Microbial Biotechnology and Biochemicals Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Thailand Science Park, Klong Luang, Pathumthani, 12120, Thailand.
Department of Computer Engineering, Faculty of Engineering, Chulalongkorn University, Phayathai Road, Wang Mai, Pathumwan, Bangkok, 10330, Thailand.
Appl Microbiol Biotechnol. 2019 Apr;103(7):3085-3097. doi: 10.1007/s00253-019-09667-5. Epub 2019 Feb 9.
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.
作为全细胞生物催化剂的武装酵母菌株的发展涉及到选择有效的锚定蛋白，以将酶和蛋白质展示在酵母细胞表面。为了筛选具有更高效率的新型锚定蛋白，开发了一种用于鉴定适合将载体蛋白附着到酿酒酵母细胞表面的糖基磷脂酰肌醇锚定细胞壁蛋白（GPI-CWP）的生物信息学管道。在这里，根据以下标准选择假定的 GPI-CWP 的 C 端序列（CTS）：序列必须包含至少 30%S/T 含量的丝氨酸/苏氨酸丰富（S/T）区域、至少 10%的苏氨酸总量、至少 130 个连续氨基酸的 S/T 延伸和至少 10 个连续的富含 T 的氨基酸。在预测的 790 种蛋白质中，从不同的酵母和真菌物种中选择了 37 种假定的 GPI-CWP 来评估它们在展示酵母增强型绿色荧光蛋白和β-葡萄糖苷酶酶方面的性能。这导致鉴定了 5 种新型锚定蛋白，与用作基准的α-凝集素相比，它们的性能更高。乳克鲁维酵母中未鉴定蛋白的 CTS，即 6_Kl，是该组中最有效的锚定蛋白。6_Kl 蛋白的 CTS 提供了高达 23.5 U/g 细胞干重的β-葡萄糖苷酶活性，比α-凝集素的 CTS 高 2.8 倍。这些鉴定出的 CTS 可能是未来构建高效武装酵母用于生物技术应用的潜在新型锚定蛋白候选物。
