###### Abstract  
Tricyclene, a tricyclic monoterpene naturally occurring in plant essential oils, holds potential for the development of medicinal and fuel applications. In this study, we successfully synthesized tricyclene in _E. coli_ by introducing the heterologous mevalonate (MVA) pathway along with _Abies grandis_ geranyl diphosphate synthase (GPPS) and _Nicotiana sylvestris_ tricyclene synthase (TS) XP_009791411. Initially, the shake-flask fermentation at 30 \({}^{\circ}\)C yielded a tricyclene titer of 0.060 mg/L. By increasing the copy number of the TS-coding gene, we achieved a titer of 0.103 mg/L. To further enhance tricyclene production, optimal truncation in the N-terminal region of TS XP_009791411 resulted in an impressive highest titer of 47.671 mg/L, approximately a 794.5-fold improvement compared to its wild-type counterpart. To the best of our knowledge, this is the highest titer of the heterologous synthesis of tricyclene in _E. coli_. The SDS-PAGE analysis revealed that lowering induction temperature and truncating the random coil N-terminal region effectively improved TS solubility, which was closely associated with tricyclene production levels. Furthermore, by truncating other TSs, the titers of tricyclene were improved to different degrees. tricyclene; tricyclene synthase; biosynthesis; transit peptide truncations; protein solubility +
Footnote †: journal: International Journal of the Society of America  
0000-000  
raw materials for renewable fuels with high energy density. Consequently, the tricyclic monoterepne tricyclene may also hold potential as a precursor for advanced biofuels. Traditionally, monoterepnes have been derived from plant biomass; however, this conventional extraction method is laborious and inefficient, necessitating the significant consumption of natural resources [15]. Therefore, the achievement of large-scale commercial production has not yet been realized. Similarly, the chemical synthesis strategy for monoterepnes was considered as a means to enhance productivity; however, it is accompanied by drawbacks such as intricate reactions, exorbitant costs, and significant environmental pollution [16; 17]. Recently, the heterologous production of monoterepnes by microbial hosts has garnered significant attention due to advancements in metabolic engineering and synthetic biology [18]. The advantages of _Escherichia coli_ include rapid growth, elucidated metabolic pathways, facile gene manipulation techniques, and well-established fermentation procedures [16; 19]. Recently, the use of _E. coli_ as a cell factory for the gram-scale synthesis of monoterepnes has been reported, such as geraniol (5.52 g/L) [20], \(\alpha\)-pinene(1.04 g/L) [10], and limonee (3.6 g/L) [21]. Therefore, constructing and engineering _E. coli_ as a cell factory for the efficient production of tricyclene is operational. [PERSON] et al. expressed heterologous terpene synthase in _E.coli_ to produce monoterepnes, where the production level of tricyclene was at least 5% of the total monoterepne production, and indicated that tricyclene has very good octane properties and oxidative stability, so it may be more suitable as a gasoline component than other monoterepnes [22]. Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are universal precursors for terpenoids [23]. The biosynthesis pathways of IPP and DMAPP include the methylerythnitol-4 phosphate (MEP) and the mevaloate (MVA) pathways. The MEP pathway is generally found in bacteria such as _E. coli_, while the MVA pathway is generally found in eukaryotes such as _Saccharomyces cerevisiae_. [PERSON] et al. successfully constructed the heterologous MVA pathway in _E. coli_ for the first time and obtained a high-yield strain of amorphadiene, which can be used as a platform host for the production of general terpen [24]. Afterward, due to the regulatory mechanisms of the native host limiting the expression of the MEP pathway, the heterologous expression of the MVA pathway in _E.