Browsing by Person "Tadele, Lea Rahel"

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    High‐level recombinant protein production with Corynebacterium glutamicum using acetate as carbon source
    (2022) Kiefer, Dirk; Tadele, Lea Rahel; Lilge, Lars; Henkel, Marius; Hausmann, Rudolf
    In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate‐utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3‐fold higher fluorescence level for acetate‐based eYFP production with T7 expression strain MB001(DE3) pMKEx2‐eyfp compared to MB001 pEKEx2‐eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass‐specific production for T7 RNAP‐dependent eYFP production using acetate as carbon source. Furthermore, high‐level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH‐coupled online feeding control, resulting in a final protein titer of 2.7 g/L and product yield of 4 g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low‐cost acetate into high‐value bioproducts, such as recombinant proteins.
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    Recombinant production of bovine αS1-casein in genome-reduced Bacillus subtilis strain IIG-Bs-20-5-1
    (2025) Biermann, Lennart; Tadele, Lea Rahel; Benatto Perino, Elvio Henrique; Nicholson, Reed; Lilge, Lars; Hausmann, Rudolf; Biermann, Lennart; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Tadele, Lea Rahel; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Benatto Perino, Elvio Henrique; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Nicholson, Reed; Motif FoodWorks, Inc., 27 Drydock Ave, Boston, MA 02210, USA;; Lilge, Lars; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Hausmann, Rudolf; Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany; (L.B.); (L.R.T.); (E.H.B.P.); (R.H.); Fouillaud, Mireille
    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 Bacillus subtilis was applied for the recombinant production of bovine αS1-casein protein. Methods: A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine αS1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant αS1-casein production. After increasing the molecular abundance of the recombinant αS1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine αS1-casein. Results: Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant αS1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis 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 mgcasein/gCDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native αS1-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of B. subtilis for the recombinant production of bovine αS1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.