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Biotechnology and the Food Supply: Proceedings of a Symposium (1988)

Chapter: New Applictations of Biotechnology in the Food Industry

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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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Suggested Citation:"New Applictations of Biotechnology in the Food Industry." National Research Council. 1988. Biotechnology and the Food Supply: Proceedings of a Symposium. Washington, DC: The National Academies Press. doi: 10.17226/1369.
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NEW APPLICATIONS OF BIOTECHNOLOGY IN THE FOOD INDUSTRY Robert H. Lawrence In the past several years, biotechnology in the food industry has been the central theme of numerous scienti- fic reviews, national and international symposia, and several major reference works (Earle, 1984; Harlander and Labuza, 1986; Jarvis and Holmes, 1982; Kirsop, 1985; Knorr, 1987; Knorr and Sinskey, 1985; Moo-Young et al., 1985; Rehm anti Reed, 1983~. Reports of significant advances have come from the full spectrum of biotech- nology research and development resources: universities and institutes as well as genetic "biotiques" and large food corporations. Important business alliances continue to be formed on a worldwide scale, linking advanced biotechnology research skills with large producers and marketers of food products, principally in the United States, Japan, the United Kingdom, and Europe. These alliances include Amgen/Kodak, CalBio/American Home Proclucts, Genentech/Lilly, Genentech/Corning (Genencor), Interferon/Anheuser-Busch, Molecular Genetics/Upjohn, Synergen/Procter & Gamble, American Cyanamid/Pioneer Hi-Bred, Dupont/Advanced Genetic Sciences, W. R. Grace/ Cetus (Agricetus), Hoechst/Harvard, Monsanto/Genentech, Monsanto/Washington University/Rockefeller University, Roche/Agrigenetics, Beatrice/Ingene, Campbell/DNA Plant Technology (DNAP), Campbell/Calgene, CPC/Enzyme Bio- systems, Kraft/DNAP, General Foods/DNAP, Kellogg/ Agrigenetics, Heinz/ARCO, McCormick/Native Plants, Inc., Molson/Allelix, R]R-Nabisco/Escagen, and Seagram/ 19

Biotechnica. Corporate boards and strategic planning groups of major food companies now understand the language of biotechnology and can perceive its utility and value; this has been the case with their corporate research departments for years. One thing is clear: The excite- ment and enthusiasm for biotechnology so characteristic of the pharmaceutical and medical areas in the early 1980s have now begun to hit the food industry with increasing force, and this momentum will likely establish this indus- try as the largest commercial arena for biotechnology. Companies involved include Archer Daniels Midland, American Home Products, Beatrice, Campbell, Cargill, Corn Products Company, Coors, Chr. Hansen's Laboratory, Firmenich, General Foods, Heinz, Hunt-Wesson, Kraft, LaBatt, McCormick, Nestle, Pillsbury, Purdue, Procter & Gamble, Ralston, RJR-Nabisco, Staley, Unilever, and Universal Foods. At least three important factors are responsible for this. First, in pharmaceuticals, the feasibility of the biotechnology promise has been established and the commer- cial reduction to practice (i.e., commercial application) is in place--in the marketplace! This was achieved by using many of the same technical concepts and strategies currently envisioned for food industry applications. Second, key advancements in technology continue to be made, principally in molecular genetics, cell technolo- gies, computer-aided protein engineering, bioreactor design, and biosensor/diagnostic technology. These advancements have substantially redefined the technical skills base and broadened the potential applications of biotechnology to foods. Third, within the food industry, reports of successful new applications of biotechnology (e.g., those reported here) add confidence to the predic- tion that biotechnology may well be the next key source of competitive leverage at the corporate and international levels, and may be the most important single technical consideration in consolidation strategies. The following paragraphs are a review of new appli- cations of biotechnology in each of the following food-related areas: enzymes, including the processing of cheese; fermentation, including brewing and wine making; agricultural raw materials (e.g., crop plants, meat, poultry, fish) with improved functionality; and plant cell bioreactors for food ingredient production. 20

