Integration of kinetic models with data from 13C-metabolic flux experiments

[摘要] A detailed mathematical description of all the processes in a cell could be aninformative tool for investigating biological function. Detailed kinetic modelscould be built either by obtaining enzyme kinetic parameters in vitro, orby obtaining them from time series analyses of metabolite data from rapidpulse experiments. A genome scale in vitro enzyme kinetic assay projectwould be prohibitively laborious with the current technologies. Further,there are still uncertainties about the importance of in vivo effects such asmetabolite channelling, spatial effects and molecular crowding which couldmake in vitro determined parameters invalid. Accordingly, there is muchinterest in in vivo experiments for kinetic modelling. In vivo experimentalmethods suffer from a number of technical and even fundamental problems.Technical problems are being solved by more sensitive metabolomics toolsand rapid sampling technologies. However, the large number of effectors ofeach enzyme reaction makes it impossible to obtain models at the level of detailpossible with the in vitro method. Ultimately, the solution to building agenome scale Silicon Cell is to make use of both strategies. As metabolomicstechnologies are rapidly improving, it would thus make sense to follow theparts-based in vitro kinetics methodology, and carry out a detailed accuracyassessment of the model with in vivo experiments. To address the problemof the fundamental limit of information from concentration time-series, otherin vivo experiments will have to be carried out as well. 13C-metabolic fluxanalysis has recently undergone vast improvements with the use of better experimentalprotocols and powerful algorithms for flux calculation. Incorporationof this type of experiment in the validation protocol is the aim of this thesis, which represents an intermediary step towards using the genome-scalestoichiometric models as platforms for building genome-scale kinetic models.It is illustrated here how kinetic models can be combined with metabolicflux data in a special way which allows correct modelling of boundary conditionsand validation using novel concepts. We used 13C-metabolic fluxanalysis and gas chromatography-mass-spectrometry to measure metabolicfluxes through the central metabolic pathways of the yeast Saccharomycescerevisiae. This data was integrated with a previously constructed detailedkinetic model of fermentative glycolysis in the yeast to illustrate our approach.Various implications for such data integration with kinetic modelswere identified and a software program was designed for this purpose.

[发布机构] Stellenbosch University