Sweet mogrosides are not only the primary bioactive ingredient in Siraitia grosvenorii fruits that exhibit anti-tussive properties and expectorate phlegm, but they are also responsible for the fruit’s sweetness. Increasing the content or proportion of sweet mogrosides in Siraitia grosvenorii fruits is significant for improving their quality and industrial production. Post-ripening is an essential step in the post-harvest processing of Siraitia grosvenorii fruits, but the underlying mechanism and condition of post-ripening on Siraitia grosvenorii quality improvement need to be studied systematically. Therefore, this study analyzed the mogroside metabolism in Siraitia grosvenorii fruits under different post-ripening conditions. We further examined the catalytic activity of glycosyltransferase UGT94-289-3 in vitro. The results showed that the post-ripening process of fruits could catalyze the glycosylation of bitter-tasting mogroside IIE and III to form sweet mogrosides containing four to six glucose units. After ripening at 35 °C for two weeks, the content of mogroside V changed significantly, with a maximum increase of 80%, while the increase in mogroside VI was over twice its initial amount. Furthermore, under the suitable catalytic condition, UGT94-289-3 could efficiently convert the mogrosides with less than three glucose units into structurally diverse sweet mogrosides, i.e., with mogroside III as the substrate, 95% of it can converted into sweet mogrosides. These findings suggest that controlling the temperature and related catalytic conditions may activate UGT94-289-3 and promote the accumulation of sweet mogrosides. This study provides an effective method for improving the quality of Siraitia grosvenorii fruits and the accumulation of sweet mogrosides, as well as a new economical, green, and efficient method for producing sweet mogrosides.

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.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Baicalein is a valuable flavonoid isolated from the medicinal plant Scutellaria baicalensis Georgi, which exhibits intensive biological activities, such as anticancer and antiviral activities. However, its production is limited in the root with low yield. In this study, In-Fusion and 2A peptide linker were developed to assemble SbCLL-7, SbCHI, SbCHS-2, SbFNSII-2 and SbCYP82D1.1 genes driven by the AtPD7, CaMV 35S and AtUBQ10 promoters with HSP, E9 and NOS terminators, and were used to engineer baicalein biosynthesis in transgenic tomato plants. The genetically modified tomato plants with this construct synthesized baicalein, ranging from 150 ng/g to 558 ng/g FW (fresh weight). Baicalein-fortified tomatoes have the potential to be health-promoting fresh vegetables and provide an alternative source of baicalein production, with great prospects for market application.

RNA silencing regulates diverse cellular processes in plants. Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) proteins are core components of RNA interference (RNAi). Despite their functional significance, the systematic identification and characterization of these families have remained largely unexplored in Siraitia grosvenorii. Using HMMER and Pfam analyses, we identified six SgAGO, four SgDCL, and six SgRDR genes. Phylogenetic analysis classified SgAGOs, SgDCLs, and SgRDRs into five, four, and four clades, respectively, all of which clustered closely with homologs from other Cucurbitaceae species, demonstrating lineage-specific evolutionary conservation. Promoter cis-element analysis revealed the significant enrichment of hormonal (methyl jasmonate, abscisic acid) and stress-responsive (light, hypoxia) elements, indicating their roles in environmental adaptation. Tissue-specific expression profiling showed that most SgAGO, SgDCL, and SgRDR genes were highly expressed in flowers and mid-stage fruits (35 days after pollination), while SgAGO10.1 exhibited stem-specific expression. By contrast, SgRDR1.2 displayed no tissue specificity. Notably, sex-biased expression patterns in dioecious flowers suggested the RNAi-mediated regulation of gametophyte development and their potential roles in reproductive and secondary metabolic processes. This study lays the foundation for further exploration of RNAi machinery’s role in coordinating mogroside biosynthesis and stress resilience in S. grosvenorii while providing potential targets for genetic improvement.

Crocins are high-value apocarotenoid pigments with broad applications in pharmaceuticals, foods, and personal-care products, and they exhibit diverse bioactivities, including antioxidant, antidepressant, and antidementia effects. In this study, we achieved the heterologous biosynthesis of crocins in Solanum lycopersicum L. by introducing the GjCCD4a, GjALDH2C3, GjUGT74F8 and GjUGT94E13 of Gardenia jasminoides J.Ellis to the binary expression vector via in-fusion technology and self-cleaving 2A peptides. Following Agrobacterium-mediated transformation, the engineered tomato plants predominantly produced main active ingredient crocin I, which accounted for 97–99% of the total crocins. The transgenic fruits displayed mixed red-and-golden colouration. These results highlight S. lycopersicum as a promising chassis for crocin I biosynthesis, helping to address supply constraints and enabling colour-trait breeding through synthetic biology.