Abstract
Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.
Highlights
Miraculin is a sweet protein produced from the fruit of Synsepalum dulcificum
Abiotic stress treatments To analyze the effect of abiotic stress on miraculin gene expression, TC3 line was treated with five stress factors including hydrogen peroxide (H2O2) at 220, 440, and 880 μM, salt (NaCl) at 50, 100, and 200 mM, and abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) at 50, 100, 200 μM
Miraculin expression was the Discussion In the present study, miraulin gene which was tagged to SWPA2 promoter, cloned carrot cell lines and transformed cell lines were used for production of recombinant protein
Summary
Miraculin is a sweet protein produced from the fruit of Synsepalum dulcificum. Miraculin protein has attracted much attention as a natural alternative sweetener because it has a taste-modifying activity that transforms sour taste into sweet taste and is low in calories (Jin et al 2013; Ezura and Hiwasa-Tanase 2018). Largescale production of miraculin for industrial purposes requires an investigation of alternative production systems using heterologous hosts (Sun et al 2007; Yano et al 2010). Plant systems have been studied as a platform for the production of recombinant proteins. Plants have low risk for mammalian pathogens and cost-effective production process as heterologous production systems (Huang and McDonald 2012).
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