ENZYMES Food Industrv Uses A recent report on the U.S. market for enzymes indi- cated total sales of $185 million in 1985, 58% of which was in the food industry (Charles Kline & Co., 1986~. Of the classes of enzymes used by the food industry, the proteases and carbobydrases account for most of this market. The predominant protease sold is rennin (chymosin), which is used in cheese-making processes to Coagulate milk to form curds. Of the carbohydrates, those used in cornstarch processing (the so-called starch enzymes a-amylase, glucoamylase, and glucose isomer- ase) account for 85% of sales. The other enzymes have diverse applications, including flavor development (e.g., lipases in cheese making) (Arbige et al., 1986), improve- ment of extractions (e.g., pectinases in juice processing) (Kilara, 1982), and modification of food functionality (e.g., a-amylases in retarding bread staling) (Boyce, 1986~. The technology for improvement of food enzyme production by genetic engineering is clearly in place (Lin, 1986~. Genes for many of the important food industry enzymes have been cloned (Meade et al., 1987), and gene transfer systems that permit introduction and expression in generally recognized as safe (GRAS) organisms have been developed (Lin, 1986~. Two recent, important applications of genetic engineering to enzyme production are a-amylase and chymosin. a-Amylase High Fructose Corn SYrun Industrv. The first petition to the Food and Drug Administration (FDA) to affirm the GRAS status of a food-processing enzyme produced by recombinant DNA techniques was for a-amylase. This landmark petition was filed by CPC International, Inc., on July 9, 1984. a-Amylase is the enzyme used in the first step in the production of high-fructose corn syrup (HFCS), a widely used nutritive sweetener derived from cornstarch. The HFCS process was first developed in the United States between 1968 and 1972 by the Clinton Corn Processing Co. (Lloyd and Horwath, 1985) and involves three sequential 21

enzymatic steps. First, raw cornstarch is liquefied by a-amylase hydrolysis to yield partially degraded starch chains called dextrins. The dextrins are then hydrolyzed by glucoamylase, which cleaves both the a-1,6 and a-1,4 glucosidic linkages to give corn syrup (glucose). This glucose hydrolysate is refined and then isomerized by immobilized glucose isomerase to give a mixture of glucose and fructose (42%) known as HFCS. This last step was commercialized in 1972 and represents the first large-scale use of an immobilized enzyme permitting a continuous process with significant cost reduction (Casey, 1977~. The 42% HFCS can then be further purified to yield second-generation syrups of 55% and 90% fructose (Coker and Venkatasubramanian, 1985~. The market for HFCS has grown dramatically. U.S. consumption increased from 2.3 kg/per person in 1975 to 20 kg/per person in 1985 (Newsome, 1986~. Today, produc- tion exceeds 4.54 billion kg annually. HFCS is used in many processed food products and is the principal nutritive sweetener of the soft drink industry. Grant (1986) recently discussed CPC's genetically engineered a-amylase and its GRAS affirmation petition with the FDA. His objective was to use Bacillus subtilis as a host system for the commercial production of a thermostable form of a-amylase that CPC had developed from Bacillus stearothermonhilus (Ishii et al., 191--an organism given GRAS status by FDA (Figure 1~. This heat- and acid-stable form of a-amylase is important for low-cost production of HFCS. Its production in B. subtilis was desired because of the ease with which B. subtilis can be used in commercial fermentations. According to CPC's petition, the strain designated as B. subtilis ATCC 39,705 was genetically derived from an asporogenic variety of B. subtilis ATCC 39,701, which lacked cx-amylase, by introducing genetic material from B. stearothermonhilus ATCC 39,709 for ~-amylase pro- duction. The genetically engineered B. subtilis contains DNA from a plasmid vector designated as pCPC720. The plasmid consists of a 2.4-kb portion of DNA comprising the ~-amylase gene from B. stearothermonhilus and a portion of DNA from plasmid pUBllO required for replica- tion of the new plasmid. pCPC720 does not contain the kanamycin resistance marker of pUB110, and transformed host cells are not resistant to kanamycin. 22

· A Petition for the Affirmation of the GRAS Status of Alpha-Amylase Derived from Bacillus subtilis ATCC 39,705 · Filed July 1984 by CPC International · Proposed commercial product: alpha-amylase Gags as a cell- free broth containing a thermostable form of enzyme B. sublilis (AMY-) ATCC 39,701 Asporogenic B. subtilis (AMY+) ATCC 39,705 Asporogenic (Thermostable alpha-amylase) B. stearothermophilus (AMY+ ) ATCC 39,709 (Thermostable alpha-amylase) pCPC720 (AMY=) ~ FIGURE 1 Genetically engineered a-amYlase. The CPC petition presents data characterizing the enzyme and recombinant organism to show that the genetically engineered enzyme is equivalent in every respect to that produced by B. stearothermonhilus and to establish the safety of the recombinant product. The enzymes would be used in the HFCS process only and would not be present in the food product. Regarding the CPC petition, Grant urged, "We need to have scientifically based regulatory decisions, and we need to have responsible industry actions.... Because this is the first of potentially many such petitions being reviewed by FDA with respect to a recombinant microorga- nism, the FDA has to be very careful and precise. The FDA ruling on our petition will set th'e policy for future rulings" (Grant, 1986, p. 22~. That is no doubt the case: CPC in September was awarded a patent covering the genetic engineering of _. subtilis to produce a thermo- stable pullulanase (Coleman and McAlister, 1986~. There are many similar developments, as shown in the following two examples. ChYmosin (Rennin): DairY IndustrY. Another enzyme that has been the focus of considerable genetic engi 23

