Core Facility Hohenheim

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Browsing Core Facility Hohenheim by Journal "Microorganisms"

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    Characterization of Bacillus velezensis UTB96, demonstrating improved lipopeptide production compared to the strain B. velezensis FZB42
    (2022) Vahidinasab, Maliheh; Adiek, Isabel; Hosseini, Behnoush; Akintayo, Stephen Olusanmi; Abrishamchi, Bahar; Pfannstiel, Jens; Henkel, Marius; Lilge, Lars; Vögele, Ralf ; Hausmann, Rudolf
    Bacillus strains can produce various lipopeptides, known for their antifungal properties. This makes them attractive metabolites for applications in agriculture. Therefore, identification of productive wild-type strains is essential for the development of biopesticides. Bacillus velezensis FZB42 is a well-established strain for biocontrol of plant pathogens in agriculture. Here, we characterized an alternative strain, B. velezensis UTB96, that can produce higher amounts of all three major lipopeptide families, namely surfactin, fengycin, and iturin. UTB96 produces iturin A. Furthermore, UTB96 showed superior antifungal activity towards the soybean fungal pathogen Diaporthe longicolla compared to FZB42. Moreover, the additional provision of different amino acids for lipopeptide production in UTB96 was investigated. Lysine and alanine had stimulatory effects on the production of all three lipopeptide families, while supplementation of leucine, valine and isoleucine decreased the lipopeptide bioproduction. Using a 45-litre bioreactor system for upscaling in batch culture, lipopeptide titers of about 140 mg/L surfactin, 620 mg/L iturin A, and 45 mg/L fengycin were achieved. In conclusion, it becomes clear that B. velezensis UTB96 is a promising strain for further research application in the field of agricultural biological controls of fungal diseases.
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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.
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    Surfactin shows relatively low antimicrobial activity against Bacillus subtilis and other bacterial model organisms in the absence of synergistic metabolites
    (2022) Lilge, Lars; Ersig, Nadine; Hubel, Philipp; Aschern, Moritz; Pillai, Evelina; Klausmann, Peter; Pfannstiel, Jens; Henkel, Marius; Morabbi Heravi, Kambiz; Hausmann, Rudolf
    Surfactin is described as a powerful biosurfactant and is natively produced by Bacillus subtilis in notable quantities. Among other industrially relevant characteristics, antimicrobial properties have been attributed to surfactin-producing Bacillus isolates. To investigate this property, stress approaches were carried out with biotechnologically established strains of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, the highest growth-reducing effects were detectable in B. subtilis and E. coli after surfactin treatment of 100 g/L with 35 and 33%, respectively, while P. putida showed no growth-specific response. In contrast, other antimicrobial biosurfactants, like rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. Since the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was analyzed by mass spectrometry, showing induction of stress proteins under control of alternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, although surfactin is associated with antimicrobial properties, relatively low growth-reducing effects could be demonstrated after the surfactin addition, challenging the general claim of the antimicrobial properties of surfactin.