Biosynthesis of squalene in the plant chassis has broad application prospects, and identifying efficient enzymes is of great importance. Here, we analyzed the function of squalene synthase genes WsSQS and WsSQS2 from Withania somnifera for squalene biosynthesis in tobacco. WsSQS and WsSQS2 shared 93.7% amino acid (aa) similarity, with divergent residues related to catalysis, NADPH binding, and membrane anchoring. Heterologous expression of WsSQS and WsSQS2 in tobacco increased squalene content by 2.05-fold and 1.68-fold, respectively, with the OE-WsSQS lines reaching 3.19 μg/g DW and the OE-WsSQS2 lines reaching 2.58 μg/g DW, compared to the control plants. Further transcriptomic assays revealed that overexpression of WsSQS induced broader transcriptional changes in the squalene metabolic pathway than WsSQS2. Specifically, the overexpression of WsSQS up-regulated AACT, HMGS, MVD, IspE, FPPS1, FPPS2, and SQS upstream of squalene biosynthesis and down-regulated GGPPS3 downstream of FPP biosynthesis, which is the direct precursor of squalene biosynthesis, while WsSQS2 exerted a more targeted impact, primarily up-regulating HMGS and the key rate-limiting enzyme gene HMGR in the squalene biosynthesis pathway. These findings are consistent with the high efficiency of WsSQS in squalene biosynthesis in tobacco. In summary, this study provides fundamental molecular and biochemical insights into the utilization of heterologous SQSs for squalene production based on the tobacco chassis.

Squalene, a high-value triterpenoid precursor widely used in pharmaceuticals and vaccine adjuvants, is primarily sourced from shark liver oil—an unsustainable practice that has driven interest in developing plant-based production alternatives. The first committed reaction in triterpenoid biosynthesis is catalyzed by squalene synthase (SQS), yet no SQS gene has been characterized in Amaranthus tricolor, a species recognized for its high squalene content. Here, we cloned and functionally characterized AtrSQS, a novel squalene synthase gene isolated from A. tricolor for the first time. Sequence analysis revealed that AtrSQS contains conserved domains and six characteristic motifs shared among plant SQSs, with high homology to orthologs from dicotyledonous species. To investigate its functional role in squalene biosynthesis, AtrSQS was overexpressed in Nicotiana tabacum under the CaMV 35S promoter. Transgenic lines exhibited significantly increased AtrSQS expression and accumulated squalene up to 6.81 μg/g dry weight, a 4.76-fold increase over wild-type plants. Additionally, the content of downstream product 2,3-oxidosqualene was also significantly elevated in the transgenic lines. Integrated transcriptomic and metabolomic analyses revealed that AtrSQS overexpression upregulated key mevalonate pathway genes (AACT, HMGS, MVD) and FPPS. Meanwhile, it suppressed competitive carotenoid biosynthesis and the production of an SQS-specific inhibitor, indicating a successful redirection of metabolic flux toward squalene production. These findings demonstrate that AtrSQS is crucial for squalene biosynthesis and provide both a valuable genetic resource and mechanistic insights for engineering plant-based squalene production systems.