• M M Yakimov
    Department of Microbiology, GBF National Research Center for Biotechnology, Braunschweig, Germany. Michail_Yakimov@sifi.it
  • L Giuliano
  • K N Timmis
  • P N Golyshin

Peptide synthetases are multi-domain proteins that catalyze the assembly, from amino acids and amino acid derivatives, of peptides and lipopeptides, some of which exhibit activities (pharmaceutical, surfactant, etc.) of considerable biotechnological importance. Although there is substantial interest in the generation of greater peptide diversity, in order to create new biotechnologically interesting products, attempts reported so far to exchange amino acid-activating minimal modules between enzymes have only yielded hybrid catalysts with poor activities. We report here the replacement of an entire first, L-Glu-, and fifth, L-Asp-incorporating modules of surfactin synthetase, to create a fully active hybrid enzyme that forms a novel peptide in high yields. Whole encoding regions of lichenysin A synthetase modules were introduced into surfactin biosynthesis operon between His140/His1185 of SrfAA and His1183/His2226 of SrfAB, the amino acid residues of a proposed active-site motif (HHXXXDG) of the condensation domains which is involved in the catalysis of nonribosomal peptide bond formation (Stachelhaus et al., 1998). When the lipopeptides produced by the recombinant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical by their fatty acid profiles. We thereby demonstrate the utility of whole module swapping for designing novel peptides, for creating peptide diversity, and for redesigning existing peptides produced in performant production strains in high yields to correspond to desired peptides produced in low yields, or from strains unsuitable for production purposes.

Keywords

  • Amino Acid Sequence, Bacillus cereus/genetics, Bacterial Proteins/biosynthesis, Base Sequence, Biotechnology, DNA Primers/genetics, Ligases/genetics, Lipopeptides, Lipoproteins/biosynthesis, Peptide Synthases/genetics, Peptides, Cyclic/biosynthesis, Recombinant Proteins/biosynthesis, Surface-Active Agents/chemistry
Original languageEnglish
Pages (from-to)217-24
Number of pages8
JournalJournal of Molecular Microbiology and Biotechnology
Volume2
Issue number2
Publication statusPublished - Apr 2000
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