Establishment of a Novel Cell Surface Display Platform Based on Natural “Chitosan Beads” of Yeast Spores

Abstract

Cell surface display technology, which expresses and anchors proteins on the surface of microbial cells, has broad application prospects in many fields, such as protein library screening, biocatalysis, and biosensor development. However, traditional cell surface display systems have disadvantages: the molecular weight of phage display proteins cannot be too large; bacterial display lacks the post-translational modification process for eukaryotic proteins; yeast display is prone to excessive protein glycosylation and misfolding of multisubunit proteins; and the compatibility of Bacillus subtilis spore display needs to be further improved. Therefore, it is extremely valuable to develop an efficient surface display platform with strong universality and stress resistance properties. Although yeast surface display systems have been extensively investigated, the establishment of a surface display platform using yeast spores has rarely been reported. In this study, a novel cell surface display platform based on natural "chitosan beads" of yeast spores was developed. The target protein in fusion with the chitosan affinity protein (CAP) exhibited strong binding capability with "chitosan beads" of yeast spores in vitro and in vivo. Moreover, this protein display system showed highly preferable enzymatic properties and stability. As an example, the displayed LXYL-P1-2-CAP demonstrated high thermostability and reusability (60% of the initial activity after seven cycles of reuse), high storage stability (75% of original activity after 8 weeks), and excellent tolerance to a concentration up to 75% (v/v) organic reagents. To prove the practicability of this surface display system, the semisynthesis of paclitaxel intermediate was demonstrated and its highest conversion rate was 92% using 0.25 mM substrate. This study provides a novel and useful platform for the surface display of proteins, especially for multimeric macromolecular proteins of eukaryotic origin.

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non‐yeast signal peptide‐mediated cell surface display

“…High‐activity mannanase was successfully displayed on cell surface of Y. lipolytica with Flo1p from S. cerevisiae 20 . The technique of yeast surface display relies on fusing enzymes and large anchor proteins (more than 100 amino acid residues, approximately 10 kDa) to facilitate the aggregation of enzymes on the cell wall, which may encounter challenges such as protein misfolding, interference with the cell wall and the need for extensive time‐consuming optimization 22 . Through our study, we made an exciting discovery that a non‐yeast, which was considerably smaller (22 amino acid residues, approximately 2.2 kDa), and a robust signal peptide can successfully guide exogenous enzymes to create a likeness surface display system, which greatly broadens the array of molecular toolboxes accessible for engineering Y. lipolytica .…”