molecules Article Identification of a Novel Specific Cucurbitadienol Synthase Allele in Siraitia grosvenorii Correlates with High Catalytic Efficiency Jing Qiao 1, Zuliang Luo 1, Zhe Gu 1, Yanling Zhang 2, Xindan Zhang 2 and Xiaojun Ma 1,* 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China; [email protected] (J.Q.); [email protected] (Z.L.); [email protected] (Z.G.) 2 Guilin GFS Monk Fruit Corp., Guilin 541006, China; [email protected] (Y.Z.); [email protected] (X.Z.) * Correspondence: [email protected]; Tel.: +86-10-57833155 Received: 7 December 2018; Accepted: 24 January 2019; Published: 11 February 2019 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. Keywords: Siraitia grosvenorii; molecular breeding; site-directed mutant; mogrosides; cucurbitadienol synthase; single nucleotide polymorphism (SNP) 1. Introduction Siraitia grosvenorii (luohanguo or monk fruit) is an herbaceous perennial of the Cucurbitaceae family. It is principally cultivated in Guilin city, Guangxi Province, China [1]. The fruit of S. grosvenorii has been used in China as a natural sweetener and as a folk remedy for the treatment of lung congestion, sore throat and constipation for hundreds of years [2]. To date, luohanguo products have been approved as dietary supplements in Japan, the US, New Zealand and Australia [3,4]. Mogrosides, the major bioactive components isolated from the fruits of S. grosvenorii, are a mixture of cucurbitane-type triterpenoid glycosides that have been proven to be powerful and zero-calorie sweeteners and can hence be used as a sucrose substitute for patients with diabetes and patients who are obese [5]. Because of their complex structures (Table A1), the chemical synthesis of these compounds is inherently difficult [6]. Currently, these valuable chemicals are mainly produced through their extraction from the fruits of S. grosvenorii. With the rapid rise in market demand, the production of Molecules 2019, 24, 627; doi:10.3390/molecules24030627 www.mdpi.com/journal/molecules Molecules 2019, 24, 627 2 of 21 luohanguo extracts has increased rapidly from two tons in 2002 to 60 tons in 2007, becoming one of Molecules 2018, 23, x FOR PEER REVIEW 2 of 23 the fastest growing industries of traditional Chinese medicine extracts [7]. However, the extraction yield of theseextraction ingredients from the is fruits limited of S. bygrosvenorii difficulties. With in theS. rapid grosvenorii rise in cultivation,market demand, including the production a requirement of for heavyluohanguo artificial pollination,extracts has increased a scarcity rapidly of appropriate from two tons cultivatable in 2002 to 60 land tons andin 2007, a high becoming purification one of cost due to thethe presence fastest growing of seeds industries [8,9]. In of addition,traditional theChines lowe medicine contents extracts of the [7]. main However, active the components extraction also yield of these ingredients is limited by difficulties in S. grosvenorii cultivation, including a result in a high cost of extraction. According to the statistics of our team, the extraction cost can be requirement for heavy artificial pollination, a scarcity of appropriate cultivatable land and a high reduced bypurification 1% when cost the due mogroside to the presence V (MV) of seeds content [8,9]. increasesIn addition, by the 0.1%. low contents Therefore, of the the main production active of high-qualitycomponentsS. grosvenorii also resultis an in urgent a high issue cost of for extrac researchtion. According and in the to production the statistics field. of our team, the In recentextraction years, cost our can team be hasreduced selected by 1% many when new theS. mogroside grosvenorii Vvarieties (MV) content with betterincreases agronomic by 0.1%. traits, such as higherTherefore, yield, the improved production resistance of high-quality to various S. grosvenorii diseases is an and urgent seedless issuefruits for research [10–13 and]. In in this the study, production field. we chose 15 S. grosvenorii varieties (E1, E2, E3, E5, E12, E23, E29, S2, S3, S10, S13, C2, C3, C6 