Effect of fruit maturation on N-glycosylation of plant-derived native and recombinant miraculin.

Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.

From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants.

The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.

Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants.

Miraculin is a taste-modifying protein isolated from the red berries of Richadella dulcifica, a shrub native to West Africa. Miraculin by itself is not sweet, but it is able to turn a sour taste into a sweet taste. This unique property has led to increasing interest in this protein. In this article, we report the high-yield production of miraculin in transgenic tomato plants. High and genetically stable expression of miraculin was confirmed by Western blot analysis and enzyme-linked immunosorbent assay. Recombinant miraculin accumulated to high levels in leaves and fruits, up to 102.5 and 90.7 microg/g fresh weight, respectively. Purified recombinant miraculin expressed in transgenic tomato plants showed strong sweetness-inducing activity, similar to that of native miraculin. These results demonstrate that recombinant miraculin was correctly processed in transgenic tomato plants, and that this production system could be a good alternative to production from the native plant.