peering research is chymosin, the active component of rennet used in the dairy industry to coagulate milk to form curds in the cheese-making process. Chymosin is an endoprotease that is highly specific in the hydrolysis of peptide in bonds in the V-fold of kappa-casein of milk, resulting in the destabilization of casein micelles and subsequent curd formation. Commercial sources are calf rennet extracted from the fourth stomach of young suckling calves and microbial rennets principally from the fungi Mucor miehei, M. ousillus, or Endothia narasitica. These fungi produce chymosin with slight differences in milk-clotting properties, and in heat and pH stability, as well as a different coagulation/proteolysis ratio from that of chymosin from calf sources. Thus, a demand exists for chymosin similar to calf rennet for use in the pro- duction of quality cheeses. The opportunity for microbial production of calf rennet chymosin has led several com- panies to develop strategies to clone the gene for chymosin from cDNA libraries derived from calf stomach mRNA and to achieve expression of the heterologous gene in various host organisms (Figure 2~. In vivo, chymosin is · Strategies Gene Clone chymosin cDNA derived from calf stomach mRNA Signal -16 sequence 43 . . 365 _ (Protein) in Viva Secreted Zymogen Active Form Host Organisms · Cheese Trials t perprochymosin | prochymosin | chymosin E. coil, B. subtilis (Fusion protein) S. cerevisiae, W - erornyces /actis Aspergillus nidulans · E. co/l- derived chymosin vs. calf rennet · Comparable cheeses · Limitation · Accumulation in cytoplasm, insoluble and inactive aggregates. · Requires solubilization and refolding. Low yield, expensive. Companies -Codon, Genex, Unilever -Collaborative Research, Gist-Brocades -Genencor FIGURE 2 Genetically engineered chymosin production. 24

produced as preprochymosin, which is secreted as the zymogen, prochymosin. In low-pH solutions, prochymosin is autocatalytically cleaved to chymosin (McGuire, 1986~. Escherichia coli-produced chymosin has several limitations (Pitcher, 1986~. The enzyme accumulates in the cytoplasm, requiring an expensive and low-yield process to derive the active enzyme. Two alternative host systems have been used: the so-called supersecretor strains of yeast (used by Collaborative Research, Inc.) and the filamentous fungi (used by Genencor, Inc.~. The Genencor strategy for Asner~illus production of bovine chymosin (Heyneker et al., 1986; W.H. Pitcher, Genencor, personal communication, 1986) involved the use of hetero- logous gene constructions in A. nidulans transformants, which secrete the gene product. Plasmid constructions consisted of the control regions of the A. nicer glucoamylase gene coupled to either bovine prochymosin or preprochymosin cDNA with a glucoamylase terminator. A. nidulans transformants secreted chymosin, which was similar to authentic bovine chymosin in molecu- lar weight and specific activity. Cheese trials using these chymosin preparations are being evaluated (Pitcher, 1986~. Thus, commercial production of chymosin similar to calf rennet appears to be technically feasible. Other applications of genetic engineering to enzyme production for the food industry include: lactase, to break down milk lactose; lipase and esterase, to develop cheese flavor; pectinase, to improve yield, reduce viscosity, and enhance clarification in fruit juice processing and wine making; protease, to serve as a malt substitute when used with barley; and carbohydrases, to facilitate carbohydrate metabolism in low-calorie beer production. FERMENTATION Brewinz Yocum (1986) of BioTechnica International has reported the development of a genetic engineering procedure suitable for polyploid industrial yeast (SaccharomYces cerevisiae) strains used in brewing. A new set of plas- mids for industrial yeast transformation was developed; 25

