Browsing by Subject "Molecular adaptation"

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Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain
(2025) Hermann, Alexander; Hiller, Eric; Hubel, Philipp; Biermann, Lennart; Benatto Perino, Elvio Henrique; Kuipers, Oscar Paul; Hausmann, Rudolf; Lilge, Lars; Hermann, Alexander; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Hiller, Eric; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, 70599 Stuttgart, Germany;; Biermann, Lennart; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Kuipers, Oscar Paul; Department of Molecular Genetics, University of Groningen, 9747 AG Groningen, The Netherlands;; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany; (A.H.); (E.H.); (L.B.); (E.H.B.P.); (R.H.); Fouillaud, Mireille
Background: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis . This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved.