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Identification and evaluation of novel anchoring proteins for cell surface display on Saccharomyces cerevisiae

Phienluphon, Apisan, Mhuantong, Wuttichai, Boonyapakron, Katewadee, Deenarn, Pacharawan, Champreda, Verawat, Wichadakul, Duangdao, Suwannarangsee, Surisa
Applied microbiology and biotechnology 2019 v.103 no.7 pp. 3085-3097
Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, beta-glucosidase, biocatalysts, bioinformatics, biotechnology, enzyme activity, green fluorescent protein, structural proteins, threonine, yeasts
The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and β-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a β-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.