Reprogramming Daptomycin and A54145 Biosynthesis to Produce Novel Lipopeptide Antibiotics

Combinatorial Biosynthesis of Cyclic Lipopeptide Antibiotics: A Model for Synthetic Biology To Accelerate the Evolution of Secondary Metabolite Biosynthetic Pathways

“…In addition to A domain specificity, other specificities are encoded in C, T, and Te domains. Three types of C domain were observed in the A21978C and A54145 gene clusters, based initially on phylogeneic analyses: (i) for coupling l -amino acids to l -amino acids (C I or L C L ); (ii) for coupling d -amino acids to l -amino acids (C II or D C L ), and (iii) for coupling fatty acids to l -amino acids (C III or F C L ). ,, There are no special C domains for coupling l -amino acids to d -amino acids because the epimerization of l -amino acids follows peptide coupling. − …”

“…The three differences are (i) dptE and dptF have a fused counterpart lptEF encoding AL and ACP or AL-ACP, respectively; (ii) the dptBC gene has split gene counterparts lptB and lptC that have been successfully fused in genetic engineering experiments; and (iii) lptA module 5 has an M domain to incorporate Sar that can be deleted to incorporate Gly . Table shows which parts of the coupling devices have been successfully engineered, and Table shows some of the specific changes and product yields from strains generated by combinatorial biosynthesis. ,,,,− Ten of the 17 devices have been engineered to give novel lipopeptides. Nine functional parts were validated in these studies.…”

“…By using this approach, the individual NRPS (and other genes) could be engineered separately and then reintroduced into the expression hosts combinatorially. To accomplish this, (i) the A21978C and A54145 gene clusters were cloned in BAC vectors that could be engineered in Escherichia coli by λ-Red-mediated recombination; (ii) sets of mutants deleted for one or more A21978C and A54145 biosynthetic genes were generated; (iii) vectors were developed for genetic manipulation in E. coli , followed by conjugal transfer into S. roseosporus and S. fradiae expression hosts and site-specific integration into bacteriophage ϕC31 of ϕBT1 attB sites or into the IS 117 att site; (iv) engineered genes were expressed under the control of the strong, constitutive ermE * promoter; (v) fermentations were carried out, and novel compounds were confirmed by isolation and mass spectrometry and evaluated for microbiological activities. ,,,,,,− Reconstruction experiments confirmed that the NRPS genes could be expressed from the ermE* promoter at ectopic loci, giving lipopeptide yields comparable to controls. , This was significant because it demonstrated that constitutive expression of giant NRPS proteins during the early growth phase was not problematic, that the individual NRPS genes could be expressed from attB sites separated from the main NRPS clusters by multiple megabases, and that the NRPS multi-enzymes can assemble efficiently without sequencial translation from a single mRNA in a cellular subspace for antibiotic assembly.…”

“…Three types of C domain were observed in the A21978C and A54145 gene clusters, based initially on phylogeneic analyses: (i) for coupling L-amino acids to L-amino acids (C I or L C L ); (ii) for coupling D-amino acids to L-amino acids (C II or D C L ), and (iii) for coupling fatty acids to L-amino acids (C III or F C L ). 11,12,35 There are no special C domains for coupling L-amino acids to D-amino acids because the epimerization of L-amino acids follows peptide coupling. 36−38 There are also three types of T domain implied from biochemical studies 31,32,39−41 and supported by BLASTP analyses (R. H. Baltz, unpublished).…”