High-level production of single chain monellin mutants with enhanced sweetness and stability in tobacco chloroplasts

Abstract

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.

Getting value from the waste: recombinant production of a sweet protein by Lactococcus lactis grown on cheese whey

“…Samples of 10 μg total protein extract were loaded on 12% SDS-PAGE and then blotted onto Immobilon-P transfer membrane (EMD, Millipore Corporation, USA). Membranes were incubated with an rabbit anti-Y65R-MNEI antibody [ 36 ] (1:200, Primmbiotech, courtesy of Dr. Nunzia Scotti) and subsequently with a HRP-conjugated anti-rabbit antibody (1:50,000, BioFX Laboratories). Detection of the His-tagged MMP-9 expression in L. lactis clpP − htrA − was also performed by western blot.…”

“…MNEI is a single chain derivative of the plant ( Dioscoreophyllum cumminsii ) protein monellin [ 33 , 34 ] and has received much attention for its high intensity sweetness. Due to this potential industrial interest, several biotechnological strategies have been devised to produce it, exploiting a variety of host systems [ 35 , 36 ]. MNEI is one of the best characterized members of the sweet proteins family and it has been object of protein engineering to improve its taste profile and physicochemical characteristics, trying to meet the needs of the food and beverage industry [ 37 – 43 ].…”