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Open Access Methodology article

Flux variability scanning based on enforced objective flux for identifying gene amplification targets

Jong Myoung Park12, Hye Min Park1, Won Jun Kim1, Hyun Uk Kim12, Tae Yong Kim12 and Sang Yup Lee123*

Author affiliations

1 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Republic of Korea

2 BioInformatics Research Center, KAIST, Daejeon, 305-701, Republic of Korea

3 Department of Bio and Brain Engineering and BioProcess Engineering Research Center, KAIST, Daejeon, 305-701, Republic of Korea

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Citation and License

BMC Systems Biology 2012, 6:106  doi:10.1186/1752-0509-6-106

Published: 21 August 2012

Abstract

Background

In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model’s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes.

Results

We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF) with grouping reaction (GR) constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via “GR constraints”. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation.

Conclusions

FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm was validated through the experiments on the enhanced production of putrescine in E. coli, in addition to the comparison with the previously reported experimental data. The FVSEOF strategy with GR constraints will be generally useful for developing industrially important microbial strains having enhanced capabilities of producing chemicals of interest.

Keywords:
Flux variability scanning based on enforced objective flux; Grouping reaction constraints; Putrescine; Escherichia coli