Heterologous Reconstruction of the Mogroside Biosynthetic Pathway in <i>Nicotiana tabacum</i>

Mogrosides are natural, zero-calorie sweeteners derived from Siraitia grosvenorii. However, conventional production based on plant extraction is constrained by low yields and long cultivation cycles, limiting large-scale application. To address this, we reconstructed the mogroside biosynthetic pathway in Nicotiana tabacum through stable heterologous expression. A multigene construct (pMogroside), containing seven key S. grosvenorii genes involved in triterpene cyclization, hydroxylation, and glycosylation (SgSQE1, SgCS, SgEPH2, SgP450, AtCPR, SgUGT269-1, SgUGT289-3), was assembled using a modular strategy with P2A peptides and introduced via Agrobacterium-mediated transformation. Transgenic lines were confirmed by PCR and qRT-PCR, and HPLC-MS/MS analysis of leaves detected mogroside accumulation. Mogroside III was the dominant product (119.48 ± 25.17 ng/g FW), with lower levels of siamenoside I (21.74 ± 10.57 ng/g) and mogroside V (5.99 ± 0.61 ng/g). This work provides a sustainable plant-based platform for mogroside production and highlights the potential of N. tabacum as a versatile chassis in plant synthetic biology. Further enzyme engineering and pathway optimization will support future commercial applications in natural sweetener manufacturing.