## 2 Materials and Methods
### Expression and Purification of Recombinant Pm Mono-TPSs  
Six Pm TPS-coding sequences were amplified as described above, and a homologous recombination insertion into the pET-32a (+) vector was employed. Homologous recombinant plasmids were verified via sequencing and transferred into BL21 (DE3) competent cells. Luria-Bertani medium (1 L) containing 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL Amp was inoculated with five individual colonies and cultured until an optical density at 600 nm (OD600) of at least 0.8 was reached at 37 \({}^{\circ}\)C and 220 rpm. Isopropyl-\(\beta\)-D-thiogalactopyranoside was added to a final concentration of 0.5 mM and induced to express at 20 \({}^{\circ}\)C and 100 rpm for 12 h. Cultures were collected via centrifugation at 13,523\(\times\)\(g\) for 15 min. The bacterial cells were collected and resuspended in 100 mL resuspension buffer (50 mM PB, 300 mM NaCl, 20 mM imidazole, pH 7.4). Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM and ultrasonically broken in an ice bath (power 200 W, work 3 s, pause 5 s, for 10 min per 30 mL of resuspended bacterial cells). After centrifugation at 13,523\(\times\)\(g\) for 30 min, the supernatant and precipitate were collected (dissolved in 8 M urea). Subsequently, 20 \(\mathrm{\SIUnitSymbolMicro L}\) of each was added to 5 \(\mathrm{\SIUnitSymbolMicro L}\) loading buffer, kept in a 100 \({}^{\circ}\)C water bath for 10 min, and then centrifuged for a short time. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis was performed on 7.5% gel (100 V, 120 min). The protein gel was stained with Coomassieie brilliant blue and photographed. Recombinant Pm mono-TPS proteins were purified to homogeneity by Ni\({}^{2+}\)-affinity chromatography and then desalted at 4 \({}^{\circ}\)C for 16 h in a dialysis bag (14 kDa) with a buffer composed of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 mM NaCl, and 1 mM dithiothreitol (DTT). The dialysis target protein was frozen at \(-80\)\({}^{\circ}\)C for 4 h and treated in freeze-drying apparatus for more than 24 h until the protein was completely dried. Dried terpene synthase protein was diluted to approximately 5 mg/mL using ddH\({}_{2}\)O.  
TPS Enzyme Assays and Gas Chromatography-Flame Ionization Detection/Gas Chromatography-Mass Spectrometry (GC-FID/GC-MS) Analysis  
The TPS enzyme activities were tested according to a previously published procedure ([PERSON] et al. 2002), with modifications. Before the assay, the frozen proteins were placed at 37 \({}^{\circ}\)C until thawing. The diluted proteins were pre-equilibrated with assay buffers (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 100 mM KC1, 10 mM MgCl\({}_{2}\), 5% (\(p/v\)) glycerol, and 5 mM DTT). Enzyme activity was assessed using 1 mL of the pre-equilibrated proteins with the addition of 25 \(\mathrm{\SIUnitSymbolMicro L}\) GPP (50 mM, with 50% \(v/v\) methanol, perform nitrogen purge before use). All enzyme assays were overlaid with 800 \(\mathrm{\SIUnitSymbolMicro L}\) of hexane (Aladdin Biological Technology Co., Ltd., Shanghai, China) and 400 ng/mL isobutyl benzene (Aladdin Biological Technology Co., Ltd.) to collect the released volatiles, then incubated at 37 \({}^{\circ}\)C, 80 rpm for 1 h, 3 h, and 5 h. The upper layer hexane (200 \(\mathrm{\SIUnitSymbolMicro L}\)) was taken into a sample vial for GC-FID/GC-MS analysis. Chromatographic grade n-hexane, (+)-a-pinene, (-)-\(\alpha\)-pinene, (-)-\(\beta\)-pinene, R-(+)-limonene, linalool, and myrcene standard were purchased from Aladdin Biological Technology Co., Ltd. The Pm mono-TPS products were analyzed on a GC-FID fitted with an HP-5 column (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\mathrm{\SIUnitSymbolMicro m}\), Agilent Technologies Inc., Santa Clara, CA, USA). The injection volume was 1 \(\mathrm{\SIUnitSymbolMicro L}\) of the hexane extract.