Isolation and Functional Characterization of a Gene Encoding Squalene Synthase from Amaranthus tricolor

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.