these plasmids integrated the G418 resistance marker and targeted it for insertion at the HO (homothallism) locus. Multiple insertions were accomplished by a process that leaves the gene of interest integrated into the HO target locus but jettisons the G418 resistance gene. Yeasts transformed in this manner contain the new genes stably integrated into their chromosomes at homologous loci but with no remaining E. cold DNA sequences. For details of the plasmid contractions, refer to Figures 1 and 2 of Yocum (1986~. The BioTechnica group has demonstrated the commercial feasibility of this genetic engineering procedure in SaccharomYces for the production of light beer. BioTech- nica cloned the gene coding for glucoamylase from A. nicer and inserted the gene into brewing yeast. The glucoamylase expressed by the yeast during fermentation breaks down the soluble starch to glucose; this is metabolized by the yeast, resulting in a lower calorie beer without requiring the use of added enzyme prepar- ations. Wine Making Snow (1985) has recently proposed a strategy for genetic engineering of industrial yeast strains used in wine making to introduce the capability for malolactic fermentation. The primary fermentation that occurs in wine making is achieved through the use of yeast to convert sugar to alcohol. A secondary fermentation may be allowed to occur, particularly during production of red wines, which is catalyzed by bacteria in the genera Leuconostoc, Lactobacillus, and Pediococcus. During this secondary fermentation, malic acid is converted to lactic acid, which causes a decrease in wine acidity, brings finished wine into better acid balance, and develops more desirable flavor complexity. Procedures used to encourage the malolactic fermentation may increase the risk of wine quality loss. They also increase the costs of wine production. In the strategy proposed by Snow (1985) and experi- mentally investigated (Williams et al., 1984), the malolactic gene of Lactobacillus delbrueckii was introduced into a laboratory yeast strain. When this yeast was used to make wine in a trial fermentation, the 26

malolactic gene was expressed and limited malate conver- sion occurred. Thus, the feasibility of this approach appears to have been demonstrated. Obviously, the yeast gene transfer system developed by BioTechnica would be of value in this approach. AGRICULTURAL RAW MATERIALS New applications of biotechnology are leading to notable improvements in yield and productivity in crop plants and animals (Jaworski, see paper in this volume). Crops may be specifically improved for functional attributes, such as nutrition, flavor, texture, and processibility. These improvements result in added value to the food processor as well as to the consumer. Cron Plants Figure 3 gives a food industry perspective of plant biotechnology. This discussion focuses on three areas: the central role of modern breeding strategies in crop development, new genetic tools and how they influence breeding strategies, and the functional attributes of crops along with the concept of utilization-side genetics and added value. Functionality Genes E s m - CL Functional Attributes ~ Culture, \< ~ Cell I GeneUC| ~ PrOtoplasts) ~ ~ "Fusion ~ if_ Transter I Traditional Added Value New Added Value FIGURE 3 Plant biotechnology--food industry perspective. 27

Genetic Imorovement Strategies. Most contemporary approaches to crop improvement are centered on modern breeding strategies that use a wide range of genetic tools and germplasm resources to generate genetic variability and diversity for traits of interest and to construct genotypes with new gene combinations from which new plant varieties are developed and then selected through a series of trials and evaluations. To be effective in their critical strategic role, contemporary plant breeders must be proficient in the application of an array of genetic techniques, including several new technologies that are only now being integrated into plant breeding programs. These technologies have brought about a dramatic reorien- tation of the plant breeders' approach to the introduction of new genes into existing varieties and have greatly expanded the potential sources from which new, useful genes can be accessed. Conventional germplasm resources, including valuable wild plant populations, will remain a primary gene source. Techniques that facilitate inter- generic gene transfer will increase the importance of these germplasm resources. However, accessibility to genes from outside the plant kingdom (e.g., from bacteria and animals) is now possible and will require that plant breeders adopt a broader, interdisciplinary perspective. New hybridization systems for production of hybrid seeds are being developed; these involve cellular level manipulation of organellar genomes for cytoplasmic male sterility (Cocking, 1985) and the introduction of genes controlling self-incompatibility (Nasrallah and Nasrallah, 1985~. New hybrid seed production schemes have also been developed; these involve cloned parent lines produced by tissue culture techniques (Lawrence and Hill, 1982, 1983~. The ability to clone plants in large scale through somatic embryogenesis (Lawrence, 1981) and encapsulation to form synthetic seeds (Lutz et al., 1985; Redenbaugh et al., 1986) allow crops to be produced from unique geno- types, which cannot be economically reproduced through seeds. Thus, new options for crop establishment must be considered in developing breeding strategies. Plant breeding programs make extensive use of trials and evaluations, typically in greenhouses and field plots, for the characterization of genotypes and for making selections that will be subject to breeding advancement 28

