Abstract
Monellin a sweet-tasting protein exists naturally as a heterodimer of two non-covalently linked subunits chain A and B, which loses its sweetness on denaturation. In this study, we validated the expression of a synthetic monellin gene encoding a single polypeptide chain covalently linking the two subunits under T7 and fruit-ripening-specific promoters in Escherichia coli and tomato fruits, respectively. Purified recombinant monellin protein retained its sweet flavour at 70 °C and pH 2. We developed 15 transgenic T0 tomato plants overexpressing monellin, which were devoid of any growth penalty or phenotypic abnormalities during greenhouse conditions. T-DNA integration and fruit-specific heterologous expression of monellin had occurred in these transgenic tomato lines. ELISA revealed that expression of monellin was 4.5 % of the total soluble fruit protein. Functional analyses of transgenic tomatoes of T2-5 and T2-14 lines revealed distinctly strong sweetness compared with wild type. Monellin a potential non-carbohydrate sweetener, if expressed in high amounts in fruits and vegetables, would enhance their flavour and quality.
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Acknowledgments
The authors are grateful to the Rapid Grant for Young Investigator (RGYI) and Biocare Grants from the Department of Biotechnology, Government of India awarded to Dr. Tanushri Kaul for funding the research.
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Supplementary Fig. 1. Regenerating transgenic tomato lines harbouring monellin gene through agro-mediated plant transformation. Different stages of tomato transformation: Tomato seed germination (A); Pre-cultured and Co-cultivated explants (B), Co-cultivated explants on regeneration medium showing induction of shoots (C-E), Shoots showing elongation (F), Root induction (G), hardening of plants (H) and control and transgenic plants transferred to pots in greenhouse (I).
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Reddy, C.S., Vijayalakshmi, M., Kaul, T. et al. Improving Flavour and Quality of Tomatoes by Expression of Synthetic Gene Encoding Sweet Protein Monellin. Mol Biotechnol 57, 448–453 (2015). https://doi.org/10.1007/s12033-015-9838-5
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DOI: https://doi.org/10.1007/s12033-015-9838-5