Site-directed modification of the adenylation domain of the fusaricidin nonribosomal peptide synthetase for enhanced production of fusaricidin analogs

• DOI: 10.1007/s10529-012-0913-8
• Publication Date: 2012-03-26T11:21:01Z
• Authors: Jae Woo Han; Eun Young Kim; Jung Min Lee; Yun Sung Kim; Eunjung Bang; Beom Seok Kim
• Keywords: 0303 health sciences; 03 medical and health sciences; Amino Acid Substitution; Depsipeptides; Mutagenesis, Site-Directed; Mutant Proteins; Peptide Synthases; Paenibacillus; Protein Structure, Tertiary

Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit L: -Tyr, L: -Val, L: -Ile, L: -allo-Ile, or L: -Phe, which diversifies the fusaricidin structures. Since the L: -Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate L: -Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain.