Browsing by Person "Senger, Jana"

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Importance of the 5’ untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007
(2025) Senger, Jana; Schulz, Adriana; Seitl, Ines; Heider, Martin; Fischer, Lutz; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Schulz, Adriana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Seitl, Ines; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Heider, Martin; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany
The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the PaprE and P43 promoters and the corresponding 5’ untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii. Expression controlled by the PaprE promoter yielded a significantly higher activity of 2515 µkat/L, compared to 56 µkat/L with the P43 promoter. Modifications on the PaprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve β-galactosidase production. Since the aprE 5’ untranslated region contributes to a high mRNA stability, it was incorporated into the P43 construct to determine whether mRNA stability is responsible for the differences observed in β-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher β-galactosidase production of 2756 µkat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the β-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.
Investigating different Bacillus subtilis expression systems for recombinant enzyme production
(2025) Senger, Jana; Fischer, Lutz
Enzymes play an indispensable role in the food industry by improving texture, color, shelf life, or nutritional value of food products. A prerequisite for the application of food enzymes is their high-yield and cost-effective production in a suitable host. The Gram-positive bacterium Bacillus subtilis is a promising host due to the organism's qualified presumption of safety status, its genetic accessibility, and short cultivation times. In addition, B. subtilis can efficiently secrete heterologous enzymes into the extracellular medium, which simplifies downstream processing. This thesis explored different B. subtilis expression systems for the recombinant production and secretion of the β-galactosidase (EC 3.2.1.23) from Paenibacillus wynnii (β-gal-Pw) and the protein-glutamine glutaminase (EC 3.5.1.44) from Bacteroides helcogenes (PGB). Both enzymes have a potential application in the food industry. The β-gal-Pw offers favorable kinetic properties for application in lactose-depleted dairy products. The PGB is used to modify the techno-functional properties of proteins, thereby improving their application in food products. The first study investigated the secretion of the cytoplasmic 120 kDa β-gal-Pw using B. subtilis SCK6. Different expression conditions were tested to find proficient conditions for secretion. Codon-optimization of the native β-gal-Pw gene and cultivation temperature reduction from 37°C to 30°C increased secretory β-gal-Pw production. Furthermore, two promoters and four signal peptides were tested in multiple combinations. Signal peptides of the Sec-pathway and the Tat-pathway enabled efficient secretion, which, however, depended on the specific combination of promoter and signal peptide used. The highest extracellular activity of 55 µkat/Lculture was achieved with the PaprE promoter and the Tat-signal peptide PhoD in shake flask cultivations. The subsequent bioreactor cultivation further improved secretory β-gal-Pw production by 1.4-fold and resulted in 21 mg/Lculture purified β-gal-Pw. The second study explored the intracellular β-gal-Pw production in the undomesticated strain B. subtilis 007. Great differences in production were observed by testing the PaprE and P43 promoter with each corresponding 5’ untranslated region (5’UTR). The use of the PaprE promoter led to an intracellular β-gal-Pw activity of 2515 µkat/Lculture, which was 45-fold higher compared to the P43 promoter. Further modification of the core PaprE promoter or the spacer sequence in the 5’UTR did not improve β-gal-Pw production. The 5’UTR in the P43 construct was replaced with the aprE 5’UTR, which significantly improved mRNA stability. In addition, β-gal-Pw production was enhanced from 56 µkat/Lculture to 2756 µkat/Lculture. The crucial role of the 5’UTR and the corresponding mRNA stability was subsequently confirmed by producing the β-glucosidase from Pyrococcus furiosus and the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus in B. subtilis 007. The third study focused on B. subtilis 007 for secretory and antibiotic-free PGB production. The genome of the undomesticated B. subtilis 007 was sequenced and provided the basis for multiple genomic integrations of the PGB expression cassette via CRISPR/Cas9. By selecting the specific integration sites, genes were simultaneously deleted to optimize the production strain and process. Four genes were targeted for the elimination of sporulation (sigF), foaming (sfp), motility (flgE), and α-amylase production (amyE). The first PGB expression cassette was integrated into sigF, which resulted in the expected asporogenic strain. An extracellular PGB activity of 4.1 µkat/Lculture was reached in bioreactor cultivations. The second expression cassette was integrated into sfp, which reduced foaming and increased the secretory PGB production by 1.3-fold. Since integration into the flgE locus did not enhance PGB production, the third PGB expression cassette was inserted into the amyE locus. The extracellular PGB activity of the respective strain was significantly increased from 5.4 µkat/Lculture to 9.5 µkat/Lculture.