Fakultät Naturwissenschaften

Permanent URI for this communityhttps://hohpublica.uni-hohenheim.de/handle/123456789/1

Biologie, Ernährungs-wissenschaften und Lebensmittelwissenschaften sind die Schwerpunkte der Fakultät. Die Forschung befasst sich mit Schlüsselthemen der Life Sciences.

Homepage: https://natur.uni-hohenheim.de/

Browse

Now showing 1 - 3 of 3

Evaluation of an oxygen‐dependent self‐inducible surfactin synthesis in B. subtilis by substitution of native promoter PsrfA by anaerobically active PnarG and PnasD
(2021) Hoffmann, Mareen; Braig, Alina; Fernandez Cano Luna, Diana Stephanie; Rief, Katharina; Becker, Philipp; Treinen, Chantal; Klausmann, Peter; Morabbi Heravi, Kambiz; Henkel, Marius; Lilge, Lars; Hausmann, Rudolf
A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis, the native promoter PsrfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters PnarG and PnasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfA expression. As hypothesized, activity of PnarG and PnasD increased with lower oxygen levels and surfactin was not produced by PsrfA::PnarG as well as PsrfA::PnasD mutant strains under conditions with highest oxygen availability. PnarG showed expressions similar to PsrfA at lowest oxygen availability, while maximum value of PnasD was more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall YP/X of 1.007 g/g. This value also surpassed the YP/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus.
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.
Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations
(2021) Treinen, Chantal; Magosch, Olivia; Hoffmann, Mareen; Klausmann, Peter; Würtz, Berit; Pfannstiel, Jens; Morabbi Heravi, Kambiz; Lilge, Lars; Hausmann, Rudolf; Henkel, Marius
Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.

Browse

Browsing Fakultät Naturwissenschaften by Journal "AMB express"