Abstract
BackgroundThe overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. However, the lack of quality data often makes it difficult to correctly identify key enzymes through control analysis. Here, we proposed a method combining in vitro metabolic pathway analysis and proteomics measurement to find the key enzymes in threonine synthesis pathway.ResultsAll enzymes in the threonine synthesis pathway were purified for the reconstruction and perturbation of the in vitro pathway. Label-free proteomics technology combined with APEX (absolute protein expression measurements) data analysis method were employed to determine the absolute enzyme concentrations in the crude enzyme extract obtained from a threonine production strain during the fastest threonine production period. The flux control coefficient of each enzyme in the pathway was then calculated by measuring the flux changes after titration of the corresponding enzyme. The isoenzyme LysC catalyzing the first step in the pathway has the largest flux control coefficient, and thus its concentration change has the biggest impact on pathway flux. To verify that the key enzyme identified through in vitro pathway analysis is also the key enzyme in vivo, we overexpressed LysC in the original threonine production strain. Fermentation results showed that the threonine concentration was increased 30% and the yield was increased 20%.ConclusionsIn vitro metabolic pathways simulating in vivo cells can be built based on precise measurement of enzyme concentrations through proteomics technology and used for the determination of key enzymes through metabolic control analysis. This provides a new way to find gene overexpression targets for industrial strain improvement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0275-8) contains supplementary material, which is available to authorized users.
Highlights
The overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement
The results indicate that LysC, one of the isoenzymes of aspartate kinase, is the key enzyme for increasing threonine synthesis flux
In vitro analysis at this period could be more helpful for identifying the true bottleneck for further improvement of threonine synthesis flux in vivo
Summary
The overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. Zhang et al Microb Cell Fact (2015) 14:86 targets [3,4,5,6]. As these methods are just based on reaction stoichiometry, all genes in a linear pathway will have equal contribution to the pathway flux and these methods will not be able to identify which enzymes are more important for increasing the pathway flux. A theoretical method called metabolic control analysis has been developed to quantitatively measure the impact of an enzyme on the pathway flux through flux control coefficients (FCCs) [7].
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