## 3 Results
### Heterologous Expression and Functional Verification of Pm Mono-TPSs  
All cDNAs of _Pm TPSs_ cloned into the pET32 (+) vector with thioredoxin and histidine (HIS) tags were successfully expressed in _E. coli_ DH5\(\alpha\) competent cells, and recombinant proteins were then purified with His 60 Ni Superflow Resin and Gravity Columns (Figure S4). Following an in vitro enzyme activity assay, Pm TPS (+)-\(\alpha\)-pinene was confirmed to encode a mono-TPS enzyme that catalyzes the formation of mostly (+)-\(\alpha\)-pinene, \(\beta\)-pinene, linalool, and a few minor R-(+)-limonene, as detected by GC-FID and GC-MS analysis (Figures 6, S5 and S6, Table S5). Pm TPS (-)-\(\alpha\)-pinene catalyzed the formation of mostly (-)-\(\alpha\)-pinene, as well as myrcene, R-(+)-limonene, and linalool. Pm TPS \(\beta\)-pinene catalyzed the formation of mostly \(\beta\)-pinene and linalool, as well as a few (+)-\(\alpha\)-pinene and R-(+)-limonene. Pm TPS limonene catalyzed the formation of mostly R-(+)-limonene and a few (-)-\(\alpha\)-pinene and linalool. The GC-MS analysis of the reaction products catalyzed by Pm TPS (+)-\(\alpha\)-pinene with GPP as a substrate showed that the reaction product concentration of (-)-\(\alpha\)-pinene was 120 ug/mL after 1 h of reaction, whereas that of Pm TPS limonene, Pm TPS (+)-\(\alpha\)-pinene, and Pm TPS \(\beta\)-pinene were 6, 1, and 1.36 ug/mL, respectively (Table S5). With the extension of the incubation time, the main product of Pm mono-TPSs varied and increased during 1-3 h of the in vitro reaction, except for Pm TPS (-)-\(\beta\)-pinene. Within 3-5 h of incubation, the concentrations of the main products of Pm TPS (-)-\(\alpha\)-pinene decreased from 125.6 to 114 ug/mL, whereas the concentrations of Pm TPS (+)-\(\alpha\)-pinene and Pm TPS limonene increased from 2.43 and 6.67 ug/mL to 2.6 and 8.57 ug/mL. The by-products of Pm TPS (+)-\(\alpha\)-pinene, linalool,  
Figure 5: Molecular docking of Pm TPSs and substrates. **(Left)** Contact surface between terpene synthase and substrate molecules. **(Right)** Specific amino acids forming hydrogen bonds with substrate. Dashed lines indicate possible hydrogen bonds.  
and (\(-\))-\(\beta\)-pinene were detected after 1 h and 3 h of incubation, respectively, and gradually increased to 2.333 and 0.393 \(\upmu\)g/mL after 5 h of incubation. Limonene was detected after 5 h of incubation. Limonene was detected after 5 h of incubation. Linalool and (\(-\))-\(\alpha\)-pinene, the by-products of Pm TPS (\(-\))-\(\beta\)-pinene, were detected after 1 h of incubation, but the content of linalool exceeded that of the main product \(\beta\)-pinene after 3 h of incubation. The content of (\(-\))-\(\alpha\)-pinene gradually increased to 0.67 \(\upmu\)g/mL after 5 h of incubation but was lower than that of the main product (\(-\))-\(\beta\)-pinene at 1.03 \(\upmu\)g/mL. Limonene was detected after incubation for 5 h. The levels of limonene and (\(+\))-\(\alpha\)-pinene, by-products of Pm TPS (\(-\))-\(\alpha\)-pinene, accounted for approximately 5% of the main product and were detected after 1 h of incubation. The levels of (\(-\))-\(\alpha\)-pinene and linalool, two by-products of Pm TPS limonene, were lower than those of Pm TPS (\(-\))-\(\alpha\)-pinene by-products, and were detected after 1 and 3 h of incubation, respectively.  
Figure 6: Representative GC–FID traces showing products of recombinant Pm mono-TPSs. The labeled numbers represent corresponding products. 1: (\(+\))-\(\alpha\)-pinene, 2: (\(-\))-\(\alpha\)-pinene, 3: (\(-\))-\(\beta\)-pinene, 4: limonene, 5: myrcene, 6: linalool, IS: internal standard isobutyl-benzene.