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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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-01-01
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| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/13/1/60 |
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| Summary: | 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. |
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| ISSN: | 2076-2607 |