Design an artificially concise biocatalytic cascade for the manufacture of Rebaudioside M from a naturally derived steviol glycosides mixture

Abstract

Rebaudioside M (Reb M) is an important natural sweetener due to its high sweetness, low calorie content, and pleasant taste, but its low natural abundance and costly, unsustainable extraction limit large-scale production. While enzymatic synthesis of Reb M offers an environmentally friendly alternative, its dependence on purified single substrates due to limited glycosyltransferase specificity, which results in high energy input, waste generation, and inefficient utilization of natural mixtures. To overcome these limitations, we design a simple two-enzyme cascade that enables the efficient bioproduction of Reb M from a naturally derived steviol glycosides mixture, leveraging enzyme promiscuity through enzyme screening and engineering. After addressing key challenges including enzyme promiscuity, rate-limiting steps, and intermodule balance, the optimized enzyme cascade system achieved high-level biosynthesis of Reb M with a final titer of 38.8 g/L and a molar yield of 85.5% from steviol glycosides mixture. This work not only demonstrates a green and scalable route for Reb M biosynthesis but also provides a generalizable strategy for the sustainable production of other high-value natural products from complex plant extracts.