## 3 Results and Discussion
### Analysis of Plasmid Presence and Antibiotic Resistance Genes
#### 3.6.6 Other Gene Clusters
This absence of key biosynthetic genes indicates that region 14 is unlikely to be involved in the biosynthesis of APEs. Consequently, we conclude that BGC region 14 does not participate in APE biosynthesis and is, instead, likely involved in a different biosynthetic or metabolic pathway. BGC Region 18 Derived from _Lentzea_ sp. JNUCC 0626  
BGC region 18 was analyzed with antiSMASH, revealing a benzastatin cluster (Bez) that exhibits 100% similarity to a previously established cluster. The newly identified cluster contains 11 open reading frames (ORFs), as shown in Figure 10. The functional annotation of the genes within the ben cluster (Table S36) revealed high similarity with those of the bez cluster previously reported by [PERSON] et al. [92]. The main difference between the ben and bez clusters is the presence of a 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase gene (protein XJ17687) in the ben cluster, which is thought to form an operon structure with BenJ. On the other hand, there is one NRPS present in the closely related upper region of the benzastatin BGC; however, the antiSMASH analysis predicted that the corresponding domain would be inactive. BGC Region 27 Derived from _Lentzea_ sp. JNUCC 0626  
The antiSMASH analysis of BGC region 27 revealed an 80% structural similarity to the C-nucleoside antibiotic minimycin BGC. This significant similarity provides important insights into the biosynthetic pathways of minimycin- and indigiodine-like compounds. Minimycin, a C-nucleoside antibiotic structurally related to pseudouridine, exhibits notable biosynthetic parallels with indigiodine, a blue pigment produced by various bacteria, despite their distinct biosynthetic pathways [93; 94]. Protein XJ10315, identified as a non-ribosomal peptide synthetase (NRPS), shares 55.6% sequence identity with minA from _Streptomyces hygroscopicus_ strain JCM 4712 and 54.9% with indC from _Streptomyces chromofiscus_ ATCC 49982. This finding aligns with previous studies indicating that the NRPS protein MinA plays a pivotal role in regulating the divergent biosynthesis of minimycin and indigiodine. These results support the hypothesis that MinA governs a biosynthetic \"switch\" between these two molecules, warranting further experimental validation. Additionally, protein XJ1J0317 exhibits 70.9% and 69.4% identity to pseudouridine-5-phosphate glycosylases MinB and IndA, respectively, further confirming the shared features of these  
Figure 10: Organization of a putative ben BGC from _Lentzea_ sp. JNUCC 0626 with the respective homologous genes from _Streptomyces_ sp. RI18 layered onto it. The original relative orientation of individual genes (indicated by the direction of the arrow) has been maintained. two biosynthetic pathways. Interestingly, protein XJJ10318, composed of 221 amino acids, differs from MinC and IndB, which contain 613 and 614 amino acids, respectively. MinC and IndB possess both an N-terminal HAD phosphatase domain and a C-terminal DUF4243 domain, whereas protein XJJ10318 contains only the N-terminal HAD phosphatase domain. This structural difference may imply a specialized function of protein XJJ10318 within the minimycin-like biosynthesis, potentially contributing to its unique regulatory mechanisms. Protein XJJ10319 shares 70.0% and 69.9% sequence identity with MinD and Orf2 (uracil phosphoribosyltransferase) from _S. chromofuscus_ ATCC 49982 (Table 1), suggesting a conserved role in nucleoside metabolism. Additionally, protein XJJ10320 encodes a transporter protein, likely responsible for exporting the antibiotic out of the producer cell. Unlike the major facilitator superfamily (MFS) transporters typically associated with minimycin and indigiodine, protein XJJ10320 belongs to the EamA family of transporters. This difference suggests that the transport mechanisms of minimycin and indigiodine biosynthesis may be distinct, raising the possibility of novel self-resistance strategies within these pathways.