## 2 Diversity of LPBSs and NRPSs from _Bacillus_ and _Pseudomonas_
### Pseudomonas
#### 2.2.3. Arthrofactin Syntheatase  
Arthrofactin is a cyclic lipoundecapeptide produced by _Pseudomonas_ sp. MIS38, which was initially misidentified as _Arthrobacter_ sp. [56], and belongs to the amphisin group. The molecule is cyclized through the formation of an ester bond between the carboxyl group of the _C_-terminal Asp and the \(\beta\)-hydroxyl group of d-_allo_-Thr [97]. Arthrofactin is one of the most effective cyclic LPBSs; it reduces the surface tension of water from 72 to 24 mN/m with a CMC of 13.5 mg/L [56]. Arthrofactin appears to be essential for swarming activity and inhibits initial attachment of the planktonic cells in biofilm formation [98]. Several Arthrofactin-like compounds with remarkable biosurfactant and antifungal properties or an enzyme inhibitor have been reported from _Pseudomonas_ spp. [99, 100, 11, 57]. Like arthrofactin, amphisin is involved in swarming motility of _Pseudomonas_ sp. DSS73 [99].  
Biosynthesis of Arthrofactin is catalyzed by the arthrofactin synthetase (Arf), which consists of three NRPS protein subunits, ArfA (234 kDa), ArfB (474 kDa), and ArfC (648 kDa), which contain two, four, and five functional modules, respectively (Figure 2) [98]. An additional C-domain was identified in the first module of ArfA, suggesting that the first amino acid could be initially acylated with a fatty acid. Site-directed mutagenesis changing the histidine residue of conserved core motif (HHXXXDG) to alanine impairs arthrofactin production [100]. This result suggested that the first C-domain is essential for biosynthesis of lipopeptide. Indeed, the \(\beta\)-hydroxydecanoyl thioester may be coupled to the activated leucine by the action of this C-domain to yield \(\beta\)-hydroxydecanoyl-L-Leu as the initial intermediate. A phylogenetic tree showed that the first C-domain of Arf belongs to _N_-acyl groups that use fatty acyl-CoA as their starter unit [70]. Although seven of the 11 amino acid residues in arthrofactin are in the d-form, Arf contains no E-domains, as found in syringomycin and syringopeptin [98]. The A-domain of d-Leu\({}_{1}\) specifically recognizes only l-Leu _in vitro_. Based on these observations, we initially hypothesized that an external racemase may be responsible for incorporation of the d-amino acids in arthrofactin. Different amino acid sequences downstream of a conserved core motif [FFELGGHSLLA(V/M)] in the T-domains were expected to reflect the recognition by external racemase. However, [PERSON] and coworkers later demonstrated that Arf contains unique dual C/E domains, which contribute to the conversion of l-amino acids to the d-form [89]. This novel C/E domain is cryptically embedded with the C-domain located downstream of the d-amino acid-incorporating modules. Dual C/E domains can be recognized by an elongated His motif (HHI/LXXXXGD). This feature was also identified in the Syr and Syp synthetases. Another unique characteristic of Arf is the presence of _C_-terminal tandem Te-domains like syringopeptin. By site-directed mutagenesis, the first Te-domain (ArfC-Te1) was shown to be essential for the completion of macrocyclization and the release of the final product. The second Te-domain (ArfC-Te2) was suggested to be involved in the evolution of Arf to improve the macrocyclization efficiency [101]. Moreover, we found that the gene encoding putative ArfA/B/C exists in the genome sequence of _Pseudomonas fluorescens_ Pf0-1 (YP_347943/YP_347944/YP_347945) [102]. Arf represents a novel NRPS architecture that features tandem Te-domains and dual C/E domains. Interestingly, another type of NRPS involved in biosynthesis of a siderophore, pyoverdine, was also identified in arthrofactin-producing _Pseudomonas_ sp. MIS38. A gene encoding NRPS for the chromophore part of pyoverdine contains a conventional E-domain [102]. This observation suggests that different NRPS systems with dual C/E domains and a conventional E-domain are both functional in _Pseudomonas_ spp.  
Figure 2: The arthrofactin biosynthesis assembly line.