## 3 Results and Discussion
### Analysis of Plasmid Presence and Antibiotic Resistance Genes
#### 3.6.1. Trepene-Related Gene Clusters
The N-terminus of protein XIJ14783, like that of protein XIJ14780.1, exhibited homology with the cAMP-binding domains of the CRP/FNR family, but with closer evolutionary ties to proteins from _Deinococcus radiodurans_, _Bacteroides thetaiotaomicron_, and _Sinorhizobium melili_. Both proteins XIJ14780 and XIJ14783 shared C-terminal similarity to the _Acinetobacter baumanni_ EncS. In conclusion, the terpene BGC in region 1 exhibits a unique genetic composition andarrangement, with two copies of EncT (XIJ14781 and XIJ14782) paired with EncS proteins (XIJ14780 and XIJ14783), alongside a distinct terpene cyclase (XIJ14779). Further investigation is needed to explore the under-researched EncS proteins, particularly focusing on the divergent N-terminal features and the conserved C-terminal characteristics of proteins XIJ14780 and XIJ14783. BGC Region 3 Derived from _Lentzea_ sp. JNUCC 0626  
The BLASTP analysis revealed that in BGC region 3 of _Lentzea_ sp. JNUCC 0626, two terpene biosynthesis-related genes, namely, geranyl diphosphate 2-C-methyltransferase transferase (2-CMT, 284 aa) and 2-methylisoborneol synthase (2-MIBS, 354 aa), are arranged in the same transcriptional direction. AlphaFold structure predictions indicate that protein XIJ15369 shows the highest similarity to 2-MIBS from _Streptomyces lasalocidi_ (UniProt: D3 KYU2), _Streptomyces ambofacialen_ (UniProt: A3 KI17), _S. coelicolor_ (UniProt: Q9F1Y6), _Streptomyces griseus_ (UniProt: Q9F1V8), and _Saccharomyces erythraaea_ (UniProt: A4 FG19), with sequence identities of 37%, 36%, 34%, 34%, and 30%, respectively. These results suggest that protein XIJ15369 may functionally resemble enzymes involved in 2-MIB biosynthesis. In contrast, structural predictions using SWISS-MODEL and HHPred indicate that the protein XIJ15369 is most similar to the 2-MIBS from _S. coelicolor_ (PDB: 3V1V) and also shares structural similarities with isoprenoid synthase from _Streptomyces hydicus_ (PDB: 4 ZQ8) and (E)-biformene synthase LrdC from _Streptomyces_ sp. strain K155 (PDB: 6 OH6). This suggests a close structural relationship with terpene biosynthesis enzymes. The biosynthesis of 2-MIB occurs in two stages. In the first stage, MT methylates geranyl pyrophosphate (GPP) to produce 2-methyl GPP. In the second stage, 2-MIBS cyclizes this intermediate to form 2-MIB [46]. Therefore, the 2-MIB BGC requires the presence of a CMT gene. Our analysis confirmed the expected presence of a corresponding CMT gene downstream of the 2-MIBS gene in a translational coupling arrangement. Additionally, AlphaFold, SWISS-MODEL, and HHPred structure predictions show that this gene exhibits high structural similarity to geranyl diphosphate 2-CMT from actinobacteria. In many bacteria, 2-MIB BGCs commonly include cyclic nucleotide-binding proteins alongside 2-MIBS and CMT. Studies on the model soil bacterium _S. griseus_ have suggested that the cyclic nucleotide-binding protein EshA encodes a conserved transcription factor. However, recent studies have revised this function, indicating that EshA functions as an EncS protein. Based on these findings, 2-MIB biosynthesis appears to be compartmentalized within protein shells. An analysis of 723 2-MIB BGCs in actinobacteria revealed that approximately 80% code for EncS proteins, while the remaining 20% contain only geranyl diphosphate 2-CMT and terpene cyclase genes without EncS proteins [47]. This suggests that while EncS proteins are generally associated with 2-MIB biosynthesis, they are not essential. Notably, the 2-MIB BGC in region 3 of _Lentzea_ sp. JNUCC 0626 does not include an EncS protein (see Figure S2 and Table S8). Figure 3: Terpene gene cluster within BGC region 1 of _Lentzea_ sp. JNUCC 0626 genome. In the lower subregion, proteins XIJ14775, XIJ14776, XIJ14777, and XIJ14778 are annotated as TetR/AcrR family transcriptional regulator, acyltransferase family protein, 1-deoxy-D-xylulose-5-phosphate synthase, and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, respectively.