Designing an artificially concise biocatalytic cascade for the manufacture of rebaudioside M from a naturally derived steviol glycoside mixture

Abstract

Rebaudioside M (Reb M) is a valuable natural sweetener with high sweetness, low-calorie content, and a pleasant taste. However, its low natural abundance and costly, unsustainable extraction limit large-scale production. While the enzymatic synthesis of Reb M offers an environmentally friendly alternative, its dependence on purified single substrates, due to limited glycosyltransferase specificity, results in high energy input, waste generation, and the 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-glycoside mixture, leveraging enzyme promiscuity through enzyme screening and engineering. After addressing key challenges, including enzyme promiscuity, rate-limiting steps, and inter-module balance, the optimized enzyme cascade system achieved the high-level biosynthesis of Reb M with a final titer of 38.8 g L⁻¹ and a molar yield of 85.5% from a steviol glycoside 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.