Essay 13.1

Engineering Fruit Aromas

Gepstein Shimon, Department of Biology, Technion–Israel Institute of Technology, Haifa, Israel; Lewinsohn Efraim, Newe Ya′ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

September, 2006

Flavors and Aromas

Apart from the sensations perceived by our sense of taste such as sweetness, bitterness, sourness and saltiness, the unique "flavors" associated with our food are contributed by aromas perceived through our sense of smell. This sense is very sensitive and characterized by an almost infinite number of different odors than we realize. Although some aromas are prominently defined by a single molecule (such as cinnamaldehyde in cinnamon), most aromas consist of complex mixtures of volatiles. The contribution of each component to the overall aroma of our foods differs according to their perception thresholds, synergism with, and masking of, other components. Interestingly, despite the vast number of chemical structures involved, the large majority of scent compounds are biosynthesized by a surprisingly small number of metabolic pathways. Parts of these metabolic pathways are ubiquitous, and have developed by small but important modifications of ancestral genes and pathways (Pichersky and Gang 2000).

Why Genetic Engineering Is Appropriate

Crop breeding is used to improve plant variety and productivity in intensive agriculture. Not only has crop breeding produced a myriad of highly productive plants, but it has improved upon the quality of these plants, in some cases. Traditionally, most of the effort in breeding plants has been directed towards the inclusion of desirable agronomical traits, such as: high yields, ease of mechanization, perfect visual appeal, plant resistance to pests and pathogens, shelf life, and other commercially important characteristics. It is unfortunate that, with the development of these excellent crop varieties, traits that affect the aroma and flavor of fruit and vegetables have often been lost. The problem exists in most agricultural crops and has resulted in often legitimate consumer complaints concerning the lack of flavor (mainly aroma) in agricultural produce.

Metabolic engineering is ideally suited to alleviate this problem, either by providing assistance in conventional breeding programs (marker-assisted breeding) or by the implementation of genetic engineering. Although the specific major flavor and aroma compounds have been identified in many fruits, the genes and enzymes involved are not yet fully understood. Thus, to implement the novel biotechnological advances in restoring the "lost" aromas of fruits, it is imperative to identify the genes that affect flavor and aroma production, and to understand their regulation and limitations.

Metabolic Pathways: Identifying Key Enzymes and Genes

Three major pathways are involved in the biosynthesis of the main aroma component in plants:

Degradation of lipids for the formation of short-chain alcohols and aldehydes, such as n-hexanol or cis-3 hexenol (compounds imparting fresh and green notes);
The shikimic acid pathway by which eugenol (cloves), t-anethole (anise) and estragole (basil) are biosynthesized; and
The terpenoid pathway by which geraniol (rose), 1,8-cineole (eucalyptus) and menthol (peppermint) are synthesized.

Although many of the volatile constituents have been identified in vegetables and fruits, many of the enzymes and genes involved in their biosynthesis are still not known. Modification of fruit aroma by genetic engineering is dependent on the availability of identified genes, which encode enzymes of key reactions that influence or divert biosynthetic pathways of known aroma components.

Several approaches aimed at isolating and characterizing genes involved in fragrance formation have been successful. These methodologies have made available dozens of genes inferred to be involved in the formation of particular aromas. A few attempts to modify aroma by genetic engineering technology have been reported. Wang et al. (1996) successfully increased the concentration of some short chain alcohols and aldehydes by overexpressing a yeast desaturase in tomato fruits. In addition, two research groups have also succeeded in changing the aroma volatiles content in tomato fruits by overexpressing an alcohol dehydrogenase—thus, altering the ratio of short-chain alcohols to aldehydes (Spires et al. 1998; Prestage et al. 1999).

Metabolic Engineering of the Terpenoid Pathway

The terpenoid pathway is partially responsible for the synthesis of many important metabolites in plants, including the phytol chain found in chlorophyll, and plant growth regulators such as gibberellins, abscisic acid, accessory photosynthetic pigments, chromophores, and such vitamins as carotenoids, and tocopherols (Figure 1) (Croteau et al. 2000). Therefore, it is not surprising that manipulations of the terpenoid pathway in plants could produce undesired results. Attempts to increase carotenoid levels by overexpressing phytoene-synthase in tomato plants under the control of the CaMV 35S promoter, resulted in dwarf phenotypes, possibly due to a reduction of key diterpene derivatives (such as gibberellic acid), and an accompanied reduction in chlorophyll levels, presumably due to lack of phytol (Fray et al. 1995). However, a major success in manipulating the carotenoid pathway has been accomplished in rice, an important world food source. Normally, the mechanically processed rice grain that is consumed as food contains poor levels of beta-carotene, a vitamin A precursor (provitamin A). By a complex process of combining daffodil and bacterial genes that are involved in the terpenoid pathway, beta-carotene was produced in the grain endosperm. The transgenic rice grains enriched in provitamin A were designated as "golden rice" and are likely to provide a solution to the vitamin A deficiency problem (Ye et al. 2000).

Figure 1 Biosynthetic pathway of terpenoids, one of the major classes of natural products in plants. Components of aroma of many flowers and fruits are synthesized via this pathway. Monoterpenes (such as S-linalool), diterpenes (such as gibberelin and tocopherols) and tetraterpenes (such as carotenoids), are generally synthesized in plastids from glyceraldehyde-3-phosphate/deoxyxylulose phosphate (DOXP) via isopentenyl diphosphate (IPP). (Click image to enlarge.)

