Direct Yeast Surface Codisplay of Sequential Enzymes with Complementary Anchor Motifs: Enabling Enhanced Glycosylation of Natural Products.

Yeast surface display is an appealing technique for constructing multienzyme cascades. This technique is commonly achieved using a scaffold for the ordered arrangement of various enzymes. However, this method is typically complicated because scaffold use may engender extra metabolic burden on the cell host. Here, we established a direct yeast surface codisplay strategy by employing two complementary anchor motifs, Agα1 and Pir1. These motifs allow for the codisplay of sequential uridine diphosphate-glycosyltransferase (UGT) and sucrose synthase (SUS) on the surface of Pichia pastoris (syn. Komagataella phaffii ) for the glycosylation of natural products. We manipulated the displayed stoichiometry, amount, and assembly order of UGT and SUS by coupling them with anchor motifs. Furthermore, their effect on enzyme activity was thoroughly investigated. The surface-codisplayed strain UGT-Pir-SUS-Agα exhibited greater thermostability than the single-displayed strains and their free counterparts. Moreover, the strain UGT-Pir-SUS-Agα was successfully applied to glycyrrhetinic acid (GA) glycosylation to produce GA-3-O-Glc, with sucrose being the sugar donor in this process. This generated 7.5- to 20- and 5.3-fold higher GA-3-O-Glc concentration compared with the free counterparts (enzyme mass loading of 20-fold in excess) and mixed single-displayed strains of UGT-Agα and SUS-Pir, respectively. This increase was due to the improved biochemical properties and substrate channeling effect of strain UGT-Pir-SUS-Agα. This controllable direct surface codisplay strategy, based on complementary anchor motifs, is readily extendable to other enzyme cascades.