title: Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production.
abstract: OBJECTIVE
  The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase.
  
  
  RESULTS
  We constructed E. coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80% mol mol(-1) from glycerol under nongrowth conditions.
  
  
  CONCLUSIONS
  The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol.
  
  
  CONCLUSIONS
  An E. coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.
authors: Institut für Industrielle Genetik, Universität Stuttgart, Stuttgart, Germany.
authors: S
authors: S
authors: Soellner
authors: M
authors: M
authors: Rahnert
authors: M
authors: M
authors: Siemann-Herzberg
authors: R
authors: R
authors: Takors
authors: J
authors: J
authors: Altenbuchner
keywords: POMP pathway; flux rerouting; gene deletion; metabolic evolution; phosphoenolpyruvate carboxylase; pyruvate kinase