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. 2023 Feb 17;6(1):191.
doi: 10.1038/s42003-023-04553-3.

Heterologous mogrosides biosynthesis in cucumber and tomato by genetic manipulation

Jingjing Liao  1   2 , Tingyao Liu  3 , Lei Xie  1 , Changming Mo  4 , Jing Qiao  1 , Xiyang Huang  5 , Shengrong Cui  1 , Xunli Jia  1 , Zuliang Luo  6 , Xiaojun Ma  7
Affiliations

Heterologous mogrosides biosynthesis in cucumber and tomato by genetic manipulation

Jingjing Liao et al. Commun Biol. .

Abstract

Mogrosides are widely used as high-value natural zero-calorie sweeteners that exhibit an array of biological activities and allow for vegetable flavour breeding by modern molecular biotechnology. In this study, we developed an In-fusion based gene stacking strategy for transgene stacking and a multi-gene vector harbouring 6 mogrosides biosynthesis genes and transformed it into Cucumis sativus and Lycopersicon esculentum. Here we show that transgenic cucumber can produce mogroside V and siamenoside I at 587 ng/g FW and 113 ng/g FW, respectively, and cultivated transgenic tomato with mogroside III. This study provides a strategy for vegetable flavour improvement, paving the way for heterologous biosynthesis of mogrosides.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cloning and analysis of candidate promoters in cucumber and tobacco.
a Recombinant plasmid with different promoters. b Histochemical GUS assay of transient expression in cucumber. c Histochemical GUS assay of transient expression was performed after 48 h of infiltration in tobacco. WT plants were used as negative controls. 35S pro: CaMV 35S promoter; AtUBQ10 pro: AtUBQ10 promoter; AtPD7 pro: AtPD7 promoter.
Fig. 2
Fig. 2. Genetic transformation of cucumber with Agrobacterium harbouring the U22p-SCE vector.
a Genetic transformation of cucumber with Agrobacterium harbouring the U22p-SCE vector. (1) Sterilized seeds. (2) 4-days-old seedlings. (3) The cotyledons were cut and used as explants. (4) The explants in the selected medium. (5) The regenerated plants in the rooting media. (6, 7) Regenerated plants. b Leaves and fruits of transgenic cucumber line U1. c PCR-based detection of U1. The lanes from left to right represent the Maker, WT, U1 fruits and U1 leaves. An image of the DNA marker (4.5 kb) is in the bottom right-hand corner of the figure. d Transcript level analysis of transgenic cucumber line U1 according to qRT-PCR. The Csactin is used as an internal control. Expression of cucumber WT plants was set to 1. The data are presented as the mean values ± SDs, n = 3 biologically independent samples.
Fig. 3
Fig. 3. Production of mogrosides in transgenic cucumber line.
a HPLC-ESI-MS/MS analysis of mogrosides in transgenic cucumber line U1. The black arrows indicate the peak of mogrosides. b Accumulation of mogrosides in transgenic cucumber line U1. n.d., not detected. The data are presented as the mean values ± SDs, n = 3 biologically independent samples.
Fig. 4
Fig. 4. The total ion chromatogram of mogrosides in transgenic cucumber line U1 and standards.
MIA-1, MII-E, MIII, SI, and MV represented the mogroside I-A1, mogroside II-E, mogroside III, siamenoside I, and mogroside V, respectively.
Fig. 5
Fig. 5. Molecular analysis and detection of mogrosides in transgenic tomato lines.
a Micro-Tom tomato wild-type plants (WT) and transgenic tomato plants. b PCR-based analysis of the transgenic tomato fruits. The lanes from left to right represent the Maker, WT, S8, S10, S14 and S17. An image of the DNA marker (4.5 kb) is in the bottom right-hand corner of the figure. c Relative expression level analysis of 6 mogrosides biosynthesis genes in transgenic tomato fruits. The Leactin is used as an internal control. Expression of tomato WT plants was set to 1. The data are presented as the mean values ± SDs, n = 3 biologically independent samples.
Fig. 6
Fig. 6. Production of mogrosides in tomato transgenic lines.
a HPLC-ESI-MS/MS analysis of mogrosides in transgenic tomato fruits. b Accumulation of MIII in transgenic tomato fruits. The black arrows indicate the peak of mogrosides. n.d., not detected. The data are presented as the mean values ± SDs, n = 3 biologically independent samples.
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