Identification of a Novel Specific Cucurbitadienol Synthase Allele in Siraitia grosvenorii Correlates with High Catalytic Efficiency

Abstract

Mogrosides, the main bioactive compounds isolated from the fruits of Siraitia grosvenorii, are a group of cucurbitane-type triterpenoid glycosides that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. However, the extraction cost is high due to their relatively low contents in plants. Therefore, molecular breeding needs to be achieved when conventional plant breeding can hardly improve the quality so far. In this study, the levels of 21 active mogrosides and two precursors in 15 S. grosvenorii varieties were determined by HPLC-MS/MS and GC-MS, respectively. The results showed that the variations in mogroside V content may be caused by the accumulation of cucurbitadienol. Furthermore, a total of four wild-type cucurbitadienol synthase protein variants (50R573L, 50C573L, 50R573Q, and 50C573Q) based on two missense mutation single nucleotide polymorphism (SNP) sites were discovered. An in vitro enzyme reaction analysis indicated that 50R573L had the highest activity, with a specific activity of 10.24 nmol min−1 mg−1. In addition, a site-directed mutant, namely, 50K573L, showed a 33% enhancement of catalytic efficiency compared to wild-type 50R573L. Our findings identify a novel cucurbitadienol synthase allele correlates with high catalytic efficiency. These results are valuable for the molecular breeding of luohanguo.

Encapsulation of β-Glucosidase within PVA Fibers by CCD-RSM-Guided Coelectrospinning: A Novel Approach for Specific Mogroside Sweetener Production

“…2,5 For example, some studies showed that the sweetest mogroside, S I, is 563 times sweeter than 5% sucrose solution, although this compound accounts for less than 5% of total MGs. 2,7,8 Moreover, unlike other types of sweet MGs with sweet profiles varying from that of sucrose, 9 the sweetness characteristics of S I were more similar to those of sucrose, without any aftertaste. 10 Several attempts have been made to develop procedures for obtaining S I, and most of them have focused on the transformation of MG V into S I.…”

“…Although there are several kinds of MGs in monk fruit, namely, MG VI, MG V, 11-oxo-MG V, MG IV, S I, MG III E, MG II, and MG I, few kinds are sweet. , For example, some studies showed that the sweetest mogroside, S I, is 563 times sweeter than 5% sucrose solution, although this compound accounts for less than 5% of total MGs. ,, Moreover, unlike other types of sweet MGs with sweet profiles varying from that of sucrose, the sweetness characteristics of S I were more similar to those of sucrose, without any aftertaste…”

“…Several attempts have been made to develop procedures for obtaining S I, and most of them have focused on the transformation of MG V into S I. The reason is because MG V accounts for 60% of the total MGs in the fruit without enrichment, and if a 100% conversion rate could be reached, large amounts of S I could be obtained. However, methods including acid hydrolysis, enzymatic treatment, and in vivo digestion all have shown little effect on generating S I from MG V due to low specificity, as the chemical structures of these two compounds only differ by one glucose moiety, where MG V has one additional glucose on the C-3 position of S I, with a β (1 → 6) linkage (Figure A). ,, Recently, the transformation of MG V using microbes has shed light on the specific production of S I from MG V. Saccharomyces cerevisiae BY4741 wild-type cells were able to produce β-glucosidase ( Sc Exg1), which acts on peripheral glucose with (1 → 6)-β- d -glucosyl linkages at the C-3 and C-24 positions of MG V, generating S I and MG IV as intermediates and MG III E as the end product .…”

Effects of Forchlorfenuron on the Morphology, Metabolite Accumulation, and Transcriptional Responses of Siraitia grosvenorii Fruit

“…Three intermediates (squalene, cucurbitadienol, and cycloartenol) were measured. The fruit samples for GC-MS analysis were prepared and the basic operational conditions were as described previously [13,33]. Helium gas was used as the carrier gas, and the carrier flow rate was 1.5 mL min −1 .…”

“…However, the low content of mogrosides in LHG cannot meet the increasing market demand. Market surveys showed that the larger the fruit, the higher the price because of the high content (1%–2%, m / m ) of mogrosides in large LHG, and the extraction cost can be reduced by 1% when the mogroside content increases 0.1% [13]. Therefore, it is an urgent issue, taking some advanced technologies to achieve high-quality LHG.…”