title: Synthesis of natural variants and synthetic derivatives of the cyclic nonribosomal peptide luminmide in permeabilized E. coli Nissle and product formation kinetics.
abstract: We used a recombinant, permeabilized E. coli Nissle strain harbouring the plu3263 gene cluster from Photorhabdus luminescens for the synthesis of luminmide type cyclic pentapeptides belonging to the class of nonribosomally biosynthesized peptides (NRP). Cells could be fully permeabilized using 1 % v/v toluene. Synthesis of luminmides was increased fivefold when 0.3 mM EDTA was added to the substrate mixture acting as an inhibitor of metal proteases. Luminmide formation was studied applying different amino acid concentrations. Apparent kinetic parameters for the synthesis of the main product luminmide A from leucine, phenylalanine and valine were calculated from the collected data. Ksappvalues ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and rmaxappwas about 3 × 10-8mmol min-1(g CDW)-1). By removing phenylalanine from the substrate mixture, the formation of luminmide A was reduced tenfold while luminmide B was increased from 50 to 500 μg/l becoming the main product. Two new luminmides were synthesized in this study. Luminmide H incorporates tryptophan replacing phenylalanine in luminmide A. In luminmide I, leucine was replaced with 4,5-dehydro-leucine, a non-proteinogenic amino acid fed to the incubation mixture. Our study shows new opportunities for increasing the spectrum of luminmide variants produced, for improving production selectivity and for kinetic in vitro studies of the megasynthetases.
authors.affiliation: Biochemical Engineering Institute, Saarland University, D-66123, Saarbrucken, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Weyler
authors.affiliation: Biochemical Engineering Institute, Saarland University, D-66123, Saarbrucken, Germany. e.heinzle@mx.uni-saarland.de.
authors.forename: Elmar
authors.identifier: http://orcid.org/0000-0002-1689-9300
authors.initials: E
authors.lastname: Heinzle
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D005810:Multigene Family; D010456:Peptides, Cyclic / Q000235:genetics / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D020637:Photorhabdus / Q000235:genetics; D014050:Toluene
chemical_list: D010456:Peptides, Cyclic; D014050:Toluene
keywords: Biocatalysis; E. coli Nissle; Luminmide; Non-ribosomal peptide; Permeabilization; Permeabilized cells