## 4 Discussion  
Tricyclene, a tricyclic monoterpene, has broad application prospects in many fields, including biofuel and medicine. However, due to the scarcity of tricyclene, its use needs to be further explored. In this study, we reported the construction of a heterologous biosynthetic pathway of tricyclene in _E. coli_ by introducing TSs from different plant species. It was determined by increasing the copy number of the TS-coding gene that the TS was the rate-limiting step for tricyclene biosynthesis. Low temperatures could reduce the inclusion bodies of _E. coli_, thereby increasing production [25]. The optimal culture temperature for tricyclene synthesis was determined to be 30 \({}^{\circ}\)C, as further reductions in incubation temperature did not yield any improvement in tricyclene titer. The low activity of tricyclene synthase leads to its low biosynthetic potency, which needs further optimization to meet the requirements of large-scale industrial production. The tricyclene titer was increased from 0.060 mg/L to 47.671 mg/L, a 794.5-fold improvement, through truncating 44 amino acids of the N-terminal random coil of TS 1411. The titers of byproducts including \(\alpha\)-pinene, \(\beta\)-pinene, sabinene, camphene, and limonene were also increased. Removing transit peptides could improve protein expression in the _E. coli_ system without affecting its enzyme activity [40]. [PERSON] et al. introduced \(\gamma\)-terpinene  
Figure 3: Product analysis of the truncated TSs. (**A**) GC-MS analysis of 1411(44) products. Peak 1, tricyclene; Peak 2, \(\alpha\)-pinene; Peak 3, camphene; Peak 4, sabinene; Peak 5, \(\beta\)-pinene; Peak 6, limonene. Blank represents a mixture of n-dodecane and ethyl acetate. (**B**) Product titers of the truncated TSs. The truncated TSs producing detectable products in this study. The error bars represent one standard deviation for three replicates. synthase in _E. coli_ to synthesize \(\gamma\)-terpinene; the results of SDS-PAGE showed that the formation of inclusion bodies could be reduced by removing the transit peptide [32]. In this study, SDS-PAGE analysis revealed that by lowering the induction temperature to 30 \({}^{\circ}\)C and truncating the transit peptide, the soluble expression level of TS was effectively improved, indicating that the enhancement of the tricyclene titer was probably caused by improved TS solubility. Our findings indicate that the appropriate truncation of plant synthase transit peptides can improve their soluble expression and product titers in _E. coli_, which lacks a mechanism for clearing plant transit peptides. According to the results above, the truncated region referred to the transit peptide segment, which impacted the soluble expression of TS. Truncating the transit peptide significantly enhanced the solubility of TS and subsequently improved tricyclene's synthetic titer. In cases where truncation was insufficient, the incomplete removal of the transit peptide still affected protein solubility, resulting in a change in tricyclene's synthetic titer due to variations in TS solubility during gradient truncation. If the truncated region was excessively long (e.g., 1411(73)), it potentially disrupted a domain within TS responsible for catalyzing tricyclene synthesis, thereby reducing its synthetic titer instead. Consequently, we hypothesized that achieving the highest tricyclene titer required optimal TS solubility without compromising the integrity of the domain involved in tricyclene synthesis. To synthesize tricyclene, we retrieved 16 synthases predicted to synthesize tricyclene from public databases (Table S3) and introduced them into _E. coli_. The fermentation results showed that only 1411 could synthesize detectable tricyclene. Due to the high similarity of the protein sequences and predicted conformations of the retrieved 16 TSs, the transit peptides of the other 15 TSs should be consistent with 1411. Therefore, the other 15 truncated TSs were subjected to shake-flask fermentation after being truncated by 44 amino acids.