Steviol glycosides, natural sweeteners extracted from Stevia rebaudiana, are widely used as sucrose substitutes due to their high sweetness and low caloric value. Among them, Rebaudioside M (Reb M) is regarded as a next-generation, high-value steviol glycoside product because of its intense sweetness and superior taste profile. However, the natural abundance of Reb M in Stevia is extremely low.
Efficient biosynthetic methods are needed to meet market demand. Until now, the key enzyme catalyzing the conversion of Rebaudioside D (Reb D) to Reb M in the biosynthetic pathway has not been identified, and it is generally assumed to be UGT76G1. However, UGT76G1 exhibits strict regioselectivity for the C13 position of steviol glycosides, while its catalytic activity at the C19 position is very weak.
In a study published in PNAS on September 17, a team led by Prof. YIN Heng from the Dalian Institute of Chemical Physics of the Chinese Academy of Sciences identified the key glycosyltransferase that catalyzes the conversion of Reb D to Reb M, and revealed the molecular mechanism underlying its substrate regioselectivity.
Researchers first identified and characterized UGT76G4, a natural variant of UGT76G1, from a high-Reb M producing Stevia cultivar. They discovered that UGT76G4 exhibited regioselectivity toward the C19 position of steviol glycosides in both in vivo and in vitro assays, thereby confirming it as the key enzyme catalyzing the conversion of Reb D to Reb M.
Then, researchers systematically elucidated the molecular basis of UGT76G4 regioselectivity, and identified residue G200 as critical for its C19 catalytic activity.
Furthermore, researchers engineered several tool enzymes with enhanced performance by applying semi-rational design. They engineered UGT76G4 variants capable of efficiently synthesizing Reb M using Rebaudioside E (Reb E) and Reb D as substrates.
"This work not only clarifies the question of which enzyme is responsible for Reb M biosynthesis, but also provides engineered biocatalysts to enable the efficient production of this next generation natural sweetener," said Prof. YIN.