and W4). In recent years, our team has selected many new S. grosvenorii varieties with better agronomic From thesetraits, varieties, such as higher elite germplasms,yield, improved which resistance constitute to various an diseases important and seedless resource fruits for [10–13].S. grosvenorii In breeding,this may study, be developed.we chose 15 S. Thegrosvenorii macroscopic varieties (E1, characteristics E2, E3, E5, E12, of E23, the E29, 15 S.S2, grosvenorii S3, S10, S13,varieties C2, C3, are very similar,C6 and especially W4). From beyond these varieties, the flowering elite germplas andms, fruiting which constitute periods. an However, important resource the quality for S. varies dramatically.grosvenorii Therefore, breeding, further may evaluations be developed. of S.The grosvenorii macroscopicgermplasms characteristics based of onthethe 15 activeS. grosvenorii component content arevarieties important are very to similar, better serveespecially molecular beyond breedingthe flowering purposes. and fruiting Because periods. all However, of these varietiesthe quality varies dramatically. Therefore, further evaluations of S. grosvenorii germplasms based on the are cultivated under the same conditions, we hypothesize that effective genetic polymorphisms in active component content are important to better serve molecular breeding purposes. Because all of functionalthese genes varieties involved are cultivated in MV biosynthesisunder the same are cond oneitions, of the we most hypothesize important that effective reasons genetic for the MV content change.polymorphisms in functional genes involved in MV biosynthesis are one of the most important To date,reasons key for genes the MV involved content change. in the biosynthesis of mogrosides have been successfully cloned and characterized,To date, including key genes genesinvolved from in the five biosynthesis enzyme families:of mogrosides the have squalene been successfully epoxidase cloned (SQE) [14], cucurbitadienoland characterized, synthase including (CS) [15 ],genes epoxide from hydrolasefive enzyme (EPH),families: cytochrome the squalene P450epoxidase (CYP450) (SQE) [14], [2,9 ] and cucurbitadienol synthase (CS) [15], epoxide hydrolase (EPH), cytochrome P450 (CYP450)[2,9] and UDP-glucosyltransferaseUDP-glucosyltransferase (UGT) (UGT) families families (Figure (Figure1) [ 16 1)]. Squalene[16]. Squalene is thought is thought to be to the be initialthe initial substrate and precursorsubstrate for triterpenoidand precursor and for triterpenoid sterol biosynthesis. and sterol SQE biosynthesis. has been SQE generally has been recognized generally recognized as the common rate-limitingas the enzyme common in therate-limiting common pathwayenzyme in from the mevalonatecommon pathway (MVA) from and mevalonate methylerythritol (MVA) phosphateand (MEP) pathways,methylerythritol catalyzing phosphate squalene (MEP) to pathways, 2,3-oxidosqualene catalyzing squalene [17,18]. to In 2,3-oxidosqualeneS. grosvenorii, the[17,18]. initial In S. step in the biosynthesisgrosvenorii of, the cucurbitane-type initial step in themogrosides biosynthesis of is cucurbitane-type the cyclization mogrosides of 2,3-oxidosqualene is the cyclization to form of the 2,3-oxidosqualene to form the triterpenoid skeleton of cucurbitadienol, which is catalyzed by CS. triterpenoid skeleton of cucurbitadienol, which is catalyzed by CS. EPH and CYP450 further oxidize EPH and CYP450 further oxidize cucurbitadienols to produce mogrol, which is glycosylated by UGT cucurbitadienolsto form MV. to produce mogrol, which is glycosylated by UGT to form MV. Figure 1. Genes involved in the mogroside biosynthesis pathway. 2,3-oxidosqualene is converted cucurbitadienol by cucurbitadienol synthase (CS), while cucurbitadienol is further converted to mogrosides through epoxide hydrolase (EPH), cytochrome P450s (CYP450) and UDP-glucosyltransferases (UGT). Single arrows represent the one-step conversions, while triple arrows represent multiple steps. Molecules 2019, 24, 627 3 of 21 Single