The potential of genetic engineering to improve fruit aroma by modifying the early steps of the terpenoid pathway has also been demonstrated. Linalool is an acyclic monoterpene alcohol that has an aroma with a sweet, floral alcoholic note. Linalool is a major component of the scent of many flowers, and is present in many edible fruits, such as guava, peach, plum, pineapple, and passionfruit. Linalool is a chiral compound, naturally appearing in two forms (S- and R-linalool) that differ in their aroma specificity. The enzyme linalool synthase, which catalyzes the formation of S-linalool from the ubiquitous monoterpene precursor geranyl diphosphate (Figure 2) has been purified (Pichersky et al. 1995), and its gene (LIS) has been cloned from the flowers of the California annual plant, Clarkia breweri.

Figure 2 Metabolic engineering of the aroma of tomato fruits is utilized to accumulate the volatile compound S-linalool. The LIS (linalool synthase) gene, under the control of the late-ripening E8 promoter, has been transferred from the Clarkia breweri flowers into the tomato fruits. Diversion of the existing plastid terpenoid pathway leading to carotenoids into the production of S-linalool in ripening tomatoes has been confirmed by GC-MS analysis (Figure 3). (Click image to enlarge.)

Figure 3 Presence of S-linalool and 8-hydroxylinalool in LIS-transgenic tomato fruits. GC-MS analysis of fresh tomato transgenic fruits has confirmed successful manipulation of the tomato fruit aroma by the LIS transgene. Top: A chromatograph of an extract from a LIS-transgenic fruit; bottom, from a non-transformed control. (Click image to enlarge.)

This gene has been suggested as a key candidate for metabolic engineering to modify the aroma of the modern tomato varieties that have lost some of their volatiles such as linalool. Since GPP is also an intermediate in the pathway to the tetraterpenoid carotenoid synthesis during fruit ripening, it was hypothesized that expressing the C. breweri LIS in the fruit during ripening would divert a portion of the GPP pool to the production of the volatile S-linalool, and possibly would improve the fruit aroma without having a significant influence on the accumulation of carotenoids (Lewinsohn et al. 2001). Indeed, tomato varieties have been transformed with the Clarkia LIS transgene under the control of the late-ripening specific E8 promoter, and accumulation of S-linalool was observed in the ripening fruits (see Figure 2).

The linalool that accumulated in the LIS-transgenic plants was virtually enantiomerically pure, exclusively accumulating S-linalool. The apparent lack of racemization might reflect a process of compartmentalization that separates linalool from the acid environment in the fruit tissues. Transgenic fruits also accumulated low levels of 8-hydroxylinalool, possibly due to allylic hydroxylation, a common reaction in monoterpene metabolism. The results suggest that such a hydroxylating activity is present in ripening tomato fruits, although the endogenous substrate is presently unknown. This finding is a cautionary example of one of the possible drawbacks of metabolic engineering. Introducing one trait, such as the production of S-linalool, can inadvertedly cause the appearance of an unexpected novel metabolite, such as 8-hydroxylinalool, due to the genetic and biochemical background of the transformed plants.

The possibility of having negative effects on the accumulation of related terpenoid compounds resulting from the manipulation of the terpenoid pathways was examined. Levels of major terpenoids such as lycopene, other carotenoids and tocopherols were unaffected in the linalool-accumulating transgenic fruits as compared to the controls. These results indicate that it is possible to significantly increase the levels of aroma components in ripening fruits (Lewinsohn et al. 2001). With the advent of other genes encoding enzymes and other biosynthetic pathways leading to the production of various volatile aroma chemicals, the potential for flavor and aroma improvement in a variety of fruits, flowers and vegetables by genetic manipulation is very promising.

References

Croteau, R., Kutchan, T. M., and Lewis, N. G. (2000) Natural products (secondary metabolites). In B. Buchanan, W. Gruissem, and R. Joneas, eds., Biochemistry and Molecular Biology of Plants. American Society of Plant Biologists, Rockville, MD, pp. 1250–1268.

B. Buchanan, W. Gruissem, and R. Jones, eds., American Society of Plant Physiologists, Rockville, MD, pp. 1250–1318.

Fray, R. G., Wallace, A., Fraser, P. D., Valero, D., Hedden, P., Bramley, P. M., and Grierson, D. (1995) Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway. Plant J. 8: 693–701.

Lewinsohn, E., Schalechet, F., Wilkinson, J., Matsui, K., Tadmor, Y., Nam, K.-H., Amar, O., Lastochkin, E., Larkov, O., Ravid, U., Hiatt, W., Gepstein, S., and Pichersky, E. (2001) Enhanced levels of the aroma and flavor compound S-Linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol. 127: 1256–1265.

Pichersky, E., and Gang, D. R. (2000) Genetics and biochemistry of secondary metabolites in plants: An evolutionary perspective. Trends Plant Sci. 205: 439–445.

Pichersky, E., Lewinsohn, E., and Croteau, R. (1995) Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch. Biochem. Biophys. 316: 803–807.

Prestage, S., Linforth, R. S. T., Taylor, A. J., Lee, E., Speirs, J., and Schuch, W. (1999) Volatile production in tomato fruit with modified alcohol dehydrogenase activity. J. Sci. Food Agric. 79: 131–136.

Speirs, J., Lee, E., Holt, K., Yong-Duk, K., Scott, N. S., Loveys, B., and Schuch, W. (1998) Genetic manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the balance of some flavor aldehydes and alcohols. Plant Physiol. 117: 1047–1058.

Wang, C., Chin, C. K., Ho, C. T., Hwang, C. F., Polashock, J. J., and Martin, C. E. (1996) Changes of fatty acids and fatty acid derived flavor compounds by expressing the yeast Delta-9 desaturase gene in tomato. J. Agric. Food Chem. 44: 3399–3402.

Ye, X., Al-Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P., and Potrykus, I. (2000) Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287: 303–305.