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.