Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production
Article first published online: 6 SEP 2013
© 2013 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 115, Issue 6, pages 1368–1378, December 2013
How to Cite
Soellner, S., Rahnert, M., Siemann-Herzberg, M., Takors, R. and Altenbuchner, J. (2013), Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production. Journal of Applied Microbiology, 115: 1368–1378. doi: 10.1111/jam.12333
- Issue published online: 15 NOV 2013
- Article first published online: 6 SEP 2013
- Accepted manuscript online: 20 AUG 2013 12:31AM EST
- Manuscript Accepted: 13 AUG 2013
- Manuscript Revised: 9 AUG 2013
- Manuscript Received: 26 MAR 2013
- Stiftung Baden-Württemberg
- flux rerouting;
- gene deletion;
- metabolic evolution;
- phosphoenolpyruvate carboxylase;
- POMP pathway;
- pyruvate kinase
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.
Methods and 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.
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.
Significance and Impact of Study
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.