04 Fakultät Energie-, Verfahrens- und Biotechnik

Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5

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Institute: search.filters.UBSInstitut.Institut für MikrobiologieAuthor: search.filters.author.Takors, Ralf

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    Synthetic mutualism in engineered E. coli mutant strains as functional basis for microbial production consortia
    (2022) Müller, Tobias; Schick, Simon; Beck, Jonathan; Sprenger, Georg; Takors, Ralf
    In nature, microorganisms often reside in symbiotic co‐existence providing nutrition, stability, and protection for each partner by applying “division of labor.” This principle may also be used for the overproduction of targeted compounds in bioprocesses. It requires the engineering of a synthetic co‐culture with distributed tasks for each partner. Thereby, the competition on precursors, redox cofactors, and energy - which occurs in a single host - is prevented. Current applications often focus on unidirectional interactions, that is, the product of partner A is used for the completion of biosynthesis by partner B. Here, we present a synthetically engineered Escherichia coli co‐culture of two engineered mutant strains marked by the essential interaction of the partners which is achieved by implemented auxotrophies. The tryptophan auxotrophic strain E. coli ANT‐3, only requiring small amounts of the aromatic amino acid, provides the auxotrophic anthranilate for the tryptophan producer E. coli TRP‐3. The latter produces a surplus of tryptophan which is used to showcase the suitability of the co‐culture to access related products in future applications. Co‐culture characterization revealed that the microbial consortium is remarkably functionally stable for a broad range of inoculation ratios. The range of robust and functional interaction may even be extended by proper glucose feeding which was shown in a two‐compartment bioreactor setting with filtrate exchange. This system even enables the use of the co‐culture in a parallel two‐level temperature setting which opens the door to access temperature sensitive products via heterologous production in E. coli in a continuous manner.
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    Stability of a mutualistic Escherichia coli co‐culture during violacein production depends on the kind of carbon source
    (2024) Schick, Simon; Müller, Tobias; Takors, Ralf; Sprenger, Georg A.
    The L‐tryptophan-derived purple pigment violacein (VIO) is produced in recombinant bacteria and studied for its versatile applications. Microbial synthetic co‐cultures are gaining more importance as efficient factories for synthesizing high‐value compounds. In this work, a mutualistic and cross‐feeding Escherichia coli co‐culture is metabolically engineered to produce VIO. The strains are genetically modified by auxotrophies in the tryptophan (TRP) pathway to enable a metabolic division of labor. Therein, one strain produces anthranilate (ANT) and the other transforms it into TRP and further to VIO. Population dynamics and stability depend on the choice of carbon source, impacting the presence and thus exchange of metabolites as well as overall VIO productivity. Four carbon sources (D‐glucose, glycerol, D‐galactose, and D‐xylose) were compared. D‐Xylose led to co‐cultures which showed stable growth and VIO production, ANT‐TRP exchange, and enhanced VIO production. Best titers were ∼126 mg L -1 in shake flasks. The study demonstrates the importance and advantages of a mutualistic approach in VIO synthesis and highlights the carbon source's role in co‐culture stability and productivity. Transferring this knowledge into an up‐scaled bioreactor system has great potential in improving the overall VIO production.
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    Polyphosphate kinases phosphorylate thiamine phosphates
    (2022) Hildenbrand, Jennie C.; Sprenger, Georg A.; Teleki, Attila; Takors, Ralf; Jendrossek, Dieter
    Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high-performance liquid chromatography tandem mass spectrometry. Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli ) was confirmed in a coupled enzyme assay. Our study shows that PPKs are promiscuous enzymes in vitro that could be involved in the formation of a variety of phosphorylated metabolites in vivo.