04 Fakultät Energie-, Verfahrens- und Biotechnik

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

Browse

Filters

Settings

Institute: search.filters.UBSInstitut.Institut für MikrobiologiePublication Type: search.filters.UBSTyp.Zeitschriftenartikel

Search Results

Now showing 1 - 10 of 28
  • Thumbnail Image
    ItemOpen Access
    A universal polyphosphate kinase : PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate
    (2020) Hildenbrand, Jennie C.; Teleki, Attila; Jendrossek, Dieter
    Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP.
  • Thumbnail Image
    ItemOpen Access
    Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250
    (1989) Engesser, Karl-Heinrich; Schulte, P.
    Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho-substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.
  • Thumbnail Image
    ItemOpen Access
    Polyphosphat : ein unterschätztes Molekül
    (2022) Jendrossek, Dieter; Hildenbrand, Jennie C.
    Polyphosphate (polyP) is an inorganic biopolymer ubiquitously present in all species. It has a variety of functions ranging from that of a reservoir for phosphorous in many microorganisms to functions in blood coagulation and plays a role in neurogenerative diseases in humans. Here, we provide a summary of the structure and functions that have been addressed to polyP in microorganisms.
  • Thumbnail Image
    ItemOpen Access
    Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates
    (2020) Frank, Celina; Teleki, Attila; Jendrossek, Dieter
    Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1AT and ppk2AT, of which only ppk1AT is essential for polyP granule formation in vivo. Biochemical characterization of the purified PPK1AT and PPK2AT proteins revealed a higher substrate specificity of PPK1AT (in particular for adenine nucleotides) than for PPK2AT. In contrast, PPK2AT accepted all nucleotides at comparable rates. Most interestingly, PPK2AT catalyzed also the formation of tetra-, penta-, hexa-, hepta-, and octa-phosphorylated nucleosides from guanine, cytosine, desoxy-thymidine, and uridine nucleotides and even nona-phosphorylated adenosine. Our data - in combination with in vivo results - suggest that PPK1AT is important for the formation of polyP whereas PPK2AT has the function to replenish nucleoside triphosphate pools during times of enhanced demand. The potential physiological function(s) of the detected oligophosphorylated nucleotides await clarification.
  • Thumbnail Image
    ItemOpen Access
    The multiple roles of polyphosphate in Ralstonia eutropha and other bacteria
    (2021) Rosigkeit, Hanna; Kneißle, Lea; Obruča, Stanislav; Jendrossek, Dieter
    An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.
  • Thumbnail Image
    ItemOpen Access
    Enrichment of dibenzofuran utilizing bacteria with high co-metabolic potential towards dibenzodioxin and other anellated aromatics
    (1989) Strubel, Volker; Rast, Hans G.; Fietz, Walter H.; Knackmuss, Hans-Joachim; Engesser, Karl-Heinrich
    Dibenzofuran degrading bacteria were enriched from various environmental sources. A mutualistic mixed culture of strain DPO 220 and strain DPO 230 was characterized. Strain DPO 220 alone showed limited growth with dibenzofuran as sole source of carbon and energy (td ≥ 4.5 h). A labile degradation product, C12H10O5, and salicylate were isolated from the culture fluid. Salicylate was found to be a central intermediate of DBF-degradation.Strain DPO 220 co-metabolized a wide range of anellated aromatics as well as heteroaromatics. High rates of co-oxidation of dibenzodioxin demonstrate analogue-enrichment to be a powerful technique for selecting enzymatic activities for otherwise non-degradable substrates.
  • Thumbnail Image
    ItemOpen Access
    (+)-4-Carboxymethyl-2,4-dimethylbut-2-en-4-olide as dead-end metabolite of 2,4-dimethylphenoxyacetic acid or 2,4-dimethylphenol by alcaligenes eutrophus JMP 134
    (1990) Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim
    2,4-Dimethylphenoxyacetic acid and 2,4-dimethylphenol are not growth substrates for Alcaligenes eutrophus JMP 134 although being cooxidized by 2,4-dichlorophenoxyacetate grown cells. None of the relevant catabolic pathways were induced by the dimethylphenoxyacetate, 3,5-Dimethylcatechol is not subject to metacleavage. The alternative ortho-eleavage is also unproductive and gives rise to (+)-4-carboxymethyl-2,4-dimethylbut-2-en-4-olide as a dead-end metabolite. High yields of this metabolite were obtained with the mutant Alcaligenes eutrophys JMP 134-1 which constitutively expresses the genes of 2,4-dichlorophenoxyacetic acid metabolism.
  • Thumbnail Image
    ItemOpen Access
    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.
  • Thumbnail Image
    ItemOpen Access
    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.
  • Thumbnail Image
    ItemOpen Access
    Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases
    (1992) Layh, Norman; Stolz, Andreas; Förster, Siegfried; Effenberger, Franz; Knackmuss, Hans-Joachim
    Bacteria were enriched from soil samples, using benzylcyanide, α-methyl-, α-ethyl- or α-methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(–)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.