In silico profiling of cell growth and succinate production in Escherichia coli NZN111.

Xingxing Jian,Qiang Hua,Ningchuan Li,Cheng Zhang

doi:10.1186/s40643-016-0125-5

Abstract

BackgroundSuccinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB, respectively. However, this double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and consequently disables proper growth under anaerobic conditions.ResultsIn this study, genome-scale metabolic core model of E. coli was reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strain E. coli NZN111. Non-optimized artificial centering hit-and-run (ACHR) method and metabolite flux-sum analysis were utilized to evaluate metabolic characteristics of strains. Thus, metabolic characteristics of the strains wild-type E. coli, ldhA mutant, pflB mutant, and NZN111 under anaerobic conditions were successfully unraveled.Conclusions We found a viewpoint contrary to the widespread realization that an NADH/NAD+ in NZN111 mainly resulted from the inactivation of PFL rather than the inactivation of LDH. In addition, the two alternative anaerobic fermentation pathways, lactate and ethanol production pathways, were blocked owing to the disruption of ldhA and pflB, resulting in insufficient NAD+ regeneration to oxidize or metabolize glucose for cell growth. Furthermore, we speculated reaction NADH16, the conversion of ubiquinone-8 (q8) to ubiquinol-8 (q8h2), as a potential amplification target for anaerobically improving cell growth and succinate production in NZN111.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0125-5) contains supplementary material, which is available to authorized users.

Highlights

Succinic acid is a valuable product due to its wide-ranging utilities
On the basis of metabolite flux-sum analysis, we found a viewpoint opposed to the widespread realization that in NZN111, NADH/NAD+ imbalance mainly resulted from the inactivation of pyruvate formate lyase (PFL) rather than the inactivation of lactate dehydrogenase (LDH), which was consistent with the experimental results and gene expression profile of the wild-type E. coli and NZN111
The average flux distributions consisted of average fluxes for wild-type E. coli, ldhA mutant, pflB mutant, and NZN111 were achieved to shape their global metabolisms

Summary

Introduction

To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB, respectively. This double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and disables proper growth under anaerobic conditions. A widely hold view is that the inactivation of the NADHdependent LDH limits the regeneration of NAD+ and hinders growth under anaerobic conditions (Ahn et al 2016). An increase of succinate yield and a decrease of pyruvate accumulation in E. coli NZN111 (Wu et al 2007) and its derivative AFP111 (Jiang et al 2010) were observed using a dual-phase fermentation mode, where a high cell density was achieved in the first aerobic growth phase, and subsequently succinate was produced in the second anaerobic production phase

Methods

Results

Conclusion