Construction Of Escherichia Coli For Succinate Production Based On Elementary Mode Analysis

Recently, several computational frameworks based on the metabolic andregulatory networks were developed for rational strain design. Elementary modeanalysis （EMA） is a powerful metabolic pathway analysis tool to identify all pathwaysexisting in cellular metabolism at steady state just based on the stoichiometricstructure and thermodynamic constraint of reactions without requiring kineticparameters and flux calculations. EMA had been successfully applied tosystematically evaluate robustness and implement rational strain design in metabolicengineering. Recently, a new Computational Approach for Strain Optimizationaiming at high Productivity （CASOP） based on EMA had been reported, in which theproduct yield and network capacity were both considered to shift the natural flux tosynthesis of the desired product with high production. We applied the CASOP strategy,which was based on elementary mode analysis, to predict the metabolic engineeringtargets for succinate production of Escherichia coli under aerobic conditions.A metabolic network of E. coli with glucose as carbon source was constructed byusing the previously described network. In order to consider effects of carboxylationreactions on succinate production comprehensively, two constraints were introducedinto this network as followed: i) the exogenous pyruvate carboxylase （pyc） reactionthat converts pyruvate to oxaloacetate （OAA） was added in the model, ii) the reactionconverting malate to pyruvate, catalyzed by malic enzyme （maeA） in E. coli, wasseparated into two irreversible reactions with opposite side. Based on the CASOPanalysis, five knockout candidates （sdhA, ackA-pta, poxB, mgsA, iclR） and fiveoverexpression candidates （aceA, aceB, pyc, ppc, maeA） were finally chosen to createa functional space with high network capacity for succinate production.To validate the model prediction, a library of mutants were constructed from widetype E.coli W1485and tested for their performances. Strain ZJG13, with the fiveknockout candidates mutated, exhibited healthy growth behavior and accumulatedsuccinate as a major product with an average specific productivity of0.50mmol gCDW^-1h^-1. Strain ZJG21, with the glyoxylate shunt genes promoter replaced by trchad no significantly effect in growth and succinate production. When pT9aceKABwas transported into ZJG13,13.9%increase in succinate yield comparing with ZJG13 were achieved which was also companied with56.3%decrease in glucose uptakerates. Overexpression of pyruvate carboxylase （pyc） demonstrated significantlyimprovement in succinate production and the effect varied corresponding to theexpression level. Whereas coexpression of glyoxylate shunt operon with pyruvatecarboxylase （pyc） or simultaneous overproduction of PEP carboxylase （ppc） and pycshowed less effects than when pyc was overexpressed alone in ZJG13. StrainZJG13^-1Y was the best one among the strains constructed in this study.Fed-batch culture of the final strain ZJG13^-1Y led to a titre of36.1g/L （306mM） succinate, with an average specific productivity of0.88mmol g CDW^-1h^-1andan overall yield of0.72mol mol^-1glucose. These results indicate that our aerobicsuccinate production system is highly efficient and the titre and yield of succinate canbe further increased by optimizing the fermentation process.