Stable expression of the sweet protein monellin variant MNEI in tobacco chloroplasts

Monellin is a naturally sweet protein that consists of two polypeptide chains and has potential uses as a highly potent non-carbohydrate sweetener. We aimed to make this protein more usable by increasing its stability and expressing it in a high-yielding system. MNEI is a modified version of the protein that consists of the two natural chains of monellin joined via a dipeptide linkage. In the thermostability analysis of MNEI variants, four mutated MNEIs, MNEI-E24L, MNEI-E24F, MNEI-E24W, and MNEI-E24A, had higher melting temperatures than wild-type MNEI and retained their sweet flavor even at temperatures above 70 °C. Our findings indicate that the increased stability of monellin allows it to retain its strong sweetness even under extreme conditions. We successfully overexpressed the thermostable MNEI mutants in tobacco chloroplasts. Here, we report that the MNEI mutants showed enhanced thermostability, and the stable forms of MNEI can be produced through plastid transformation in tobacco.