Genetic Engineering of Acidic Lipopeptide Antibiotics

The term combinatorial biosynthesis embraces a broad set of methodologies—including genetic engineering, the use of mutants blocked in specific biosynthetic steps, and the exploitation of natural variations in substrates and feeding unnatural substrates—to expand the numbers of compounds generated beyond what can be achieved by genetic engineering alone. For relatively small antibiotic gene clusters, it is possible to isolate complete biosynthetic gene clusters on individual cosmids. Another approach is to clone antibiotic gene clusters in bacterial artificial chromosome (BAC) vectors, which can accommodate DNA inserts of >100 kb. The methods discussed in this chapter include some that are specific to nonribosomal peptide synthetases (NRPSs) engineering, including (i) splicing at intermodule or interpeptide linker sites; (ii) recognizing and exploiting the correct type of C domain when coupling fatty acids to L-amino acids (FCL), D-amino acids to L-amino acids (DCL), or L-amino acids to L-amino acids (LCL); and (iii) maintaining the integrity of T-TE didomains when engineering terminal modules. The chapter points out that both S. roseosporus and S. fradiae can be readily manipulated by the genetic engineering methods described. As such, they may be useful hosts for the expression and engineering of other secondary metabolic pathways, particularly other NRPS pathways. For example, the A54145 gene cluster was expressed successfully in an S. roseosporus strain deleted for daptomycin biosynthetic genes.