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 x 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. x 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.

肥胖和 2 型糖尿病的流行促使消费者对植物性低热量甜味剂产生了浓厚的兴趣。三叶苷是一种甜味剂，在一系列海棠 ( Malus ) 物种的叶子中被发现，但在驯化的苹果 ( Malus x Domestica ) 叶子中却没有发现，因为后者含有三叶苷的苦味位置异构体根皮苷。三叶苷含量的变异被映射到由驯化苹果和海棠杂交产生的分离群体中 LG 7 上的三叶苷基因座。通过活性定向蛋白质纯化和差异基因表达分析，在三叶苷含量高但根皮苷含量低的样品中鉴定出根皮素糖基转移酶 2 ( PGT2 )。为PGT2开发的标记与Trilobatin基因座严格共分离。生化分析表明 PGT2 有效催化 4'-哦-根皮素糖基化为三叶苷以及3-羟基根皮素糖基化为西博丁。双键还原酶、查耳酮合酶和PGT2基因的瞬时表达重建了本塞姆氏烟草中三叶苷生产的苹果途径。过度表达PGT2 的转基因M. x Domestica植物在幼叶中产生高浓度的三叶苷。转基因植物表型正常，与在模拟田间条件下生长的野生型植物相比，没有观察到疾病易感性的差异。感官分析表明，来自PGT2转基因的苹果叶茶很容易与对照叶茶区分开，并且被认为明显更甜。 PGT2的鉴定允许开发标记辅助选择来培育含有三叶苷的苹果，并通过酵母中的生物制药和代谢工程生产大量这种天然低热量甜味剂。