## 3 Results
### Truncation of TS 1411  
In order to elucidate the rate-limiting steps of the pathway, we introduced an additional copy of the 1411-coding gene, resulting in strain ZMJ02. This modification led to a significant improvement in titer from 0.060 mg/L to 0.103 mg/L, corresponding to a 0.67% enhancement (Figure 1F). Furthermore, it has been reported that the heterologous pathway could increase the titer of monoterpenes such as \(\alpha\)-pinene and myrcene to 104.6 mg/L [33] and 510.38 mg/L [37], respectively, suggesting that TS was probably the rate-limiting step for the biosynthesis of tricyclene. Therefore, further improvements in the titer were conducted through reconstructions of the TSs.  
Plant synthases often possess a transit peptide in the N-terminal region, which facilitates protein translocation to specific plastid compartments prior to proteolytic cleavage. Transit peptides typically consist of 30 to 80 amino acids and are characterized by an abundance of basic, slightly hydrophobic, and hydroxylated amino acids. Due to the absence of mechanisms in _E. coli_ for eliminating plant transit peptides, there is a potential risk of protein misfolding. Therefore, the truncation of the transit peptide is frequently employed as a strategy to enhance synthase activity [38].  
The protein structure of 1411 was predicted using Uni-Fold (Figure 2A) [39]. There was a random coil at the N-terminal of 1411 from L1 to W73, probably causing protein misfolding in _E. coli_. To determine the appropriate truncation site, the transit peptide of 1411 was predicted using the online tool TargetP 2.0 ([[https://services.healthtech.dtu.dk/services/TargetP-2.0/](https://services.healthtech.dtu.dk/services/TargetP-2.0/)]([https://services.healthtech.dtu.dk/services/TargetP-2.0/](https://services.healthtech.dtu.dk/services/TargetP-2.0/)), accessed on 20 March 2024.). As a result, 1411 was predicted to have an N-terminal thylakoid luminal transfer peptide, with a cleavage site at H37. Therefore, 1411 was truncated at five different positions of P19, P24, H37, I49, and W73 at the N-terminal, obtaining 1411(19), 1411(24), 1411(37), 1411(49), and 1411(73), respectively (Figure 2B). As shown in Figure 2C, the strain containing 1411(73) was not able to produce detectable tricyclene, while the remaining four truncated 1411 synthases resulted in increased tricyclene titers at different levels, compared with wild-type 1411. The highest tricyclene titer of 38.331 mg/L was obtained for the strain containing 1411(49). Furthermore, because H37 was the predicted cutting site and the truncation at I49 led to the highest titer, 13 positions from H37 to I49 were truncated by one amino acid. The corresponding titers are shown in Figure 2D. The highest titer of tricyclene was obtained for the strain containing 1411(44), which was 47.671 mg/L, an approximately 794.5-fold improvement compared with wild-type 1411. The strains containing 1141(45), 1141(48), and 1141(49) also exhibited significantly elevated tricyclene titers of 40.708 mg/L, 38.907 mg/L, and 38.331 mg/L, respectively. This result indicated that the appropriate truncation of TS 1411 helped improve the tricyclene titer.