College of Biotechnology and Bioengineering, Zhejiang University of Technologygrid.469325.f, Zhejiang, China.
Institute of Fermentation Technology, Zhejiang University of Technologygrid.469325.f, Zhejiang, China.
Appl Environ Microbiol. 2022 Sep 13;88(17):e0102722. doi: 10.1128/aem.01027-22. Epub 2022 Aug 11.
Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its / is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced / value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.
橙皮苷是一种富含于柑橘皮中的类黄酮，可使用 CGTase 进行酶糖化，从而显著提高其水溶性。然而，野生型 CGTase 催化的反应效率相当低，表现在产率和收率都较差。通过聚焦于糖苷配基攻击步骤，选择了七个残基进行突变，以提高转糖苷效率。由于缺乏针对所研究反应的高通量筛选技术，我们开发了一种大小/极性导向的三码策略，以缩小文库规模。选择的残基被三种大小或极性发生改变的合理选择的氨基酸所取代，从而构建出一个仅有 32 个突变体的极度浓缩文库，以进行筛选。构建的突变体中有 25%被证明是阳性的，这表明构建文库的质量很高。对最佳突变体 Y217F 的特异性转糖苷活性进行了测定，结果为 935.7 U/g，其 / 值比野生型高 6.43 倍。同源建模和对接计算表明，催化效率显著提高的原因是配体转移和结合效应的结合。本研究开发了一种大小/极性导向的三码策略，一种新型的半理性诱变策略，并将其用于工程 CGTASE 的糖苷配基攻击位点。筛选压力设定为提高橙皮苷葡萄糖苷的合成能力，通过筛选仅 32 个突变体获得了 8 个阳性突变体。设计文库的高质量证实了所开发策略的有效性，可能对未来的诱变研究具有潜在价值。解释了产生积极效果的机制。最佳突变体表现出 6.43 倍增强的 / 值，被证实是一种具有潜在应用价值的合成橙皮苷葡萄糖苷的优良全细胞催化剂。
