Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling

Abstract

Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man8GlcNAc2 to Man5GlcNAc2 efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.

Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica

“…All of these tools have been successfully used in Y. lipolytica to produce such proteins as xylanase, lipase, leucine aminopeptidase II, human interferon, α2b endoglucanase II, and cellobiohydrolase II [ 6 , 9 , 14 , 19 , 20 ]. Past studies have also identified at least three sequences that can be used to optimize protein secretion in Y. lipolytica : preLip2, preXpr2, and preSuc2 [ 6 , 14 , 21 – 24 ].…”

Hydrolytic secretome engineering in Yarrowia lipolytica for consolidated bioprocessing on polysaccharide resources: review on starch, cellulose, xylan, and inulin

“…The discussed above studies aimed at expanding native substrate range of the species to highly abundant polysaccharides, i.e., starch, cellulose, xylan, and inulin. Due to the review capacity, at least several highly promising issues had to be neglected, like acquired capacity to decompose pectins by overexpression of polygalacturonase (Muller et al 1998), or glucomannan, through surface display of mannosidase (Moon et al 2013). Based on conducted literature search and own experience, the key challenge in developing Y. lipolytica-based consolidated biocatalysts is balancing optimal conditions for the host growth and the enzyme activity.…”

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement

“…This C-terminal part of CWP1 gene corresponds to a GPI anchor domain, namely a signal for the posttranslational addition of a GPI (glycosylphosphatidylinositol) structure to the secreted protein and for its covalent linking to β-1,6 glucans from the yeast cell wall (surface display). As reviewed previously [ 20 ], a few other GPI anchoring signals from Y. lipolytica cell wall proteins have also been applied to surface display in this yeast [ 250 , 251 , 252 ], but the OUC/INRA zeta-based auto-cloning pINA1317-CWP110 surface display expression vector [ 221 ] remains up to now the more widely used tool for constructing arming Y. lipolytica cells.…”