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

Abstract

SummaryMiraculin 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 µ g/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.

Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits

“…The transgenic tomato line 56B was produced in our previous work (Sun et al 2007). In brief miraculin encoding 660-bp DNA fragment was inserted into the XbaI/SacI sites of the plant transformation vectors pBI121 (Sun et al 2006).…”

“…Therefore, we decided to produce recombinant miraculin in transgenic tomatoes. We developed transgenic tomato plants that accumulate significant levels of miraculin protein (Sun et al 2007), and the protein produced had almost same level of tastemodifying activity as native miraculin from miracle fruits. Furthermore, we characterized the miraculin accumulation pattern in the transgenic tomato fruits in detail and found that miraculin accumulation was highest in the exocarp (Kim et al 2010).…”

“…Nevertheless, commercial production of miraculin is limited because the natural source is a tropical plant that is difficult to cultivate and is not very productive. Efforts to produce miraculin in other organisms such as Escherichia coli (Kurihara 1992), yeast (Kurihara and Nirasawa 1997), tobacco (Kurihara and Nirasawa 1997), lettuce (Sun et al 2006), tomato (Sun et al 2007), and strawberry (Sugaya et al 2008) have been going on since 1990. Recently, a research group reattempted to produce miraculin in E. coli, but the recombinant protein exhibited lower taste-modifying activity than the native miraculin because the recombinant miraculin was not glycosylated (Matsuyama et al 2009).…”

“…The extracts were centrifuged at 15,000ϫg for 20 min at 4°C. The resulting supernatant was used for immunoblot analysis and ELISA, which were performed according to Sun et al (2007) and Kim et al (2010), respectively. …”

Genetic Manipulation of Tomato (Solanum lycopersicum) cv. PKM-1 using cry2AX1 Gene for Insect Resistance

“…when these modified genes are expressed in them (Mandaokar et al, 2000). Tomato is an excellent plant for stably expressing transgenes, indicating a good target for genetic engineering (Kobayashi et al, 2003;Omura et al, 2007;Sun et al, 2007). These characteristics make it an attractive research target for practical application in food and agricultural industries.…”