Yeast Surface Display of Artificial Multienzyme Complexes for Enhanced Production of Rebaudioside M

Rebaudioside M (Reb M) is an ideal low‐calorie natural sweetener with excellent sensory properties and well‐established safety profile. Industrial production of Reb M is limited by low natural abundance and the bottlenecks of existing biomanufacturing strategies, including low productivity, intermediate accumulation and high enzyme costs. Here, a novel biosynthetic platform based on yeast surface display of artificial multienzyme complexes was established for efficient Reb M production. The artificial protein scaffold containing multiple orthogonal cohesin modules was displayed on the surface of Pichia pastoris via the AGA1 anchoring motif. Through orthogonal cohesin‐dockerin interactions, Reb M biosynthetic pathway enzymes (PgUGT Mut , AtSUS1 and UGT76G1 Mut ) with the corresponding dockerin modules were assembled on the artificial protein scaffold to form multienzyme complexes. The catalytic activity of crude glycosyltransferases was improved by isozyme screening and expression optimization with molecular chaperones. This surface‐displayed system demonstrated multiple advantages. First, the multienzyme complex assembled on the yeast surface showed higher catalytic efficiency than free enzymes. Additionally, fructose was consumed by P. pastoris to drive the reaction toward Reb M biosynthesis. Furthermore, the whole‐cell catalyst showed promising reusability. Ultimately, this platform enabled the production of 36.88 g/L Reb M with a yield of 91.9% from rebaudioside A (Reb A) in a 10 mL reaction system. This work provided a feasible strategy to address the key bottlenecks in Reb M biomanufacturing and offered a reference for producing other high‐value natural products via multi‐enzyme cascade catalysis.