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. 2016 Nov 22;113(47):E7619-E7628.
doi: 10.1073/pnas.1604828113. Epub 2016 Nov 7.

The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii

Maxim Itkin  1 , Rachel Davidovich-Rikanati  2 , Shahar Cohen  1 , Vitaly Portnoy  2 , Adi Doron-Faigenboim  1 , Elad Oren  2 , Shiri Freilich  2 , Galil Tzuri  2 , Nadine Baranes  2 , Shmuel Shen  1 , Marina Petreikov  1 , Rotem Sertchook  3 , Shifra Ben-Dor  4 , Hugo Gottlieb  5 , Alvaro Hernandez  6 , David R Nelson  7 , Harry S Paris  2 , Yaakov Tadmor  2 , Yosef Burger  2 , Efraim Lewinsohn  2 , Nurit Katzir  2 , Arthur Schaffer  8
Affiliations

The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii

Maxim Itkin et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway.

Keywords: functional genomics; metabolic pathway discovery; mogrosides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic diagram of the proposed pathway for mogroside biosynthesis in fruit of S. grosvenorii. The left portion of the schematic represents the steps leading to the nonglycosylated tetra-hydroxycucurbitane, mogrol. The right side indicates the successive glucosylations. Enzyme names and numbers are described in the text.
Fig. 2.
Fig. 2.
Cucurbitadienol synthase yields both cucurbitadienol and epoxycucurbitadienol. (A) Extracted ion chromatogram of CDS activity. The control panel shows the yeast GIL77 erg7− accumulation of both 2,3 oxidosqualene (peak 6) and 2,3;22,23-diepoxysqualene (peak 4) in the absence of lanosterol synthase activity. In the presence of SgCDS (Lower) both cucurbitadienol (peak 7) and 24,25-epoxycucurbitadienol (peak 3) are accumulated. Corroborative results with transgenic tobacco plants expressing SgCDS are presented in SI Appendix, Fig. S4. Mass spectra of compounds are presented in SI Appendix, Fig. S5, and NMR results for cucurbitadienol and 24,25-epoxycucurbitadienol are presented in SI Appendix, Table S8. (B) Modeling of CDS with epoxycucurbitadienol. Calculated affinities for cucurbitadienol and epoxycucurbitadienol are, respectively, −12.3 and −12.5 kcal/mol. Detailed docking model is presented in SI Appendix, Fig. S6.
Fig. 3.
Fig. 3.
EPH expression, activity, and protein-docking model. (A) Hierarchial cluster heat map of expression patterns of the eight epoxide hydrolase genes expressed in the developing Siraitia fruit. The five stages of fruit development presented are 15, 34, 51, 77, and 103 DAA. Genes EPH1, EPH 2, and EPH 3 that showed high expression in young fruit were functionally expressed in yeast (chromatograms presented in SI Appendix, Fig. S7). (B) Relative levels of di-hydroxycucurbitadienol and epoxycucurbitadienol in the control and EPH-expressing yeast lines. Metabolites were identified by LC-MS as described in SI Appendix, Methods, and quantification is presented as peak area of the chromatograms in SI Appendix, Fig. S7. Three independent cultures were analyzed, and results are presented as means ± SD. (C) Docking modeling of SgEPH protein with 24(R),25-epoxycucurbitadienol showing strong affinities and perfect matches with the epoxide oxygen positioned just between the two tyrosine residues and the nucleophile asp-101 in close proximity (3.0 Å) to the C24 and C25 positions. Detailed description of the docking model is provided in SI Appendix, Fig. S8.
Fig. 4.
Fig. 4.
CYP450 family: expression and activity with cucurbitadienol. (A) Phylogenetic tree of the cytochrome P450 genes (expandable version in SI Appendix, Fig. S9). Protein sequences used are presented in Dataset S2. (B) LC-MS analysis of extracts of yeast coexpressing SgCDS with CYPs showing cucurbitadienol-hydroxylating activity. The extracted ion chromatogram (m/z = 407–444) represents relevant triterpenoid compounds and derivatives accumulated in the yeast. Yeast coexpressing SgCDS with CYP87D18 (Middle chromatogram) produced mainly 11-hydroxy cucrbitadienol (peak 5), which coeluted with 11-oxo-cucurbitadienol. Yeast coexpressing SgCDS with CYP88L4 (Bottom chromatogram) produced mainly 19-hydroxycucurbitadienol (peak 11). A chromatogram from yeast harboring SgCDS alone is illustrated in the Upper chromatogram as negative control. (C) Production of mogrol in yeast extracts expressing SgCDS, SgEPH3, SgCYP87D18, and AtCPR in presence (+) and absence (−) of lanosterol synthase inhibitor R0 48–8072 at 24 and 48 h after addition of the inhibitor. Lower chromatogram shows the mogrol standard (peak 1), and the mogrol can be identified at 48 h after inhibitor addition (48 h+). Peak numbers are as follows: 1—mogrol; 2—24,25-dihydroxycucurbitadienol; 3—unidentified C30H48O3; 4—unidentified C30H52O3; 5—11-hydroxycucurbitadienol; 6—24,25-epoxycucurbitadienol; 7—2,3;22,23-diepoxysqualene; 8—2,3-oxidosqualene; 9—cucurbitadienol; 10—lanosterol; and 11—19-hydroxycucurbitadienol. MS spectra and NMR results are represented in SI Appendix, Fig. S5 and Table S8, respectively.
Fig. 5.
Fig. 5.
Siraitia UGTs involved in mogroside glucosylations. (A) Phylogenetic tree of Siraitia UGTs (expandable version in SI Appendix, Fig. S13). UGTs referred to in this study are boxed in red. Protein sequences used are presented in Dataset S2. (B) Schematic summary of primary glucosylation reactions using the various substrate precursors, as described in SI Appendix, Fig. S1 and Methods. The schematic representation of the mogroside compounds comprises two blue ovals, representing two cyclic rings each of the tetracyclic cucurbitane skeleton; red circles represent glucosyl moieties in the substrate, and green circles represent the newly glycosylated positions due to the reaction. In the case of branched glycosylations, a 1–6 glucosyl arrangement is represented with the attached circle pointing upwards, while a 1–2 arrangement is represented by the attached circle pointing downwards. (C) Chromatograms showing the single glucosylation performed by UGT720-269–4 (Upper chromatogram) and the double glucosylation performed by UGT720-269–1 (Bottom chromatogram). (D) Schematic summary of branched glucosylations using various substrates, described in SI Appendix, Table S1. Chromatograms and MS data are presented in SI Appendix, Fig. S15.
Fig. 6.
Fig. 6.
Modeling of the mogroside branching UGTs. (A) Docking model of UGT94-289–3 with a glucosylated mogroside residing in the substrate pocket, shown in blue shading. The model shows a deep pocket behind the catalytic His14 (stick figure) that easily accommodates one or two glucose moieties. The wall pocket is created by two helices (colored in yellow) that create a polar interface suitable for glucose binding. The Glu193 is positioned behind the space-filled mogroside. The UDP-glu donor molecule is represented as a stick and ball model. (B) Alignment of the region containing the three characteristic polar residues of the branching UGT94 enzymes (listed as 289–1,2,3 and colored in blue) of representative UGT proteins listed in Dataset S2. Complete protein alignment is presented in SI Appendix, Fig. S22.
Fig. 7.
Fig. 7.
Hierarchical clustering of the expression patterns of the members of the five gene families responsible for mogroside biosynthesis: SE, EPH, CDS, CYPs, and UGTs. The cluster containing the genes of the pathway is outlined in blue and is expanded to the right. The genes shown to be involved in mogroside biosynthesis are marked by an asterisk. An expandable version of the entire heat map is presented in SI Appendix, Fig. S24 in which the additional genes identified in this paper are marked. Genes with numbers containing decimal points are derived from the UGT-containing scaffolds, and the number following the decimal indicates its position in the tandem arrangement. Genes with numbers containing a lower hyphen are derived from the CYP-containing scaffolds, and the last number indicates its position in the tandem arrangement. Genes beginning with the letter “S” are derived from the genomic scaffolds. All genes are described in Dataset S2. The CYP87D18 coding for the C11-hydroxylating enzyme is listed as S623.1, closely clustering with the CDS gene.
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