Expression, purification and characterization of a novel double-sites mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability.

The sweet protein monellin has high sweet potency with limited stability. In this study, 3 double-sites mutants (E2N/E23A, E2N/Y65R and E23A/Y65R) of the single-chain monellin (MNEI) were constructed. The proteins were expressed in E. coli BL21 and purified to homogeneity by nickel affinity chromatography with yields above 10 mg/L cell culture. Introduction of a sweeter mutant E2N into E23A or Y65R (E2N/E23A and E2N/Y65R) led to about 3-fold increase of sweetness, while addition of a more stable mutant E23A into E2N or Y65R (E2N/E23A and E23A/Y65R) resulted in improved thermal stability (about 10 °C). The results indicate that residues E2 and E23 mediate the sweetness and thermal stability of the protein, respectively. Multiple mutations of different residues (E2N/E23A) led to an additive performance with both improved sweetness and stability, suggesting that the sweetness and stability could be modulated by the independent molecular mechanism. The sweeter and thermal stable variant has a potential in further industrial applications.

Computational design towards a boiling-resistant single-chain sweet protein monellin.

Sweet proteins offer a promising solution as sugar substitutes by providing a sugar-like sweetness without the negative health impacts linked to sugar or artificial sweeteners. However, the low thermal stability of sweet proteins has hindered their applications. In this study, we took a computational approach utilizing ΔΔG calculations in PyRosetta to enhance the thermostability of single-chain monellin (MNEI). By generating and characterizing 21 variants with single mutation, we identified 11 variants with higher melting temperature (Tm) than that of MNEI. To further enhance the thermal stability, we conducted structural analysis and designed an additional set of 14 variants with multiple mutations. Among these variants, four exhibited a significant improvement in thermal stability, with an increase of at least 20 °C (Tm > 96 °C) compared to MNEI, while maintaining their sweetness. Remarkably, these variants remained soluble even after being heated in boiling water for one hour. Moreover, they displayed exceptional stability across alkaline, acidic and neutral environments. These findings highlight the tremendous potential of these variants for applications in the food and beverage industry. Additionally, this study provides valuable strategies for protein engineering to enhance the thermal stability of sweet proteins.

A Super Stable Mutant of the Plant Protein Monellin Endowed with Enhanced Sweetness.

Sweet proteins are a class of proteins with the ability to elicit a sweet sensation in humans upon interaction with sweet taste receptor T1R2/T1R3. Single-chain Monellin, MNEI, is among the sweetest proteins known and it could replace sugar in many food and beverage recipes. Nonetheless, its use is limited by low stability and high aggregation propensity at neutral pH. To solve this inconvenience, we designed a new construct of MNEI, dubbed Mut9, which led to gains in both sweetness and stability. Mut9 showed an extraordinary stability in acidic and neutral environments, where we observed a melting temperature over 20 °C higher than that of MNEI. In addition, Mut9 resulted twice as sweet than MNEI. Both proteins were extensively characterized by biophysical and sensory analyses. Notably, Mut9 preserved its structure and function even after 10 min boiling, with the greatest differences being observed at pH 6.8, where it remained folded and sweet, whereas MNEI lost its structure and function. Finally, we performed a 6-month shelf-life assessment, and the data confirmed the greater stability of the new construct in a wide range of conditions. These data prove that Mut9 has an even greater potential for food and beverage applications than MNEI.