High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts
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Plastid-based MNEI protein mutants retain the structure, stability and sweetness of their bacterial counterparts, confirming the attractiveness of the plastid transformation technology for high-yield production of recombinant proteins.
The prevalence of obesity and diabetes has dramatically increased the industrial demand for the development and use of alternatives to sugar and traditional sweeteners. Sweet proteins, such as MNEI, a single chain derivative of monellin, are the most promising candidates for industrial applications. In this work, we describe the use of tobacco chloroplasts as a stable plant expression platform to produce three MNEI protein mutants with improved taste profile and stability. All plant-based proteins were correctly expressed in tobacco chloroplasts, purified and subjected to in-depth chemical and sensory analyses. Recombinant MNEI mutants showed a protein yield ranging from 5% to more than 50% of total soluble proteins, which, to date, represents the highest accumulation level of MNEI mutants in plants. Comparative analyses demonstrated the high similarity, in terms of structure, stability and function, of the proteins produced in plant chloroplasts and bacteria. The high yield and the extreme sweetness perceived for the plant-derived proteins prove that plastid transformation technology is a safe, stable and cost-effective production platform for low-calorie sweeteners, with an estimated production of up to 25–30 mg of pure protein/plant.
KeywordsGreen factory Plastid transformation Nicotiana tabacum Sweet proteins Protein structure Low-calorie sweeteners
This work was partially funded by Fondazione con il Sud, Project 2011-PDR-19, and POR Campania FESR207-2013, Project “Bio Industrial Processes (BIP)”. Technical assistance of Mr. G. Guarino, Ms. S. Arcari and Mr. R. Nocerino (CNR-IBBR, Portici, Italy) with artworks and plant growth is gratefully acknowledged. The authors declare that they have no conflict of interests.
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