Xu Luping, Bai Xingjian, Jeong Deokyeol, Lee Dahye, Semidey Fransheska, Li Chenhai, Oh Eun Joong
Department of Food Science, Purdue University, West Lafayette, IN, 47907, USA.
Whistler Center for Carbohydrate Research, Purdue University, West Lafayette, IN, 47907, USA.
Microb Cell Fact. 2025 Apr 1;24(1):76. doi: 10.1186/s12934-025-02702-3.
Saccharomyces boulardii (Sb) has gained significant attention for its potential therapeutic application as a probiotic yeast strain. Current approaches often leverage its secretion and display capabilities to deliver therapeutic agents aimed at alleviating intestinal disorders. However, relatively few studies have focused on optimizing its display efficiency. In this study, we evaluated two surface display systems, Aga2- and Sed1-based, for use in Sb by systematically modifying display cassette components and the host strain. Initially, both systems were tested in Saccharomyces cerevisiae (Sc) and Sb to validate their design. Sc consistently outperformed Sb in both display expression and efficiency, highlighting the need for further optimization in Sb. To enhance the display efficiency in Sb, we investigated specific modifications to the display cassette, including the use of linker sequences for Aga2 and variations in anchor length for Sed1. These experiments identified key factors influencing display performance. Subsequently, we engineered a modified Sb strain, LIP02, by overexpressing AGA1 and deleting cell wall-related genes (CCW12, CCW14, and FYV5). These modifications were expected to expand the available docking sites for the protein of interest (POI) and improve overall protein secretion and display efficiency. As a result, the modified strain exhibited a significant enhancement in display capacity compared to the wild-type Sb strain. Furthermore, genome integration of the display cassette in LIP02 enhanced both stability and expression compared to plasmid-based systems. Importantly, the functionality of β-glucosidase displayed on LIP02 was preserved, as demonstrated by improved enzymatic activity and robust growth on cellobiose as the sole carbon source. These findings establish LIP02 as a superior host for surface display applications in Sb, offering a more stable and efficient platform for the expression of therapeutic proteins and other functional biomolecules.
布拉氏酵母菌（Sb）作为一种益生菌酵母菌株，因其潜在的治疗应用而备受关注。目前的方法通常利用其分泌和展示能力来递送旨在缓解肠道疾病的治疗剂。然而，相对较少的研究关注于优化其展示效率。在本研究中，我们通过系统地修饰展示盒组件和宿主菌株，评估了两种基于Aga2和Sed1的表面展示系统在Sb中的应用。最初，这两种系统在酿酒酵母（Sc）和Sb中进行了测试，以验证其设计。在展示表达和效率方面，Sc始终优于Sb，这突出了在Sb中进一步优化的必要性。为了提高Sb中的展示效率，我们研究了对展示盒的特定修饰，包括使用Aga2的接头序列和Sed1锚定长度的变化。这些实验确定了影响展示性能的关键因素。随后，我们通过过表达AGA1和删除细胞壁相关基因（CCW12、CCW14和FYV5）构建了一种改良的Sb菌株LIP02。这些修饰预期会扩大目的蛋白（POI）的可用对接位点，并提高整体蛋白质分泌和展示效率。结果，与野生型Sb菌株相比，改良菌株的展示能力有了显著提高。此外，与基于质粒的系统相比，LIP02中展示盒的基因组整合增强了稳定性和表达。重要的是，LIP02上展示的β-葡萄糖苷酶的功能得以保留，这通过提高的酶活性和在以纤维二糖为唯一碳源的培养基上的稳健生长得到了证明。这些发现确立了LIP02作为Sb表面展示应用的优良宿主，为治疗性蛋白质和其他功能性生物分子的表达提供了一个更稳定、高效的平台。
