## 3 Results and Discussion
### Analysis of Plasmid Presence and Antibiotic Resistance Genes
#### 3.6.6 Other Gene Clusters
Unlike conventional NRPSs, this \(\varepsilon\)-PL-producing NRPS lacks condensation and thioesterase domains, instead containing adenylation and thiolation domains, along with six transmembrane and three soluble domains [98; 99]. The BLASTP analysis revealed that this \(\varepsilon\)-PL synthase shares significant sequence identity (56.03%) and similarity (67.42%) with _Streptomyces nouvesi \(\varepsilon\)-_PL synthase (WP_205368663). Adjacent to this NRPS is protein XJ12647, encoding an M1 family metallopeptidase, which is part of a zinc-dependent enzyme group known as aminopeptidase N (APN). This enzyme class typically cleaves amino acids from the N-terminus of peptides and contains a conserved zinc-binding motif (HEXCHK18E), essential for its catalytic activity [100; 101]. Protein XJ12647 also possesses this motif (\({}^{321}\)HELAH325X18E) and exhibits amino acid identity (40.57%) and similarity (51.02%) with _S. nouvesi_ M1 family metallopeptidase (WP_205368662.1). The accumulation of \(\varepsilon\)-PL is regulated by both the NRPS and \(\varepsilon\)-PL-degrading enzymes. The activation of the degrading enzyme inhibits \(\varepsilon\)-PL accumulation, while the suppression of its expression increases \(\varepsilon\)-PL levels. This suggests that strategies to enhance \(\varepsilon\)-PL production could involve inhibiting the degrading enzyme or increasing NRPS expression via genetic modification [102]. Furthermore, four serine hydrolase genes (XJ12654-XJ12657) were found to be located seven proteins upstream of the \(\varepsilon\)-PL and M1 metallopeptidase genes. These enzymes belong to a group characterized by a serine residue in their catalytic site and are responsible for hydrolyzing ester, amide, and peptide bonds [103]. All four serine hydroxlases in region 29 showed high homology to D-alanyl-D-alanine carboxypeptidases, which are involved in bacterial cell wall synthesis. While no direct link between these hydrolases and \(\varepsilon\)-PL resistance or degradation has been established, the functional interaction between these enzymes remains a potential area for further investigation. BGC Region 30 Derived from _Lentza_ sp. JNUCC 0626  
An analysis of the KnownClusterBlast results for BGC region 30 indicated that it exhibits approximately 3% lower structural similarity compared to the LL-D49194\(\alpha\)1 BGC [104]. The protein encoded by gene XJ12721 shares 51.42% identity and 65.91% similarity with lldRg2, a YafY family regulator of LL-D49194\(\alpha\)1, while the protein encoded by gene XJ12722 shows 51.43% identity and 62.86% similarity with lldO1, an extradiol dioxygenase from the VOC family (Figure S28, Table S40). However, no significant similarities were identified among the remaining genes located in region 30, including those related to LL-D49194\(\alpha\)1 and other BGCs. BGC Region 32 Derived from _Lentza_ sp. JNUCC 0626  
The proteins XJ14006 and XJ14007 located in BGC region 32 exhibit low levels of amino acid homology to the IucA/IucC family proteins and IucA/IucC family siderophore biosynthesis proteins, which are components of the aerobactin biosynthesis operon iucABCD, as determined by BLASTP analysis (Figure S29, Table S41). IucA and IucCare known to catalyze distinct steps in the biosynthesis of aerobactin from N \(\varepsilon\)-acetyl-N \(\varepsilon\)-hydroxyllysine and citrate [105]. According to recent studies, the enzymatic synthesis of aerobactin was reconstituted biochemically through the heterologous expression of the four genes in _E. coli_. The biosynthesis of aerobactin requires the activity of the acetyltransferase transferase IucD and hydroxylase IucB to form N6-acetyl-N6-hydroxyllysine (ahLys), followed by the sequential addition of two molecules of the hydroxamate intermediate ahLys to the primary carboxyl groups of citrate, catalyzed by IucA and IucC, to generate aerobactin [106; 107]. However, no candidate genes corresponding to IucB and IucD are present in BGC region 32.