Elucidation of the Biosynthesis of Asiaticoside and Its Reconstitution in Yeast

Asiaticoside is a triterpenoid in Centella asiatica with wound healing and anti-inflammatory activities. De novo production of asiaticoside remains incompletely resolved. To determine the asiaticoside biosynthetic pathway, a variety of metabolic engineering modifications were made to achieve a high level of synthesis of asiatic acid in Saccharomyces cerevisiae. Transcriptome analysis of C. asiatica identified 51 glycosyltransferases that are potentially involved in the glycosylation of asiatic acid to form asiaticoside. Functional analysis of these glycosyltransferases revealed that CaUGT73C7 and CaUGT73C8 function in the glucosylation of asiatic acid monoglycoside at the C-28 position and five rhamnose glycosyltransferases convert asiatic acid diglucoside to asiaticoside. To achieve the de novo biosynthesis of asiaticoside in S. cerevisiae, glycoside hydrolase EGH1 that degrades asiaticoside was knocked out and key pathway enzymes were introduced. After a series of metabolic engineering modifications, the de novo biosynthesis of asiaticoside was achieved with a titer of 772.3 μg/L in a 5 L fermenter. This is the first time the complete pathway of asiaticoside has been identified and de novo synthesis in microorganisms. The results provide a valuable reference for the biosynthesis of asiaticoside and other similar triterpenoid saponins.