Heterologous expression of TPSs in E. coli and in vitro enzymatic assay

The shortened sequences of terpene synthases (TPS) lacking the anticipated signal peptide were inserted into a pDE2 vector containing a recombinant C-terminal and poly-histidine tag using the pDE2 Directional Expression Kit Ver. 2 (TSINGKE, China). The resulting constructs were then transformed into E. coli BL21 (DE3), and the recombinant proteins were induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a concentration of 1.0 mM for approximately 8 h. Subsequently, the proteins were purified using a His-Tagged Gravity Column (Merck Millipore). To confirm successful purification, sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) was performed.

For the in vitro enzymatic assay to assess TPS activity, purified proteins (approximately 20 µL) were mixed with 500 µL of buffer (25 mM HEPES, pH 7.3; 10 mM MgCl₂; 10% glycerol; 10 mM DTT) and 10 µg of either farnesyl diphosphate (FPP), neryl diphosphate (NPP), or geranyl diphosphate (GPP) (Sigma‒Aldrich). The enzymatic assay protocol described by Chen et al. [24] was followed. After vortexing, the mixtures were incubated at 30 °C for 2 h. Subsequently, 250 mL of hexane was added and vortexed for 1 min. The upper layers were then centrifuged at 1200 g and 4 °C for 30 min, followed by transfer to 2 mL glass vials for gas chromatography‒mass spectrometry (GC‒MS) analysis. Heat-inactivated recombinant proteins were used as a negative control in the experimental procedure.