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. 2022 Sep 9;23(18):10422.
doi: 10.3390/ijms231810422.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides

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

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides

Jingjing Liao et al. Int J Mol Sci. .

Abstract

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

Keywords: mogrosides; multigene assembly; natural sweetener; plant chassis; synthetic biology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The Flow Chart of mogrosides biosynthetic pathway in Siraitia grosvenorii fruits. Mogrosides synthase genes which were transformed in this study are marked in blue, including SgSQE1, squalene epoxidases; SgCS, curbitadienol synthase; SgEPH2, epoxide hydrolases; SgP450, cytochrome P450 mono-oxygenase; SgUGT269-1 and SgUGT289-3, UDP-glucosyltransferases; The substrate, 2,3-oxidosqualene is shown with a yellow background. MI-A1, mogroside I-A1; MII-E, mogroside II-E; MIII, mogroside III; SI, siamenoside I; MV, mogroside V (in the dotted bordered rectangle).
Figure 2
Figure 2
Molecular analysis of transgenic tobacco lines. (A) PCR-based analysis of the transgenic tobacco lines. The lanes from left to right represent the WT, N16, N22, N30, N31, N32, N45, N46, N47, and N48. An image of the DNA marker (4.5 kb) is shown in the upper-left corner of the figure. (B) Relative expression level analysis of 6 mogrosides biosynthesis genes in transgenic tobacco lines (N16, N22, N30, N32, N45, N47). The Nbactin is used as an internal control. Expression of tobacco WT plants was set to 1. The data are presented as the mean values ± SDs, n = 3 biologically independent samples, ** represents significant difference at p < 0.01 (LSD test).
Figure 3
Figure 3
Molecular analysis of transgenic Arabidopsis lines. (A) Arabidopsis WT plants and transgenic lines. (B) PCR-based analysis of transgenic Arabidopsis lines. The lanes from left to right represent the WT, AA3, AA5, AA6, AA7, AU6, AU7, AU8, AU10, AU11, AU12, and AU13. DNA marker (4500 bp) is used in the figure. (C) RT-PCR detection of 6 mogrosides biosynthesis genes in the WT and transgenic Arabidopsis lines AA3, AA5, AA6, AU7, AU10, AU11, A12 (from left to right). The Atactin is used as an internal control.
Figure 4
Figure 4
Production of mogrosides in transgenic tobacco lines. (A) HPLC-MS/MS analysis of MIII and MII-E in transgenic tobacco lines. (B) Accumulation of MIII in transgenic tobacco lines. (C) Accumulation of MII-E in transgenic tobacco lines. n.d., not detected. The data are presented as the mean values ± SDs, n = 3 biologically independent samples. The black arrows indicate the peak of MIII and MIIE. (D) Full-scan product ion of MIII and MII-E.5.0 × 104.
Figure 4
Figure 4
Production of mogrosides in transgenic tobacco lines. (A) HPLC-MS/MS analysis of MIII and MII-E in transgenic tobacco lines. (B) Accumulation of MIII in transgenic tobacco lines. (C) Accumulation of MII-E in transgenic tobacco lines. n.d., not detected. The data are presented as the mean values ± SDs, n = 3 biologically independent samples. The black arrows indicate the peak of MIII and MIIE. (D) Full-scan product ion of MIII and MII-E.5.0 × 104.
Figure 5
Figure 5
Production of mogrosides in transgenic Arabidopsis lines. (A) HPLC-MS/MS analysis of MIII in transgenic Arabidopsis lines. (B) HPLC-MS/MS analysis of SI in transgenic Arabidopsis lines. The black arrows indicate the peak of MIII and SI. (C) Full-scan product ion of SI.
Figure 6
Figure 6
Accumulation of mogrosides in transgenic Arabidopsis lines AA3, AA6, and AU7. n.d., not detected. The data are presented as the mean values ± SDs, n = 3 biologically independent samples.
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References

    1. Food and Drug Administration, U.S.A GRAS Notice 000693: Generally Recognized as Safe (GRAS) Status of D-Allulose (D-Psicose) [(accessed on 2 February 2017)]; Available online: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory.
    1. Food and Drug Administration, U.S.A GRAS Notice 000253: Rebaudioside A for Use as a General Purpose Sweetener. [(accessed on 14 March 2008)]; Available online: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory.
    1. Food and Drug Administration, U.S.A GRAS Notice 000301: Luo Han Fruit. [(accessed on 15 January 2010)]; Available online: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory.
    1. Sun-Waterhouse D., Wadhwa S.S. Industry-relevant approaches for minimising the bitterness of bioactive compounds in functional foods: A review. Food Bioprocess Technol. 2013;6:607–627. doi: 10.1007/s11947-012-0829-2. - DOI
    1. Pawar R.S., Krynitsky A.J., Rader J.I. Sweeteners from Plants-with Emphasis on Stevia Rebaudiana (Bertoni) and Siraitia Grosvenorii (Swingle) Anal. Bioanal. Chem. 2013;405:4397–4407. doi: 10.1007/s00216-012-6693-0. - DOI - PubMed

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