title: In silico profiling of cell growth and succinate production inEscherichia coliNZN111.
abstract: BACKGROUND
  Succinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation,Escherichia coliNZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genesldhAandpflB, 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.
  
  
  RESULTS
  In this study, genome-scale metabolic core model ofE. coliwas reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strainE. coliNZN111. 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-typeE. coli,ldhAmutant,pflBmutant, 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 ofldhAandpflB, 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Xingxing
authors.initials: X
authors.lastname: Jian
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Ningchuan
authors.initials: N
authors.lastname: Li
authors.affiliation: Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China ; Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCICBT), Shanghai, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Hua
keywords: Artificial centering hit-and-run (ACHR); Metabolite flux-sum analysis; NADH/NAD+; NZN111; Sample solution space; Succinate