Birgitta Ebert

RWTH Aachen University, Biology, Post-Doc

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My research focusses on the rational, model-guided engineering of microbial production strains and processes for industrial biotechnology. Towards this goal, we are following a systems-level based approach consisting of the three major steps: metabolic analysis, modeling and simulation, and strain engineering.

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Martin Siemann

Simone Aparecida Schmitz

Ralf Takors

vicente bernal

Sushil Gaykawad

Delft University of Technology

Repsol

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Papers by Birgitta Ebert

Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Applied and …, Jan 1, 2011

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Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis

Antioxidants & Redox Signaling, Jan 1, 2010

Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to... more Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to perform synthetically interesting reactions. Besides hydrolases, oxidoreductases represent the most applied enzyme class in industry. Oxidoreductases are attributed a high future potential, especially for applications in the chemical and pharmaceutical industries, as they enable highly interesting chemistry (e.g., the selective oxyfunctionalization of unactivated C-H bonds). Redox reactions are characterized by electron transfer steps that often depend on redox cofactors as additional substrates. Their regeneration typically is accomplished via the metabolism of whole-cell catalysts. Traditionally, studies towards productive redox biocatalysis focused on the biocatalytic enzyme, its activity, selectivity, and specificity, and several successful examples of such processes are running commercially. However, redox cofactor regeneration by host metabolism was hardly considered for the optimization of biocatalytic rate, yield, and=or titer. This article reviews molecular mechanisms of oxidoreductases with synthetic potential and the host redox metabolism that fuels biocatalytic reactions with redox equivalents. The tools discussed in this review for investigating redox metabolism provide the basis for studies aiming at a deeper understanding of the interplay between synthetically active enzymes and metabolic networks. The ultimate goal of rational whole-cell biocatalyst engineering and use for fine chemical production is discussed. Antioxid. Redox Signal. 13, 349-394.

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Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

Febs Journal, Jan 1, 2008

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically fu... more A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logPow solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using 13C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only ∼ 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

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Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint‐based modeling and experimental verification

Biotechnology and Bioengineering, Jan 1, 2008

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, ... more Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts. Biotechnol. Bioeng. 2008;100: 1050–1065. © 2008 Wiley Periodicals, Inc.

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Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand

Applied and …, Jan 1, 2011

Download

Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis

Antioxidants & Redox Signaling, Jan 1, 2010

Download

Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

Febs Journal, Jan 1, 2008

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Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint‐based modeling and experimental verification

Biotechnology and Bioengineering, Jan 1, 2008

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