Transgenics with Monellin

Abstract

Sweet proteins are potential substitutes to artificial sweeteners as they are natural and low calorie sweeteners. Monellin a naturally sweet protein from Dioscoreophyllum cumminsii elicits sweet flavour as a carbohydrate has been characterized to reveal that the native structure is essential for its sweet taste. It is a heterodimeric protein of 94 amino acid residues with a molecular weight of 10.5 kDa that loses its activity during denaturation. Monellin is proposed to be a promising sweetener that tastes sweet at pH 2–9, whereas high pH or heating beyond 70 °C or more can denature this protein. Structural studies showed that linking the two subunits of this heterodimer monellin confers stability than the native counterpart. Moreover, monellin comprised of a single polypeptide exhibits identical conformation and sweetness in flavour as the double-chain monellin. Monellin has dual applications as a flavour enhancer and a high-intensity sweetener and has been recommended for use in some countries. As provision of naturally existing monellin is limited, it has led to indepth research into its synthesis via transgenic organisms. Efforts have been directed towards production of this recombinant protein in different expression systems i.e., bacterial, yeast and transgenic plants. Production of monellin in transgenic fruits and vegetables offers a viable approach to improve their flavour and quality. We report the stable and enhanced expression of synthetic sweet protein monellin in prokaryotic (E. coli) and eukaryotic (tomato) systems. The recombinant monellin protein was thermostable and retained strong sweetness over a temperature range (up to 70 °C) and extreme pH levels. Transgenic tomato plants revealed stable gene integration in T1 generation as verified by PCR analysis. Transcript profiling of T2 lines revealed enhanced monellin expression, which correlated positively to its protein expression profiles as highlighted by western blots. Heterologous expression of monellin driven by the fruit-specific promoter (E8) did not cause any phenotypic anomalies either in vegetative or fruit growth parameters in the transgenic tomato lines. RT-PCR was performed and highest levels of transcript expression were observed in T2-5 and T2-14 lines. ELISA studies were ratified by immuno-blot analysis via comparison of the signal for each variant to that of purified monellin. Transgenic lines T2-5 and T2-14 showed maximum protein levels of 60 lg/mg (±2.06) and 54 lg/mg (±2.58), respectively. Thus, it is beneficial to earmark suitable monellin expression systems for large-scale production of this potent food supplement.