or released for agricultural use. Under development are several diagnostic tools that permit evaluations and selections to be made in the laboratory (Frey, 1984~. These tools include isozyme analysis and protein electro- phoresis; DNA probes, molecular markers, and restriction fragment-length polymorphisms (RFLPs); and immunodiag- nostics. Applications of these techniques in plant breeding are of value in the attainment of several objectives: (1) breeding for Quantitative traits; (2) varietal identification and purity checks of seed lots; (3) screening for qualitative traits through marker linkage; (4) variety and genotype characterization for patent and Plant Variety Protection Certificate appli- cations; (5) predictions of combining ability to improve breeding productivity; and (6) characterization of the expression of genes introduced by molecular techniques (Frey, 1 984~. ~, ~do, , New Genetic Technioues. Several of the new genetic techniques currently being applied to plant breeding significantly extend the potential to manipulate crops genetically with greater efficiency and precision. These technologies include somaclonal variation, somatic cell genetics, gamete culture, protoplast fusion, and molecular approaches to gene transfer (Figure 3~. ., . ~. , . Although a considerable research effort in fundamental cellular and molecular biology has been required to develop these techniques as practical genetic tools, their strategic value in breeding must be considered within the context of the specific breeding objectives for a particular crop. A successful crop improvement program will generally require a balance in the use of more ... .. . . . . _ conventional approaches with predictable outcome, combined with more advanced tools with higher risks and less predictable utility. Both require a clear definition of traits targeted for improvement and a careful assessment of their commercial value. All the following techniques rely heavily on the universal capability of plant cells and tissues to be grown and manipulated In vitro. Literature on this subject constitutes an extensive knowledge base. The value of plant cell and tissue culture lies in the ability not just to access molecular and cellular genetic strate- gies, but also to use them in a practical way. The key is 29

the capability to regenerate intact plants containing new genetic capabilities that can be linked back into conven- tional plant breeding--the mechanism for achieving commercial value. Somaclonal variation is a commonly observed phenomenon in plants regenerated from cells or tissues cultured In vitro. The genetic variability obtained is believed to be a combination of genetic changes that occurred in the original plant tissues or mutations induced in the tissue culture cycle. Evans and Sharp (1986) have reviewed the unique aspects of the somaclonal variation process and its practical utility in plant breeding. These aspects include the following: (1) The frequency of genetic variation is significantly higher than spontaneous mutation. (2) Genetic mosaics occur at a low frequency. (3) A somaclonal variant can generally be genetically stabilized in one generation. (4) Deleterious genetic changes are usually eliminated by the stringency of the regeneration event. (5) Cytoplasmic genetic changes have been observed. (6) Dominant as well as recessive mutant alleles are generated. Somaclonal variation thus appears to be an efficient method to generate useful genetic variability in several crop plants, notably tomatoes (Evans and Sharp, 1983) and wheat (Larkin et al., 1984~. Recently, Scowcroft and colleagues (1985) reported on the genetic and molecular analysis of somaclonal variants in wheat. They observed many genetic changes: increase in ploidy, gross chromosomal rearrangements, transloca- tions, and single nucleotide substitutions. New variants not obtainable by other approaches were observed for several genetic loci. In addition, a high frequency of translocation among chromosomes was shown to be a useful mechanism to introgress alien genes into commercial varieties. Obviously, under certain conditions, the cell culture cycle can be like a genomic earthquake and may generate a wide array of variations. Somatic cell genetic methods also rely on cell culture-generated variability. An additional step is included to permit selection for specific traits at the cellular level. This approach has been useful in the generation of traits for tolerance to toxic selection agents, such as herbicides (Miller and Hughes, 1980; 30

