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

Author:
Phienluphon, Apisan, Mhuantong, Wuttichai, Boonyapakron, Katewadee, Deenarn, Pacharawan, Champreda, Verawat, Wichadakul, Duangdao, Suwannarangsee, Surisa
Source:
Applied microbiology and biotechnology 2019 v.103 no.7 pp. 3085-3097
ISSN:
0175-7598
Subject:
Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, beta-glucosidase, biocatalysts, bioinformatics, biotechnology, enzyme activity, green fluorescent protein, structural proteins, threonine, yeasts
Abstract:
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.
Agid:
6360266