A t -anol/isoeugenol synthase responsible for converting coumaryl acetate to t -anol in Illicium verum

Objective: Illicium verum is an endemic plant of southern China, which is the primary country for its production. Trans-anethole ( t -anethole) is the key component of the volatile aromatic essence in I. verum , and it has therapeutic effects such as anti-cancer and anti-diabetes. However, its biosynthetic pathway in I. verum is rarely reported. Methods: In the present study, we cloned and expressed the cDNA encoding t -anol/isoeugenol synthase (IvAIS1) in Escherichia coli . The characteristics of the IvAIS1 were determined and its gene expression in different tissues was measured by real-time polymerase chain reaction. Results: The IvAIS1 protein is 76% identical to Schisandra chinensis isoeugenol synthase, and the two proteins were clustered closely together in the clade of IGS and EGS. IvAIS1 exhibits NADPH-dependent enzyme activity and dual product specificity, and it converts coumaryl acetate and coniferyl acetate to t -anol (the precursor of t -anethole) and isoeugenol, respectively. The Km values for coniferyl acetate and coumaryl acetate were 438.4 ± 44.3 μM and 480.30 ± 86.61 μM, respectively. The catalytic efficiency of IvAIS1 for coniferyl acetate was found to be higher than that for coumaryl acetate. The gene expression profiles showed that IvAIS1 accumulated in the roots, leaves, and fruits, but the levels were relatively low in the stems and flowers of I. verum . Conclusions: This study showed a putative t -anol/isoeugenol synthase responsible for converting coumaryl acetate to t -anol in I. verum. It expands our current knowledge of the enzymes involved in t -anethole biosynthesis.