Improving the Soluble Expression of Sweet Protein Thaumatin II through Directed Evolution in Escherichia coli

Thaumatin is a natural sweet protein valued for its intense sweetness, long-lasting effect, low-calorie content, and safety. However, limited solubility and low yields hinder microbial fermentation. To overcome these challenges, we engineered Escherichia coli to express thaumatin II and implemented three strategies to improve its solubility. Coexpression with molecular chaperones proved to be the most effective in significantly enhancing the soluble expression of thaumatin II. Furthermore, we applied directed evolution to further refine thaumatin II, resulting in the M-882 mutant, which achieved a thaumatin titer of 42 mg/L, representing a 45% increase compared to the original protein yield. Structural analysis revealed that disruption of two adjacent disulfide bonds in the M-882 mutant minimized mispairing during peptide folding, thereby improving protein solubility. Additionally, molecular dynamics simulations showed that the M-882 variant enhanced solvent accessibility and structural flexibility, both of which contributed to improved solubility. The interaction between thaumatin II and sweet taste receptors, as well as the analysis of surface positive charges, indicated that the M-882 variant is capable of binding to the receptors, thereby retaining its sweetness. Overall, this study not only offers valuable insights into optimizing thaumatin expression but also establishes a foundation for engineering other sweet proteins with broad industrial applications.索马甜（thaumatin）是一种天然甜味蛋白，因其强烈甜味、持久风味、低热量属性与良好安全性而备受推崇。但其可溶性较差且产量较低，限制了其通过微生物发酵的规模化生产。为克服上述挑战，我们对大肠杆菌（Escherichia coli）进行基因工程改造以表达索马甜II，并采用三种策略提升其可溶性。实验证实，与分子伴侣（molecular chaperones）共表达是最为有效的手段，可显著提升索马甜II的可溶性表达水平。此外，我们通过定向进化（directed evolution）技术对索马甜II进行优化，获得了M-882突变体，其索马甜效价达到42 mg/L，较原始蛋白产量提升45%。结构分析显示，M-882突变体中两个相邻二硫键的断裂，最大限度减少了肽链折叠过程中的错配，进而改善了蛋白可溶性。另外，分子动力学（molecular dynamics）模拟结果表明，M-882变体提升了溶剂可及性与结构柔性，二者共同助力可溶性提升。针对索马甜II与甜味受体的相互作用分析，以及表面正电荷分析结果均显示，M-882变体仍可与受体结合，故而保留了其甜味活性。综上，本研究不仅为索马甜的表达优化提供了重要参考，也为工程化改造其他具备广阔工业应用前景的甜味蛋白奠定了坚实基础。