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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
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.
Model-based process design for surfactin production with Bacillus subtilis
(2025) Hiller, Eric; Off, Manuel; Dittmann, Holger; Perino, Elvio Henrique Benatto; Lilge, Lars; Hausmann, Rudolf; Hiller, Eric; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Off, Manuel; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Dittmann, Holger; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Perino, Elvio Henrique Benatto; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany
Bacillus subtilis is one of the most important production organisms in industrial biotechnology. However, there is still limited knowledge about the kinetics of fed-batch processes in bioreactors, as well as a lack of biological performance indicators, such as production yields, particularly regarding their variation over time. Understanding these kinetics and changes is crucial for optimizing the productivity in fed-batch processes. Fed-batch bioreactor cultures of Bacillus subtilis BMV9 in high cell density processes for surfactin production have been characterized with a kinetic model composed of first-order ordinary differential equations, describing the time course of biomass, substrate, surfactin and acetate. This model contributes to understanding critical restrictions and the knowledge gained was used to design and implement a model-based process. The model integrates biomass growth based on Monod kinetics, substrate consumption, surfactin synthesis and formation of the by-product acetate. After the model was parameterized for B. subtilis BMV9 using 12 different fed-batch bioreactor experiments, the kinetic model was able to accurately describe biomass accumulation, substrate consumption, product formation rates and, to some extent, the overflow metabolism involving acetate. Based on this, the kinetic model was used for a process design, in which the batch was omitted, which led to a product titre of 46.33 g/L and a space–time-yield of 2.11 g/(L*h) was achieved. The kinetic model developed in this study enables the description of the time course of biomass growth, substrate consumption and product formation and thus significantly improves process understanding. The computation of process parameters, which are not analytically accessible at any time, could be realized. A sensitivity analysis identified the maximum specific growth rate, substrate-related maintenance and the maximum acetate formation rate as key parameters influencing model outputs.
Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii
(2025) Bechtel, Anna; Kettner, Lucas; Hessenberger, Jan; Vlassakakis, Kenny; Fischer, Lutz; Bechtel, Anna; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Kettner, Lucas; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Hessenberger, Jan; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Vlassakakis, Kenny; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany; Fischer, Lutz; Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599, Stuttgart, Germany
The diamine oxidase from Glutamicibacter halophytocola (DAO-GH) was recombinantly produced in K. phaffii using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter for methanol-free production. Firstly, K. phaffii clones were generated for intracellular and secretory DAO-GH production that still possessed antibiotic resistance due to the cloning procedure. For intracellular production, a maximum intracellular DAO activity of 15,404 nkat/Lculture was achieved in fed-batch bioreactor cultivations, while for secretory production, the highest extracellular DAO activity of 6,078 nkat/Lculture was achieved using the αMF signal peptide without its EAEA sequence. The intracellularly produced DAO-GH was partially purified in several purification steps with a yield of 80%, a purification factor of about 10 and specific DAO activity of 16.7 nkat/mgprotein. The secretory DAO-GH production resulted in a specific DAO activity of 15.4 nkat/mgprotein already in the cell-free culture supernatant at the end of cultivation without further purification steps. The food industry aims to avoid the use of antimicrobial resistance in enzyme production, therefore, a new cassette plasmid with self-excisable antibiotic resistance markers was constructed for secretory DAO-GH production. The antibiotic-resistance-free K. phaffii clone generated with this plasmid achieved a maximum extracellular DAO activity of 4,770 nkat/Lculture in a fed-batch bioreactor cultivation. The DAO-GH obtained in this cultivation was spray-dried, resulting in a storable powder with 23 nkat/gpowder DAO activity and a water activity value of 0.12. This study demonstrated the secretion of recombinant DAO in a microbial host such as K. phaffii for the first time and provides a strategy for generating antibiotic-resistance-free K. phaffii clones.

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