## 5 Genetic Engineering of NRPS to Create Novel Products  
A large number of LPBSs are synthesized by NRPSs that share similar modular architecture of their repetitive catalytic units and a similar assembly-line mechanism. However, recent genome analysis of the lipopeptide synthetases from both _Bacillus_ and _Pseudomonas_ strains revealed novel NRPS architecture that does not completely conform to the co-linearity rule, which encompasses multifunctional hybrid enzymes that can directly incorporate D-amino acid residues. In addition, most of the recently identified lipopeptide synthetases in _Pseudomonas_ spp lack the cognate E-domains, but contain a dual C/E domain and also feature tandem Te-domains. These variations demonstrate natural versatility in evolving complex pathways for lipopeptide biosynthesis and may allow for artificial alteration of the protein template with the aim of reprogramming it to create novel compounds with improved properties. Figure 4: The model of gene regulation involved in lipopeptide biosynthesis by _Pseudomonas_. Closed-head arrows indicate positive regulation whereas closed circles indicate negative regulation. Recently, new strategies for engineering NRPSs have been developed. The first successful report examined exchanging or replacing the minimal module (A- and T-domains) with the last Leu\({}_{7}\) module within SrfA-C, a single-module with the simplest structure [14]. This module was deleted and replaced with several bacterial and fungal A/T-domains by homologous recombination. Construction of the gene encoding hybrid SrfA leads to the production of surfactin variants that retain their activity (Table 3). Using the same strategy, A/T-domain exchange was applied to multi-modular SrfA. Swapping of the Leu\({}_{2}\) module results in an altered product whose peptide chain is shorter than that of wild-type [154]. However, this minimal module replacement resulted in a very low yield of variants. Therefore, [PERSON] and coworkers (2000) developed a whole-module replacement within SrfA. A complete set of C/A/T-domains of the lichenysin A synthetase Gln\({}_{1}\) module was introduced into the Glu\({}_{1}\) module of SrfA. An altered product, surfactin [Gln\({}_{1}\)], was produced by the recombinant _B. subtilis_ at a level similar to that of wild-type surfactin and it also exhibits stronger surface activity than surfactin [155]. The SrfA could also be modified by translocation of the _C_-terminal Te-domain to the end of the internal domains, thus resulting in new linear surfactin analogs [156]. Furthermore, the use of recombinant Te-domain to catalyze regiospecific cyclization of synthetic peptides or lipopeptides _in vitro_ is a powerful approach for generating libraries of novel compounds with improved properties [157, 158]. Another powerful approach for the genetic manipulation of NRPS templates is the directed mutation of the substrate specificity within the A-domain based on its specificity-conferring code [159]. Specificity-conferring codes of the Asp\({}_{5}\) module within SrfA were adapted for the recognition of Asn. The engineered _B. subtilis_ produces the new lipoheptapeptide, surfactin [Asn\({}_{5}\)] [13]. Along the same line, directed mutation of the Asp\({}_{7}\) A-domain to Asn\({}_{7}\) within CDA synthetase from _Streptomyces coelicolar_ leads to production of the expected lipoundecapeptide containing Asn\({}_{7}\) and an unexpected linear lipohexapeptide intermediate [160]. A reduction or increase in the number of peptide residues is an alternate approach to generate structural diversity of lipopeptides and glycoptides. Deletion of an entire Leu\({}_{2}\) module in SrfA causes secretion of the predicted surfactin variant with a smaller cyclic peptide. Furthermore, a novel lipohexapeptide is produced with a significantly high yield and enhanced antimicrobial activities [161, 162]. Amino acid insertion by module extension has been performed within the vancomycin-type glycoptide antibiotic, balhimycin.