Browsing by Subject "Microaerobic"

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Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440
(2025) Grether, Jakob; Dittmann, Holger; Willems, Leon; Schmiegelt, Tabea; Benatto Perino, Elvio Henrique; Hubel, Philipp; Lilge, Lars; Hausmann, Rudolf; Grether, Jakob; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Dittmann, Holger; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Willems, Leon; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Schmiegelt, Tabea; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Benatto Perino, Elvio Henrique; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hubel, Philipp; Core Facility Hohenheim, Mass Spectrometry Core Facility, University of Hohenheim, Ottilie-Zeller-Weg 2, 70599, Stuttgart, Germany; Lilge, Lars; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany; Hausmann, Rudolf; Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599, Stuttgart, Germany
Background: In biomanufacturing of surface-active agents, such as rhamnolipids, excessive foaming is a significant obstacle for the development of high-performing bioprocesses. The exploitation of the inherent tolerance of Pseudomonas putida KT2440, an obligate aerobic bacterium, to microaerobic conditions has received little attention so far. Here low-oxygen inducible promoters were characterized in biosensor strains and exploited for process control under reduction of foam formation by low aeration and stirring rates during biosynthesis of rhamnolipids. Results: In this study, homologous promoters of P. putida inducible under oxygen limitation were identified by non-targeted proteomic analyses and characterized by fluorometric methods. Proteomics indicated a remodeling of the respiratory chain and the regulation of stress-related proteins under oxygen limitation. Of the three promoters tested in fluorescent biosensor assays, the promoter of the oxygen-sensitive cbb3-type cytochrome c oxidase gene showed high oxygen-dependent controllability. It was used to control the gene expression of a heterologous di-rhamnolipid synthesis operon in an auto-inducing microaerobic two-phase bioprocess. By limiting the oxygen supply via low aeration and stirring rates, the bioprocess was clearly divided into a growth and a production phase, and sources of foam formation were reduced. Accordingly, rhamnolipid synthesis did not have to be controlled externally, as the oxygen-sensitive promoter was autonomously activated as soon as the oxygen level reached microaerobic conditions. A critical threshold of about 20% oxygen saturation was determined. Conclusions: Utilizing the inherent tolerance of P. putida to microaerobic conditions in combination with the application of homologous, low-oxygen inducible promoters is a novel and efficient strategy to control bioprocesses. Fermentation under microaerobic conditions enabled the induction of rhamnolipid production by low oxygen levels, while foam formation was limited by low aeration and stirring rates.
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.