Relative enzyme activity assays

OsCyc1 mutants used in relative enzyme activity assay were full-length and purified by Ni affinity chromatography followed by Superdex^TM200 HR 10/300 gel filtration except for proteins that were used in new product identification experiments. OsCyc1 proteins were collected in buffer A (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM DTT), except H275L, I311V, Y317F, H357W and L400F which were collected in buffer B (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM DTT, 5 mM MgCl₂). The relative enzyme activity experiments were performed in a 100 μL solution A, except for H275L, I311V, Y317F, H357W and L400F which were performed in solution B.

For the determination of the relative enzyme activity of the mutant variants used for enzyme activity comparison, solutions were supplemented with 2.5 μg GGPP and 50 μg protein, and the reactions were allowed to proceed for 2 h at 16 °C. For the determination of the relative enzyme activity of H275L/H357W/L400F, solutions were supplemented with 10 μg GGPP and 300 μg protein, and the reactions were allowed to proceed for 2 h at 16 °C. For the determination of the relative enzyme activity of mutants in Supplementary Fig. 18a–c and 18e (except for OsCyc1), solutions were supplemented with 30 μg GGPP and 600 μg protein, and the reactions were allowed to proceed for 2 h at 16 °C.

Two units of alkaline phosphatase (calf intestinal (CIP) (NEB)) were used for dephosphorylation for 2 h. Then the reaction solution was extracted with 600 μL n-hexane twice. The n-hexane extract was evaporated to dryness under a gentle nitrogen flow and then resuspended by 80 μL n-hexane.

For the determination of relative enzymatic activity of OsCyc1 under varying concentrations of magnesium ions, 2.5 μg GGPP and 40 μg of protein were incubated for 2 hours at 16 °C. Different magnesium ion concentrations were achieved by adding additionally 0 mM (control group), 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, and 50 mM magnesium chloride to buffer A. For both the control group and reactions with added magnesium ions, each reaction was performed in triplicate. Alternatively, the enzymatic activity was assessed with 5 μg GGPP and 100 μg of protein in the presence of buffer A with the additional supplementation of 1 mM, 5 mM, and 10 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA). Reactions involving the addition of EDTA were performed once for each reaction.

The identification of new product of OsCyc1^5Mu was carried out in buffer B, containing 30 μg GGPP and 300 μg OsCyc1^5Mu, at 16 °C overnight. After being dephosphorylated by alkaline phosphatase, the product was extracted three times, with each extraction using 900 μL n-hexane.

200 μg MDS^D611A and 200 μg SmKSL were utilized to catalyze 10 μg GGPP, and the resulting product was used as a standard for miltiradiene. Similarly, 300 μg TwCPS3 and 200 μg AtKS were used to catalyze 10 μg GGPP, and the product was served as a standard for ent-kaurene.

For the identification of the new product of OsCyc1^5Mu, two separate reactions were conducted. In the first reaction, 600 μg OsCyc1^5Mu and 600 μg SmKSL were used to catalyze 100 μg GGPP in buffer B overnight, with a total volume of 500 μL. In the second reaction, 600 μg OsCyc1^5Mu and 600 μg AtKS were used to catalyze 100 μg GGPP under the same conditions. After each reaction, the product was extracted three times, with each extraction using 900 μL of n-hexane.

In the above experiments, the n-hexane extract was evaporated to dryness under a gentle nitrogen flow and then resuspended in 80 μL of n-hexane to obtain the test sample. GC-MS analysis was carried out on a DB-5MS column using helium as a carrier gas. 5 μL samples were injected. Initial oven temperature was set at 50 °C for 2 min followed by a 20 °C /min gradient to 300 °C and held for 10 min.