Fig. 2: Subdomain swap strategies.
From: Gene editing enables rapid engineering of complex antibiotic assembly lines
a The structure of a ligand-bound GrsA Phe-A domain (PDB: 1AMU)32. The flavodoxin-like subdomain (FSD) is highlighted in yellow and ligands phenylalanine and adenosine monophosphate (AMP) are shown in blue and red, respectively. Residues HKGISNLKVFFENSLNV which form an alpha helix have been removed for greater visibility of the ligands. b Alignment of the grsA Phe-A domain with the endA Thr[2]-A domain, with the secondary structural features indicated and nine of the ten amino acid substrate binding residues (except Lys517) marked with red asterisks20. The FSD sequence is highlighted in yellow and the black triangles show cut sites for the subdomain swap. c Single-step CRISPR-Cas9 strategy for exchanging subdomains at the native locus. sgRNA-guided Cas9 cleaves within the Thr[2] A domain of endA. DNA repair utilizes a plasmid-borne sequence containing endA possessing a Thr[2] to Ser[12] subdomain swap, resulting in seamless exchange of the Thr[2] FSD with a Ser[12] FSD at its native locus within the BGC. d Schematic for conventional multi step gene knockout and complementation strategy. The wild type endA is replaced with an apramycin resistance cassette. The resultant ∆endA strain is complemented with an integrative plasmid containing endA (Swap 1) where the subdomain sequence of the Thr[2] A domain is replaced with the subdomain from the Ser[12] A domain. Insertion of endA (Swap 1) occurs at the ΦC31 site outside of the enduracidin biosynthetic gene cluster (BGC).
