Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbial Systems
Plant Genetic Engineering: Applications
Modification of Fruit Taste and Appearance

Alteration of Appearance

Changes in the color of harvested fruits and vegetables pose serious marketing challenges. One approach to mitigating food discoloration involves The Use of various food additives; however, this introduces other concerns. Recently, for instance, questions have been raised regarding the safety of one such additive Class—sulfites.

Discoloration in fruits and vegetables begins with The oxidation of monophenols and o-diphenols to o-Quinones. The process is catalyzed by polyphenol oxidase Enzymes, which are encoded by nuclear DNA, possess a Molecular Weight of approximately 59,000, and are localized in the membranes of METABOLISM/14.html">Chloroplasts and Cell/35.html">Mitochondria. The hypothesis that inhibiting polyphenol oxidase could solve Structure/149.html">The problem of fruit discoloration was tested using transgenic potatoes carrying various polyphenol oxidase cDNA constructs. Vectors were constructed containing either a fragment or the full-length cDNA of potato polyphenol oxidase in a "sense" or "antisense" orientation, placed under the control of one of three promoters: the cauliflower mosaic virus 35S promoter, the granule-bound starch synthase Gene promoter, or the patatin gene promoter (Fig. 18.18). The latter two promoters are specific to potato tubers. Two commercial potato cultivars transformed with these constructs exhibited high resistance to blackspot bruise (enzymatic browning), with resistance levels far exceeding those achievable through conventional breeding. Transgenic Plants harboring the polyphenol oxidase cDNA were intentionally bruised and subsequently evaluated for blackspot resistance. Most transgenic plants carrying the antisense polyphenol oxidase gene under the control of either the cauliflower mosaic virus 35S promoter or the granule-bound starch synthase gene promoter were significantly more resistant than untransformed controls. The activity of the patatin gene promoter in potato tubers appeared to be only partially expressed, and polyphenol oxidase accumulation was not blocked. All plants containing sense constructs synthesized higher levels of polyphenol oxidase and were more susceptible to damage than the controls. Although these results remain strictly preliminary, the described approach could prove valuable for controlling enzymatic browning in various commercially important crops.

Table 18.6. Transgenic canola cultivars with altered seed fatty acid composition¹⁾

¹⁾ Based on Murphy, Trends Biotechnol. 14: 206–213, 1996.

Fig. 18.18. Constructs with "sense" and "antisense" orientations of the polyphenol oxidase gene. Transcription in both cases was driven by one of the promoters: the cauliflower mosaic virus 35S promoter (p35S), the granule-bound starch synthase gene promoter (pGBSS), or the patatin 1 gene promoter (pPATATIN), along with the nopaline synthase gene transcription termination signal (tNOS). LB and RB denote the left and right T-DNA border sequences, respectively. (Adapted from Bachem et al., Bio/Technology 12: 1101-1105, 1994.)

Alteration of Flavor

Unpalatable fruits and vegetables are unlikely to find consumer demand, regardless of their high nutritional value. Of course, the taste of food products can be enhanced during preparation by adding salt, sugar, flavorings, or other additives; however, from an economic standpoint, it would be preferable for food products to naturally possess the required flavor qualities and a more appetizing appearance.

The fruit of the African plant Dioscorephyllum cumminsii Diels contains the protein monellin, which is approximately 100,000 times sweeter than sucrose on an equimolar basis. This protein could well serve as a sugar substitute, with the added advantage that, being a non-carbohydrate, it should not exert adverse metabolic effects.

Monellin is a two-chain dimer; the A-chain consists of 45 amino acid residues, and the B-chain of 50. The chains are held together by weak non-covalent bonds, which limits its application as a sweetener because it readily dissociates and loses its flavor properties upon heating during food preparation or under the action of acids (such as citric or acetic acid). The Challenge of engineering transgenic plants or microorganisms capable of synthesizing monellin is complicated by the need to clone and coordinately express two separate genes. To overcome this problem, a synthetic monellin gene was constructed that encodes the A- and B-chains as a single polypeptide. Transgenic tomato and lettuce plants synthesizing this chimeric protein were successfully generated using two different promoters. In tomatoes, this was the fruit-specific E8 promoter, which activates early in ripening. In lettuce plants, the gene was placed under the control of the cauliflower mosaic virus 35S promoter. In both cases, nopaline synthase transcription termination/polyadenylation signals from a Ti plasmid were utilized. The synthetic monellin gene was introduced into plant Cells via A. tumefaciens infection, employing a cointegrative Ti plasmid vector system. Monellin was detected in ripe and partially ripe fruits as well as in lettuce leaves, but not in green tomatoes; its levels in tomatoes increased sharply upon a surge in the concentration of the plant hormone Ethylene. Reports on comprehensive taste evaluations of genetically sweetened foods are not yet available, but if the results prove favorable, this method of fruit sweetening could be applied to numerous crops.