-Shaner and Anderson, 1985), amino acid analogs (Harms et al., 1982), or toxins from pathogens (Carlson, 1973; Gengenbach et al., 1977~. Horsch et al. (1987) recently demonstrated the value of the somatic cell genetic approach in the genetic engineer- ing of plant resistance to the herbicide glyphosate. When isolated, as a result of a 20-fold amplification of the gene, a glyphosate-tolerant cell of petunia hybrid was shown to overproduce the enzyme (EPSP synthase) responsi- ble for tolerance. A cDNA clone encoding the enzyme was isolated from a cDNA library of the tolerant cell line and used to transform herbicide-sensitive lines into tolerant cell lines. Gamete culture can be used to generate haploids and doubled haploids for rapid development of homozygous breeding lines (Baenziger et al., 1984~. Several new varieties of rice, tobacco, and barley have been developed with other culture techniques. Protoplast fusion techniques are used to produce somatic hybrids by circumventing the usual sexual barriers among species and thus generating novel gene combinations (e.g., intergeneric combinations). Dudits and Praznovszky (1985) have shown that the production of asymmetric hybrids by fusion with irradiated protoplasts is an effective technique to create new gene combinations among distantly related plant species. The use of protoplasts to substitute or exchange cytoplasms to generate cytoplasmic male sterile (CMS) lines is of great practical value to plant breeders. This approach is a workable alternative to the generations of backcrossing usually required to convert a fertile line to a CMS line. Fusion techniques permit other genetic manipulation approaches to organelles (e.g., recombination); organelles are for the most part inaccessible for genetic improvement with conventional breeding methods. Gene transfer technology offers the most precise manipulation of genetic traits. Most crop plants can now be efficiently transformed either through Aerobacterium- mediated gene transfer (Horsch et al., 1985), direct DNA transfer by uptake into protoplasts (Potrykus et al., 1985), or microinjection (Crossway et al., 1986~. The 31

practical utility of the molecular transfer of specific genes and their stable integration into the plant genome has been clearly demonstrated (laworski, see paper in this volume). This technology significantly extends the range of sources for genes beyond reach of conventional breeding and adds a new dimension to crop improvement strategies, these sources include viral genes (e.g., the tobacco mosaic virus coat protein) expressed in plants to confer resistance, bacterial genes (e.g., EPSP synthase) for herbicide tolerance (Coma) et al., 1983), and insect genes (e.g., luciferase) for visual tracking and expression of transferred genes In situ (Ow et al., 1986~. The ability to control the proper expression of introduced genes is critical to the usefulness of this technology. Several recent reports are encouraging in that regard. A seed storage protein gene of bean transferred to tobacco was shown to be properly expressed in a tissue-specific manner in tobacco seeds (Sengupta-Gopalan et al., 1985~. Regu- latory sequences of seed storage proteins from legumes and a nonseed protein gene cluster have been shown to operate correctly when transferred to tobacco. It is safe to assume that the routine introduction and expression of foreign genes in plants is close at hand. What is not so well advanced is knowledge of the genes that determine functional attributes in crop plants for food use. Functional Attribute Genetics! There has been a serious neglect of research on the functional attributes of crops. By far the lion's share of both basic and applied research for crop improvement has been related to the production side: dealing with the agronomic traits, including disease and insect resistance, biochem- ical factors influencing yield, and stress and herbicide tolerance. This production-side or supply-side genetics affects supply, availability, and cost of raw materials. On the other hand, utilization-side or value-added genetics determines the processibility, nutrition, convenience, and quality of our raw materials and food products. The food industry has traditionally started with commodity raw materials, e.g., wheat, corn, and rice (Figure 3), and added value through processing technology to develop consumer products. We are now entering an era in the food industry when more emphasis will be placed on adding value further back in the food chain. Although this will be facilitated by the new genetic tools 32

discussed above, it is severely limited by our current lack of understanding of functional attributes at the biochemical level. Also, many of the functional attribute traits are multigenic, which complicates genetic strate- gies for their improvement. Despite these limitations, we can expect to see a much greater emphasis on raw materials from crops that will be genetically tailored for the food processor and the consumer; the result will be "noncom- modity" (Klausner, 1986), differentiated raw materials. A clear demonstration of this has been the recent work with high-solids tomatoes. Increasing the solids content of tomatoes from 5% solids to a level of 6% solids has a value in the processed tomato industry worth $80-100 million per year. Several approaches are being pursued, but to date only the somaclonal variant line of DNA Plant Technology, called DNAP-9 (Certificate of Plant Variety Protection No. 8400146), has been released. DNAP-9 was derived from UCS2B, a standard, open-pollenated processing variety, by regenerating plants from cultured tissues. Data submitted with the DNAP application filed in August 1984 indicate that the only principal difference is an increase in soluble solids of approximately 20% compared to that of UCS2B. Although this appears to demonstrate the utility of the somaclonal variation approach, DNAP-9 may not be competitive with recently developed F1 hybrids, which have higher yields and a roughly equivalent content of solids. DNAP and other companies are evaluating other somaclonal variant-derived hybrids. Molecular genetic approaches to increasing soluble solids in tomatoes have not demonstrated progress as yet. The effect of mutant alleles in carbohydrate metabolism is well documented (Shannon and Garwood, 1984~. With the genetic technologies being developed, it should be possi- ble to specifically manipulate carbohydrate metabolism in cereal crops at the molecular level. Examples of food industry applications are improved texture and cooking properties of rice, enhanced sweetness and mouthfeel, e.g., creaminess of sweet corns, and antistaling char- acteristics of wheat flours for baked goods. From a somewhat different perspective, it should be possible to improve the texture of fruits and vegetables by inhibiting the expression of cellulase and pectinase during ripening (Wasserman et al., 1986~. 33

