Browsing by Person "Senger, Jana"

Now showing 1 - 5 of 5

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
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.
Recombinant production of acidophilic L-arabinose isomerase from Lentilactobacillus parakefiri in Bacillus subtilis
(2025) Weber, Nathanael; Götz, Sebastian; Senger, Jana; Lutz-Wahl, Sabine; Fischer, Lutz; Weber, Nathanael; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Götz, Sebastian; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Lutz-Wahl, Sabine; 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
Background: The monosaccharide D-tagatose is a promising alternative to sucrose because of its similar sweetness and lower glycemic index. A novel L-arabinose isomerase (L-AI) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) has been biochemically characterized and used to isomerize D-galactose to D-tagatose in skim milk ultrafiltration permeate at pH 4.5 and 6.5. However, like most L-AIs described in the literature, this enzyme has only been produced recombinantly in Escherichia coli. This study aimed to systematically investigate the intracellular recombinant production of L-AI-Lp in Bacillus subtilis, which has qualified for a presumption of safety (QPS) designation from the European Food Safety Authority. Results: The influence of four promoters on L-AI-Lp production in B. subtilis 007 was investigated in shake flask cultivations. Among these, the PAprE promoter yielded the highest volumetric L-AI activity of 69.2 ± 7.4 µkatGal, 65 °C/LCulture. The production yield was further increased to 147.7 ± 1.0 µkatGal, 65 °C/LCulture by using the nonsporulating, surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF, which was constructed by deleting sigF and sfp in B. subtilis 007. Furthermore, the influence of pH and dissolved oxygen (DO) on bioreactor cultivations of B. subtilis 007 ∆sfp ∆sigF was analyzed. In bioreactor cultivations, the highest L-AI activity of 88.6 ± 2.4 µkatGal, 65 °C/LCulture was measured under unregulated pH and low oxygen conditions (DO ≤ 5%), representing a 3.2-fold increase compared with previous recombinant production in E. coli. The L-AI-Lp was subsequently partially purified by heat treatment and precipitation methods, resulting in a 7.8-fold increase in specific activity to 128.2 nkatGal, 65 °C/mg and a yield of 84%. Conclusions: The L-AI-Lp was recombinantly produced for the first time in a microbial species with QPS status using the nonsporulating and surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF. The L-AI-Lp was subsequently partially purified via nonchromatographic methods, providing a basis for a low-cost downstream process. These results represent an important step toward potential industrial application of L-AI-Lp and highlight the potential of B. subtilis 007 ∆sfp ∆sigF as an expression host for the recombinant production of L-AIs compared with previously used hosts from the order Lactobacillales.
Secretion of the cytoplasmic and high molecular weight β-galactosidase of Paenibacillus wynnii with Bacillus subtilis
(2024) Senger, Jana; Seitl, Ines; Pross, Eva; Fischer, Lutz
Background: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. Results: In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. Conclusion: For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.
Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain
(2026) Senger, Jana; Keutgen, Mario; Roth, Nicole; Seitl, Ines; Fischer, Lutz; Senger, Jana; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Keutgen, Mario; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany; Roth, Nicole; 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; Fischer, Lutz; Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Garbenstr 25, 70599, Stuttgart, Germany
Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.Key points• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.• Increased PGB production reaching 9.5 µkat/Lculture.