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

Author:

Martinić Cezar Tea,Lozančić Mateja,Novačić Ana,Matičević Ana,Matijević Dominik,Vallée Béatrice,Mrša Vladimir,Teparić Renata,Žunar Bojan

Abstract

AbstractSurface display co-opts yeast’s innate ability to embellish its cell wall with mannoproteins, thus converting the yeast’s 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 isoform’s N-terminus, e.g., through a short GPI-independent protein anchor. 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 β-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 of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms. Graphical abstract

Funder

The Croatian Science Foundation

Publisher

Springer Science and Business Media LLC

Subject

Applied Microbiology and Biotechnology,Bioengineering,Biotechnology

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