Several strategies are being pursued to improve the essential amino acid balance of cereal grains and legume crops required in human and animal nutrition. Typically, cereals are deficient in lysine, and legumes are defi- cient in sulfur amino acids, methionine, and cysteine. Schaeffer (1986) and Hibberd et al. (1986) have reported the use of somatic cell genetics to select for cells that are resistant to amino acid analogs and that overproduce the deficient amino acids. Corn and rice plants regen- erated from these cells have been shown to have higher levels of the specific amino acids in the grain. Molecular approaches are also making progress. Larkins (1987) reported that several laboratories are modifying seed storage protein genes by inserting specific sequences or making specific base substitutions to produce endoge- nous seed storage proteins containing higher levels of the limiting amino acids. Another approach involves either enhancing the expression of endogenous genes coding for nutritionally rich proteins or introducing seed storage protein genes from heterologous species to improve the amino acid balance (Rao and Singh, 1986~. (The latter strategy is also being attempted for genes coding for the production of animal proteins, such as ovalbumin.) To date, none of these strategies has yielded an improved grain. In wheat, seed storage proteins control dough quality for baked goods, and these are amenable to similar genetic strategies. Obviously, there are many other opportunities to improve the functional attributes of crops for the food industry. Several groups (Rattray, 1984; Sharp, 1986) are manipulating lipid biosynthesis to improve oil content and to modify triglyceride composition to enhance value (e.g., the producticn of coconut type oils in soybean or rapeseed). Unilever, Sime Darby, and the DNAP/United Fruit joint venture are currently establishing plantations of elite oil palm selections based on tissue culture cloning (Sharp, longs. A 25 to 30% increase in yield of oil is predicted. In plant biotechnology, there have been several very important advances that will affect our ability to modify functional attributes. The most important is the recent report of Ecker and Davis (1986) that antisense RNA (minus-strand ROSA) inhibits specific gene expression in 34

plants. Similar observations have been reported in bacterial and animal systems (Green et al., 1986~. The practical importance of the antisense RNA approach to (in effect) generating instant mutant alleles is considera- ble. Not only can specific genes be blocked in a manner similar to mutations, but one antisense gene could also be used to block a multigene family. In addition, the antisense approach could be used to simulate a homozygous mutant allele, which would be of great value in polyploid species such as common bread wheat, which is hexaploid. In plant cell technology, methods to transform cereal crops such as rice and corn (Fromm et al., 1986; Potrykus et al., 1985) and to regenerate plants from protoplasts (Abdullah et al., 1986) were recently reported. Soybeans, which have proven to be very resistant to cell culture manipulation, can also be regenerated (Collins et al., 1985~. Thus, key techniques are now in place for most important food crops. Production-Side Versus Utilization-Side Genetics In this volume, Senator Albert Gore, Jr. (D-Tenn) has addressed the serious issue of the net effect of new applications of biotechnology on U.S. agriculture. Regarding the risks, he stated, "Given our record in <leafing with agriculture, we are far more likely to accidentally drown ourselves in a sea of excess grain, and pointed out that we may further advance an age of intractable overproduction, promoting large-scale agribusiness farming at the expense of small farms. He asked, "Is the family farm about to be genetically altered out of existence?" And he concluded that with such an outcome, "Biotechnology will be a hollow victory for science or for society...." These are valid points that reflect a perception shared by many of Senator Gore's colleagues who are involved in agricultural planning. This perception is based largely on the historically unbalanced agricultural research and development emphasis on production- or supply-side genetics (i.e., a focus on the production component of agriculture to achieve higher yields and lower costs of production). The majority of crop biotechnology is being applied to improvement of agronomic traits. This is especially true for both large corporations and small biotech companies, whose primary customer is the farmer. 35

