title: Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol.
abstract: : Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200-300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.
authors.affiliation: Institute for System Dynamics, University of Stuttgart, Pfaffenwaldring 9, 70569 Stuttgart, Germany. ronny.feuer@isys.uni-stuttgart.de.
authors.forename: Ronny
authors.initials: R
authors.lastname: Feuer
authors.forename: Katrin
authors.initials: K
authors.lastname: Gottlieb
authors.forename: Gero
authors.initials: G
authors.lastname: Viertel
authors.forename: Johannes
authors.initials: J
authors.lastname: Klotz
authors.forename: Steffen
authors.initials: S
authors.lastname: Schober
authors.forename: Martin
authors.initials: M
authors.lastname: Bossert
authors.forename: Oliver
authors.initials: O
authors.lastname: Sawodny
authors.forename: Georg
authors.initials: G
authors.lastname: Sprenger
authors.forename: Michael
authors.initials: M
authors.lastname: Ederer