Dihydrochalcones as natural sweeteners against "modern" diseases

Humans love the taste of sweet foods, a craving likely due to our natural instinct to search for high calorie foods. However, obesity, diabetes and high blood pressure have become common health issues in the modern world, and therefore there is increasing demand for foods containing decreased calories and sugar levels. Fruit trees such as citrus have been bred over thousands of years to bear high sugar content fruit, and thus, while apple fruit is considered healthy for all in some respects (fiber, antioxidants), the high sugar content is a problem for a growing worldwide population suffering from "modern" diseases. One possible strategy/solution is to develop fruits to contain natural low-calorie sweeteners (e.g. dihydrochalcone), which would allow breeding for reduced fruit sugar levels while maintaining sweetness. Dihydrochalcone-glycosides are a family of sweet tasting naturally occurring bicyclic aromatic compounds. For instance, phloretin, a simple dihydrochalcone found in plants, is glycosylated at the 4' position to obtain the sweet tasting trilobatin. Phloretin and trilobatin accumulate in low levels in Malus species, in some tropical citrus species, and in a few other plants (Gosch et al., 2010). Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the proposed dihydrochalcone precursor, p-dihydrocoumaroyl-CoA, are unknown. Here we describe the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6 µM, 92.9 µM and 101.3 µM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds