Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping

Abstract

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

“…The resulting chimera, EntF-SyrE-A1, exhibited a 30-fold loss of activity compared with wild-type EntF as measured by a previously described in vitro assay of enterobactin production. This loss of activity is consistent with previous reports of significantly impaired chimeric NRPS domains (9,10).…”

“…The modular organization of protein domains in NRPSs has stimulated efforts to swap genes encoding heterologous domains into NRPSs with the goal of producing nonnatural variants of the original small-molecule product (3,4). Although limited success has been achieved with the production of nonribosomal peptide (NRP) variants (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), the resultant chimeric NRPSs are usually nonfunctional or heavily impaired, often yielding too little of the new product to make production feasible. It is not yet known why chimeric NRPSs suffer large reductions in activity, although it is likely that domain swapping disrupts quaternary interactions between protein domains in the assembly line (15).…”

Rapid Surface Motility in Bacillus subtilis Is Dependent on Extracellular Surfactin and Potassium Ion

“…The protocols used by Shirk et al (23) were followed. Primer sequences were obtained by using the published srfA-A sequences of Schneider et al (21). The forward primer 3Ј-srfA-BamHI (5Ј-CGCGGAT CCGTGAGCTTGATGTTGAAAGG-3Ј) and the reverse primer 3Ј-srfA-EcoRI (5Ј-GCGGAATTCGAGTACGAATG TAAGCCC-3Ј) were located within the 10,764-bp wild-type gene, generating a 1,040-bp DNA fragment.…”

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

“…Despite the production of the predicted surfactin derivatives, the productivity of the engineered synthetases was dramatically reduced, which could be explained by the high selectivity of C-domains in the acceptor site for cognate side chains (7). However, further attempts to obtain other surfactin variants by domain swapping were unsuccessful (103). This can most probably be ascribed to improper artificial fusion of domains, thus indicating that a more precise definition of the domain borders was necessary.…”