Department of Animal Resources Science, Dankook University, 119 Dandae-ro, Cheonan, 31116, Republic of Korea.
Department of Food Science and Technology, Chungbuk National University, Cheongju, 361-763, Republic of Korea.
BMC Microbiol. 2022 Aug 3;22(1):190. doi: 10.1186/s12866-022-02608-9.
Recent studies have demonstrated the potential of surface display technology in therapeutic development and enzyme immobilization. Utilization of lactic acid bacteria in non-GMO surface display applications is advantageous due to its GRAS status. This study aimed to develop a novel, non-GMO cell wall anchoring system for lactic acid bacteria using a cell-surface hydrolase (CshA) from Lactiplantibacillus plantarum SK156 for potential industrial and biomedical applications. Analysis of the CshA revealed that it does not contain any known classical anchor domains. Although CshA lacks a classical anchor domain, it successfully displayed the reporter protein superfolder GFP on the surface of several lactic acid bacteria in host dependent manner. CshA-sfGFP fusion protein was displayed greatest on Limosilactobacillus fermentum SK152. Pretreatment with trichloroacetic acid further enhanced the binding of CshA to Lm. fermentum. The binding conditions of CshA on pretreated Lm. fermentum (NaCl, pH, time, and temperature) were also optimized, resulting in a maximum binding of up to 10 CshA molecules per pretreated Lm. fermentum cell. Finally, this study demonstrated that CshA-decorated pretreated Lm. fermentum cells tolerates gastrointestinal stress, such as low pH and presence of bile acid. To our knowledge, this study is the first to characterize and demonstrate the cell-surface display ability of CshA. The potential application of CshA in non-GMO antigen delivery system and enzyme immobilization remains to be tested.
近期研究表明，表面展示技术在治疗开发和酶固定化方面具有潜力。由于其 GRAS 地位，利用乳酸菌进行非转基因表面展示应用具有优势。本研究旨在开发一种新型的非转基因细胞壁锚定系统，用于乳酸杆菌，使用植物乳杆菌 SK156 的细胞表面水解酶 (CshA) 用于潜在的工业和生物医学应用。对 CshA 的分析表明，它不含有任何已知的经典锚定域。尽管 CshA 缺乏经典的锚定域，但它成功地以宿主依赖的方式在几种乳酸菌表面展示了报告蛋白超折叠 GFP。CshA-sfGFP 融合蛋白在发酵乳杆菌 SK152 上的显示量最大。三氯乙酸预处理进一步增强了 CshA 与 Lm 的结合。还优化了 CshA 在预处理 Lm.发酵菌（NaCl、pH 值、时间和温度）上的结合条件，结果表明预处理 Lm.发酵菌细胞上的最大结合量高达 10 个 CshA 分子。最后，本研究表明 CshA 修饰的预处理发酵乳杆菌细胞能够耐受胃肠道应激，如低 pH 值和胆汁酸的存在。据我们所知，本研究首次对 CshA 的细胞表面展示能力进行了表征和验证。CshA 在非转基因抗原递呈系统和酶固定化中的潜在应用仍有待测试。
