Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1
Background: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous...
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2025-01-01
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| author | Lennart Biermann Lea Rahel Tadele Elvio Henrique Benatto Perino Reed Nicholson Lars Lilge Rudolf Hausmann |
| author_facet | Lennart Biermann Lea Rahel Tadele Elvio Henrique Benatto Perino Reed Nicholson Lars Lilge Rudolf Hausmann |
| author_sort | Lennart Biermann |
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| description | Background: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced <i>Bacillus subtilis</i> was applied for the recombinant production of bovine α<sub>S1</sub>-casein protein. Methods: A plasmid-based gene expression system was established in <i>B. subtilis</i> allowing the production of his-tagged codon-optimized bovine α<sub>S1</sub>-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α<sub>S1</sub>-casein production. After increasing the molecular abundance of the recombinant α<sub>S1</sub>-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α<sub>S1</sub>-casein. Results: Non-sporulating <i>B. subtilis</i> strain BMV9 and genome-reduced <i>B. subtilis</i> strain IIG-Bs-20-5-1 were applied for recombinant α<sub>S1</sub>-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced <i>B. subtilis</i> strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg<sub>casein</sub>/g<sub>CDW</sub> after reducing the <i>B. subtilis</i> protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α<sub>S1</sub>-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of <i>B. subtilis</i> for the recombinant production of bovine α<sub>S1</sub>-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins. |
| format | Article |
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| institution | Kabale University |
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| spelling | doaj-art-2ae3059a9638492eaa02f87048c0cfd82025-01-24T13:42:28ZengMDPI AGMicroorganisms2076-26072025-01-011316010.3390/microorganisms13010060Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1Lennart Biermann0Lea Rahel Tadele1Elvio Henrique Benatto Perino2Reed Nicholson3Lars Lilge4Rudolf Hausmann5Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, GermanyInstitute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, GermanyInstitute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, GermanyMotif FoodWorks, Inc., 27 Drydock Ave, Boston, MA 02210, USAInstitute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, GermanyInstitute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, GermanyBackground: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced <i>Bacillus subtilis</i> was applied for the recombinant production of bovine α<sub>S1</sub>-casein protein. Methods: A plasmid-based gene expression system was established in <i>B. subtilis</i> allowing the production of his-tagged codon-optimized bovine α<sub>S1</sub>-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α<sub>S1</sub>-casein production. After increasing the molecular abundance of the recombinant α<sub>S1</sub>-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α<sub>S1</sub>-casein. Results: Non-sporulating <i>B. subtilis</i> strain BMV9 and genome-reduced <i>B. subtilis</i> strain IIG-Bs-20-5-1 were applied for recombinant α<sub>S1</sub>-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced <i>B. subtilis</i> strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg<sub>casein</sub>/g<sub>CDW</sub> after reducing the <i>B. subtilis</i> protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α<sub>S1</sub>-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of <i>B. subtilis</i> for the recombinant production of bovine α<sub>S1</sub>-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.https://www.mdpi.com/2076-2607/13/1/60<i>Bacillus subtilis</i>genome reductionalternative protein sourcesα<sub>S1</sub>-caseinmicrobial expression systemsfood proteins |
| spellingShingle | Lennart Biermann Lea Rahel Tadele Elvio Henrique Benatto Perino Reed Nicholson Lars Lilge Rudolf Hausmann Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 Microorganisms <i>Bacillus subtilis</i> genome reduction alternative protein sources α<sub>S1</sub>-casein microbial expression systems food proteins |
| title | Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 |
| title_full | Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 |
| title_fullStr | Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 |
| title_full_unstemmed | Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 |
| title_short | Recombinant Production of Bovine α<sub>S1</sub>-Casein in Genome-Reduced <i>Bacillus subtilis</i> Strain IIG-Bs-20-5-1 |
| title_sort | recombinant production of bovine α sub s1 sub casein in genome reduced i bacillus subtilis i strain iig bs 20 5 1 |
| topic | <i>Bacillus subtilis</i> genome reduction alternative protein sources α<sub>S1</sub>-casein microbial expression systems food proteins |
| url | https://www.mdpi.com/2076-2607/13/1/60 |
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