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Institute: search.filters.UBSInstitut.Institut für BioverfahrenstechnikAuthor: search.filters.author.Sprenger, Georg A.

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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.
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    Synthetic co-culture in an interconnected two-compartment bioreactor system : violacein production with recombinant E. coli strains
    (2024) Müller, Tobias; Schick, Simon; Klemp, Jan-Simon; Sprenger, Georg A.; Takors, Ralf
    The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such ‘one pot’ approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli - E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h) -1 at 25 °C which holds great promise for future applications.