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Article
2025
Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain
Genetic code expansion for controlled surfactin production in a high cell-density Bacillus subtilis strain
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.
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Microorganisms, 13 (2025-02-06), 2.
https://doi.org/10.3390/microorganisms13020353.
ISSN: 2076-2607
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Hermann, A., Hiller, E., Hubel, P., Biermann, L., Benatto Perino, E. H., Kuipers, O. P., Hausmann, R., & Lilge, L. (2025). Genetic Code Expansion for Controlled Surfactin Production in a High Cell-Density Bacillus subtilis Strain. Microorganisms, 13(2). https://doi.org/10.3390/microorganisms13020353
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English
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660 Chemical engineerin
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University bibliography
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Sustainable Development Goals
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@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},
}