However, in the food industry the recent increase in research in crop biotechnology is building momentum on the other side of the equation--utilization-side or value- added genetics. Here, research is oriented toward the end use of agricultural raw materials, with a focus on added value and higher return. There are great opportunities to extend the value-added food industry component back to the farm (Figure 3~. Genetic improvement of specific crops for maximal return according to end use creates differ- entiated or noncommodity raw materials. The key to progress in this area will be research to develop a knowledge of the functionality of raw materials that is sufficient to be translated to genetic manipulation strategies. Companies that are successful in these efforts may generate an important new factor in technical leverage. The same may also be true at the international level. Utilization-side or value-added genetics will bring about a certain degree of restructuring in agricultural practices (e.g., emphasizing contract crop production and raw material identity channels). Whether these changes will favorably affect the family farm is an important question to be addressed by agricultural economists. Regardless of which side of the crop improvement equation is pursued, the issue remains: Which specific genes or traits are to be selected as economically feasible targets? Plant Cell Bioreactors Thirty years ago, Routier and Nickell (1956) at Pfizer received a U.S. patent for the use of plant cell cultures for the industrial production of natural products. Since that time, considerable progress has been made in several areas: (1) in the number of plant species that can be grown in culture, (2) in the production of a wide array of secondary metabolites (Dougall, 1985), (3) in our understanding of the biochemical pathways involved and their regulation, and (4) in bioreactor design and culture protocols (Shuler and Hallsby, 1985~. Despite these advances, there are only two commercial applica- tions, and these are very high-value medicinals and cosmetic ingredients--shikonin (Tabata and Fujita, 1985) and ginsengoside (Ushlyama et al., 1986~. 36

Long-range projections for inexpensive production of secondary metabolites, based on future technical develop- ments, have been reported (Sahai and Knuth, 1985~. However, the outlook for economical production of food ingredients by cell culture is not optimistic for the near term. This is due primarily to their relatively low value, low levels of production by cells, and the high cost of the plant cell bioreactor approach. The most recent estimate at the current state of the technology is roughly $3,000/kg (Drapeau et al., 1987~. This appears to be a prime area for applying rDNA techniques to achieve the dramatic improvements required for commercial success. ANIMAL BIOTECHNOLOGY Most of the current applications of animal biotech- nology relate to the production side (Evans and Hollaender, 1986~; these include bovine growth hormone work, vaccine production, disease prevention, and embryo manipulations (sex selection, twinning, embryo storage, and transfer). Transgenic farm animals are still in the future. However, one area that relates to functional attributes for food is worth mentioning: genetic engi- neering of bovine milk proteins--the caseins. These are perhaps one of the most important and well-characterized groups of food proteins besides the seed storage proteins. Molecular work has advanced to the stage where systematic structure/function studies can be conducted on this class of proteins; this can lead to better under- standing of food protein functionality. Tom Richardson's group at the University of California, Davis, recently proposed a strategy involving protein engineering to change cascin structure to improve function in food products (e.g., caseins with additional chymosin sites to accelerate the rate of texture development) (Kang et al., 1986~. Commercial application of this strategy, however, must await progress in gene transfer and · · · ~ expression In animals. CONCLUSIONS Biotechnolo~v Research in the Food IndustrY The food industry is characteristically conservative in the amount it invests in research (typically, 1.5% or less 37

of sales) and in its adoption of rapidly emerging areas of technology. As it now exists, biotechnology research is supported primarily by such typical power bases as DuPont and Monsanto and also by an increasing number of small research companies, the biotiques. Most of these small companies are quite skilled in their areas of expertise and are highly motivated, responsive, and productive. They represent the best of what we have come to recognize as the entrepreneurial spirit. What we are seeing in food industry biotechnology is the effective use of research alliances between large food processors and the biotiques. This has allowed food companies to quickly attain critical mass in specialized areas of research and has served to accelerate develop- ments in this area. Several of the examples used in this paper were the result of such alliances. This trend will continue. However, we are beginning to enter a new phase. As food companies become more familiar with the technology and begin to experience its success in the marketplace, we will see internalization of research skills and the full integration of biotechnology into the well-established food research disciplines. Consolidation in the Food Industry Acquisitions and mergers are common phenomena in the [ood industry. The result is the development of large corporations that are horizontally integrated across a broad spectrum of food sectors. As this occurs, along with the internationalization of biotechnology research skills, we will experience a strong movement toward vertical integration in the direction of our raw material base, which will position the "value-added cascade" to begin further back in the system, at the genetic level. This may represent the emergence of an important new fulcrum of competitive leverage in the food industry and may very well bring genetic biotechnology into its most productive arena. REFERENCES Abdullah, R., E.C. Cocking, and J.A. Thompson. 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technol. 4:1087- 1 090. 38

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