Boosting N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins

Abstract

Surface display co-opts yeasts innate ability to embellish its cell wall with mannoproteins, thus converting the yeasts outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoforms N-terminus. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the {beta}-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display than the full-length Hsp150 and a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of any protein into its N-terminally anchored isoform. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/538238v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@909e7aorg.highwire.dtl.DTLVardef@9480ceorg.highwire.dtl.DTLVardef@1945bdcorg.highwire.dtl.DTLVardef@11ae505_HPS_FORMAT_FIGEXP M_FIG C_FIG