Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain
dc.contributor.author | Hermann, Alexander | |
dc.contributor.author | Hiller, Eric | |
dc.contributor.author | Hubel, Philipp | |
dc.contributor.author | Biermann, Lennart | |
dc.contributor.author | Benatto Perino, Elvio Henrique | |
dc.contributor.author | Kuipers, Oscar Paul | |
dc.contributor.author | Hausmann, Rudolf | |
dc.contributor.author | Lilge, Lars | |
dc.contributor.corporate | 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.) | |
dc.contributor.corporate | 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.) | |
dc.contributor.corporate | Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, 70599 Stuttgart, Germany; | |
dc.contributor.corporate | 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.) | |
dc.contributor.corporate | 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.) | |
dc.contributor.corporate | Kuipers, Oscar Paul; Department of Molecular Genetics, University of Groningen, 9747 AG Groningen, The Netherlands; | |
dc.contributor.corporate | 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.) | |
dc.contributor.corporate | 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.) | |
dc.contributor.editor | Fouillaud, Mireille | |
dc.date.accessioned | 2025-07-31T09:09:49Z | |
dc.date.available | 2025-07-31T09:09:49Z | |
dc.date.issued | 2025 | |
dc.date.updated | 2025-03-12T11:59:49Z | |
dc.description.abstract | 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. | |
dc.description.sponsorship | This work was funded by the German Research Foundation (DFG) with the grant numbers 501222601 and 501222797 and by the Federal Ministry of Education and Research (BMBF), within the frame of the “Biosurfactants Alliance: Function-optimized biosurfactants based on regionally available raw materials using optimized biotechnological processes” (funding code: 031B1059F). The Exploris 480 mass spectrometer was funded in part by the German Research Foundation (DFG-INST 36/171-1 FUGG). | |
dc.description.sponsorship | German Research Foundation (DFG) | |
dc.description.sponsorship | Federal Ministry of Education and Research (BMBF) | |
dc.description.sponsorship | German Research Foundation | |
dc.identifier.uri | https://doi.org/10.3390/microorganisms13020353 | |
dc.identifier.uri | https://hohpublica.uni-hohenheim.de/handle/123456789/17441 | |
dc.language.iso | eng | |
dc.rights.license | cc_by | |
dc.subject | Bacillus subtilis | |
dc.subject | Surfactin | |
dc.subject | Biosurfactants | |
dc.subject | Genetic code expansion | |
dc.subject | Non-canonical amino acid | |
dc.subject | Bioprocess engineering | |
dc.subject | Bioreactor | |
dc.subject | Molecular adaptation | |
dc.subject | Proteomics | |
dc.subject.ddc | 660 | |
dc.title | Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain | en |
dc.type.dini | Article | |
dcterms.bibliographicCitation | Microorganisms, 13 (2025-02-06), 2. https://doi.org/10.3390/microorganisms13020353. ISSN: 2076-2607 | |
dcterms.bibliographicCitation.issn | 2076-2607 | |
dcterms.bibliographicCitation.issue | 2 | |
dcterms.bibliographicCitation.journaltitle | Microorganisms | |
dcterms.bibliographicCitation.originalpublishername | MDPI | |
dcterms.bibliographicCitation.originalpublisherplace | Basel | |
dcterms.bibliographicCitation.volume | 13 | |
local.export.bibtex | @article{Hermann2025-02-06, doi = {10.3390/microorganisms13020353}, author = {Hermann, Alexander and Hiller, Eric and Hubel, Philipp et al.}, title = {Genetic Code Expansion for Controlled Surfactin Production in a High Cell-Density Bacillus subtilis Strain}, journal = {Microorganisms}, year = {2025-02-06}, volume = {13}, number = {2}, } | |
local.title.full | Genetic Code Expansion for Controlled Surfactin Production in a High Cell-Density Bacillus subtilis Strain |
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