High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts
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.
- Castiglia D, Sannino L, Marcolongo L, Ionata E, Tamburino R, De Stradis A, Cobucci-Ponzano B, Moracci M, La Cara F, Scotti N (2016) High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass. Biotechnol Biofuels 9:154CrossRefPubMedPubMedCentralGoogle Scholar
- Kaul T, Reddy CS, Pandey S (2018) Transgenics with monellin. In: Mérillon JM, Ramawat KG (eds) Sweeteners. Pharmacology, biotechnology and applications. Springer, Cham, pp 211–222Google Scholar
- Lamphear BJ, Barker DK, Brooks CA, Delaney DE, Lane JR, Beifuss K, Love R, Thompson K, Mayor J, Clough R, Harkey R, Poage M, Drees C, Horn ME, Streatfield SJ, Nikolov Z, Woodard SL, Hood EE, Jilka JM, Howard JA (2005) Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. Plant Biotechnol J 3:103–114CrossRefPubMedGoogle Scholar
- Lenzi P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti L, Grillo S, Buonaguro FM, Maliga P, Cardi T (2008) Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res 17:1091–1102CrossRefPubMedGoogle Scholar
- Temussi PA (2011a) New insights into the characteristics of sweet and bitter taste receptors. In: Jeon KW (ed) International Review of Cell and Molecular Biology, vol 291. Academic Press, San Diego, pp 191–226Google Scholar