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. K s app values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and r max app was about 3 × 10−8 mmol 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.
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Acknowledgments
We gratefully thank Dr. Xiaoyng Bian, Daniel Sauer and Prof. Rolf Müller from Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) for supplying the strain E. coli Nissle 1917 plu3263 and the purified luminmide A and B standards used in this study as well as for analytical support. We thank Prof. Rolf Müller for reading and commenting the manuscript.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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This study was funded by the BMBF (Federal Ministry of Education and Research) Project MECAT, FKZ 031P7238 within the initiative, Biotechnologie 2020+: Basistechnologien für eine nächste Generation biotechnologischer Verfahren”.
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The authors declare that they have no conflict of interest.
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Weyler, C., Heinzle, E. Synthesis of natural variants and synthetic derivatives of the cyclic nonribosomal peptide luminmide in permeabilized E. coli Nissle and product formation kinetics. Appl Microbiol Biotechnol 101, 131–138 (2017). https://doi.org/10.1007/s00253-016-7770-7
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DOI: https://doi.org/10.1007/s00253-016-7770-7