Nicola Zamboni

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..The approach is flexible and paves the way to large-scale fluxome profiling of any biological system and condition...

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..With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline...

..In the future, this practice will be complemented by novel modeling frameworks to assay particular fluxes within a network by stable isotopic tracer for targeted validation of well-defined hypotheses...

..The presented protocols take 5-10 d, have been used extensively in the past decade and are exemplified here for the central metabolism of Escherichia coli...

..Therefore, suitable internal standards, such as (13)C-labeled biomass extracts, are mandatory for quantitative metabolomics with any methods...

..This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture...

..As a consequence, higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands, suggesting a transhydrogenase-like mechanism to close the balance of reducing equivalents...

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..Presumably as a consequence of the unfavorable kinetics of an ATP-synthesizing anaplerotic PEP carboxykinase reaction, such pyruvate carboxylase mutants grow slowly or, as in the case of wild-type 168, not at all...

..Hence, alteration in one network constituent can be efficiently buffered by converse alterations in the other constituent, implying a passive mechanism to maintain metabolic homeostasis upon perturbations in enzyme capacity...

..A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes...

..Since the product-corrected biomass yields were identical in mutants and parent, the results show that efficient ATP generation is not overly important for exponential growth of B. subtilis in batch culture...

..So far, however, widespread application of NET analysis in metabolomics labs was hindered by the absence of suitable software...

..coli shake flasks and 1-ml deep-well microtiter plates. Due to its simplicity, speed, and robustness, 13C-constrained metabolic flux balancing is promising for routine and high-throughput analysis on a miniaturized scale...

..Integration of metabolomics, (13)C-flux analysis and other data enable the condition-dependent characterization of regulatory circuits that ultimately govern the metabolic phenotype...

..In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design...

..Finally, we present an exemplary screen with Escherichia coli deletion mutants to show the exquisite capacity of the platform to identify lesions in primary metabolism at high throughputs...

..5-50 mg dry cell weight. We recommend this method for routine and yet comprehensive quantification of primary metabolites in a wide variety of biological matrices...

..The described workflow allows the production of large amounts of metabolome samples within a day, is compatible with virtually all liquid extraction protocols, and paves the road to quantitative screens...

..The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures...

..coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion...

..We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory...