Development of New Multi-Glycosylation Routes to Facilitate the Biosynthesis of Sweetener Mogrosides from Bitter Immature Siraitia Grosvenorii Using Engineered Escherichia coli

Abstract

Mogrosides, which have various pharmacological activities, are mainly extracted from Siraitia grosvenorii (Luo Han Guo) and are widely used as natural zero-calorie sweeteners. Unfortunately, the difficult cultivation and long maturation time of Luo Han Guo have contributed to a shortage of mogrosides. To overcome this obstacle, we developed a highly efficient biosynthetic method using engineered Escherichia coli to synthesize sweet mogrosides from bitter mogrosides. Three UDP-glycosyltransferase (UGT) genes with primary/branched glycosylation catalytic activity at the C3/C24 sites of mogrosides were screened and tested. Mutant M3, which could catalyze the glycosylation of nine types of mogrosides, was obtained through enhanced catalytic activity. This improvement in β-(1,6)-glycosidic bond formation was achieved through single nucleotide polymorphisms and direct evolution, guided by 3D structural analysis. A new multienzyme system combining three UGTs and UDP-glucose (UDPG) regeneration was developed to avoid the use of expensive UDPG. Finally, the content of sweet mogrosides in the immature Luo Han Guo extract increased significantly from 57% to 95%. This study not only established a new multienzyme system for the highly efficient production of sweet mogrosides from immature Luo Han Guo but also provided a guideline for the high-value utilization of rich bitter mogrosides from agricultural waste and residues.