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Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

ABSTRACT: Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up- or down-regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine-1-carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three-fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.

SUBMITTER: Bitzenhofer NL

PROVIDER: S-EPMC10832525 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

Bitzenhofer Nora Lisa NL Höfel Carolin C Thies Stephan S Weiler Andrea Jeanette AJ Eberlein Christian C Heipieper Hermann J HJ Batra-Safferling Renu R Sundermeyer Pia P Heidler Thomas T Sachse Carsten C Busche Tobias T Kalinowski Jörn J Belthle Thomke T Drepper Thomas T Jaeger Karl-Erich KE Loeschcke Anita A

Microbial biotechnology 20230712 1

Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the r ...[more]

PMID: 37435812

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Project description:BackgroundIn 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.ResultsIn 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.ConclusionsUtilizing 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.

Dataset Information

Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

Publications

Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

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