in a loss in affinity to the antigen. Also, it is not clear that the problem of unwanted immunogenicity can be totally removed because repeated doses of even a fully humanized antibody may elicit an anti-idiotype response, that is directed against the antigen-binding site. However, these developments in humanized therapeutic antibodies have allowed the introduction of a range of products against specific human diseases