Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440
Abstract 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 microaer...
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2025-01-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-025-00478-z |
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| author | Jakob Grether Holger Dittmann Leon Willems Tabea Schmiegelt Elvio Henrique Benatto Perino Philipp Hubel Lars Lilge Rudolf Hausmann |
| author_facet | Jakob Grether Holger Dittmann Leon Willems Tabea Schmiegelt Elvio Henrique Benatto Perino Philipp Hubel Lars Lilge Rudolf Hausmann |
| author_sort | Jakob Grether |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-d864f3eab217417487c316d8cc6b3837 |
| institution | Kabale University |
| issn | 1754-1611 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
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| series | Journal of Biological Engineering |
| spelling | doaj-art-d864f3eab217417487c316d8cc6b38372025-01-19T12:26:32ZengBMCJournal of Biological Engineering1754-16112025-01-0119111510.1186/s13036-025-00478-zBioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440Jakob Grether0Holger Dittmann1Leon Willems2Tabea Schmiegelt3Elvio Henrique Benatto Perino4Philipp Hubel5Lars Lilge6Rudolf Hausmann7Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimCore Facility Hohenheim, Mass Spectrometry Core Facility, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimDepartment of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of HohenheimAbstract 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.https://doi.org/10.1186/s13036-025-00478-zPseudomonas putidaBiosensorMicroaerobicAuto-inductionBioprocess designRhamnolipid |
| spellingShingle | Jakob Grether Holger Dittmann Leon Willems Tabea Schmiegelt Elvio Henrique Benatto Perino Philipp Hubel Lars Lilge Rudolf Hausmann Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 Journal of Biological Engineering Pseudomonas putida Biosensor Microaerobic Auto-induction Bioprocess design Rhamnolipid |
| title | Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 |
| title_full | Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 |
| title_fullStr | Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 |
| title_full_unstemmed | Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 |
| title_short | Bioprocess exploitation of microaerobic auto-induction using the example of rhamnolipid biosynthesis in Pseudomonas putida KT2440 |
| title_sort | bioprocess exploitation of microaerobic auto induction using the example of rhamnolipid biosynthesis in pseudomonas putida kt2440 |
| topic | Pseudomonas putida Biosensor Microaerobic Auto-induction Bioprocess design Rhamnolipid |
| url | https://doi.org/10.1186/s13036-025-00478-z |
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