Epidemics of
obesity and
type 2 diabetes drive strong consumer interest in plant-based low-calorie
sweeteners.
Trilobatin is a
sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain
trilobatin's bitter positional isomer
phloridzin. Variation in
trilobatin content was mapped to the
Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples.
Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed
protein purification and differential gene expression analysis in samples high in
trilobatin but low in
phloridzin. Markers developed for PGT2 cosegregated strictly with the
Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4'-o-glycosylation of
phloretin to
trilobatin as well as
3-hydroxyphloretin to sieboldin. Transient expression of double bond
reductase,
chalcone synthase, and PGT2 genes reconstituted the apple pathway for
trilobatin production in Nicotiana benthamiana Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of
trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in
disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing
trilobatin, and for high amounts of this natural low-calorie
sweetener to be produced via biopharming and metabolic engineering in yeast.