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      Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors

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          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 (H 2O 2) 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 H 2O 2 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.

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          Most cited references20

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          Light-regulated transcriptional networks in higher plants.

          Plants have evolved complex and sophisticated transcriptional networks that mediate developmental changes in response to light. These light-regulated processes include seedling photomorphogenesis, seed germination and the shade-avoidance and photoperiod responses. Understanding the components and hierarchical structure of the transcriptional networks that are activated during these processes has long been of great interest to plant scientists. Traditional genetic and molecular approaches have proved powerful in identifying key regulatory factors and their positions within these networks. Recent genomic studies have further revealed that light induces massive reprogramming of the plant transcriptome, and that the early light-responsive genes are enriched in transcription factors. These combined approaches provide new insights into light-regulated transcriptional networks.
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            Hydrogen peroxide signalling.

            S. Neill (2002)
            Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis.
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              Plant cell cultures for the production of recombinant proteins.

              The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.
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                Author and article information

                Contributors
                soypark7@cbnu.ac.kr
                Journal
                AMB Express
                AMB Express
                AMB Express
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                2191-0855
                13 August 2020
                13 August 2020
                2020
                : 10
                : 140
                Affiliations
                [1 ]GRID grid.254229.a, ISNI 0000 0000 9611 0917, Department of Horticulture, Division of Animal, Horticultural and Food Sciences, , Chungbuk National University, ; Cheongju, 28644 Republic of Korea
                [2 ]GRID grid.418977.4, ISNI 0000 0000 9151 8497, Department of Forest Genetic Resources, , National Institute of Forest Science, ; 39 Onjeong-ro, Suwon, 16631 Republic of Korea
                [3 ]GRID grid.411968.3, ISNI 0000 0004 0642 2618, Department of Horticultural Life Science, , Hankyong National University, ; Anseong, 17579 Republic of Korea
                [4 ]GRID grid.444416.7, Department of Botany, , Karnatak University, ; Dharwad, 580003 India
                Article
                1079
                10.1186/s13568-020-01079-3
                7426344
                32789704
                a0f37166-c78a-453c-ba2a-5f5b69aa7b76
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 5 March 2020
                : 8 August 2020
                Funding
                Funded by: Next Generation BioGreen 21 Program
                Award ID: PJ013689
                Award Recipient :
                Categories
                Original Article
                Custom metadata
                © The Author(s) 2020

                Biotechnology
                agrobacterium-mediated transformation,alternative sweetener,carrot callus cultures,miraculin,plant cell culture,recombinant protein

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