%0 Journal Article %1 ISI:000326939800011 %A Soellner, S. %A Rahnert, M. %A Siemann-Herzberg, M. %A Takors, R. %A Altenbuchner, J. %C 111 RIVER ST, HOBOKEN 07030-5774, NJ USA %D 2013 %I WILEY-BLACKWELL %J JOURNAL OF APPLIED MICROBIOLOGY %K myown %N 6 %P 1368-1378 %R 10.1111/jam.12333 %T Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production %U https://doi.org/10.1111/jam.12333 %V 115 %X AimsThe 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 ResultsWe 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\% molmol(-1) from glycerol under nongrowth conditions. ConclusionsThe 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 StudyAn 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.

@article{ISI:000326939800011, abstract = {{AimsThe 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 ResultsWe 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\% molmol(-1) from glycerol under nongrowth conditions. ConclusionsThe 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 StudyAn 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.}}, added-at = {2018-06-08T13:08:27.000+0200}, address = {{111 RIVER ST, HOBOKEN 07030-5774, NJ USA}}, affiliation = {{Altenbuchner, J (Reprint Author), Univ Stuttgart, Inst Ind Genet, Allmandring 31, D-70569 Stuttgart, Germany. Soellner, S.; Altenbuchner, J., Univ Stuttgart, Inst Ind Genet, D-70569 Stuttgart, Germany. Rahnert, M.; Siemann-Herzberg, M.; Takors, R., Univ Stuttgart, Inst Bioverfahrenstech, D-70569 Stuttgart, Germany.}}, author = {Soellner, S. and Rahnert, M. and Siemann-Herzberg, M. and Takors, R. and Altenbuchner, J.}, author-email = {{josef.altenbuchner@iig.uni-stuttgart.de}}, biburl = {https://puma.ub.uni-stuttgart.de/bibtex/260e8ebd21cbf00b1cc6f966c45b8821b/ralftakors}, da = {{2018-01-26}}, doc-delivery-number = {{251OP}}, doi = {{10.1111/jam.12333}}, eissn = {{1365-2672}}, funding-acknowledgement = {{Stiftung Baden-Wurttemberg}}, funding-text = {{We would like to thank Dr. Oliver Vielhauer and Mira Lenfers-Lucker for assistance with HPLC and GC analytics. The authors further thank the Stiftung Baden-Wurttemberg for financial support.}}, interhash = {9b52af74e04922f5a2cf6487da0f6070}, intrahash = {60e8ebd21cbf00b1cc6f966c45b8821b}, issn = {{1364-5072}}, journal = {{JOURNAL OF APPLIED MICROBIOLOGY}}, journal-iso = {{J. Appl. Microbiol.}}, keywords = {myown}, keywords-plus = {{PHOSPHOENOLPYRUVATE CARBOXYLASE; ENGINEERED STRAINS; GENE-EXPRESSION; ACID PRODUCTION; FERMENTATION; ENZYME; SUCCINICIPRODUCENS; DIHYDROXYACETONE; OVEREXPRESSION; REPLACEMENT}}, language = {{English}}, month = {{DEC}}, number = {{6}}, number-of-cited-references = {{48}}, oa = {{gold}}, pages = {{1368-1378}}, publisher = {{WILEY-BLACKWELL}}, research-areas = {{Biotechnology \& Applied Microbiology; Microbiology}}, times-cited = {{4}}, timestamp = {2018-06-08T11:08:27.000+0200}, title = {{Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production}}, type = {{Article}}, unique-id = {{ISI:000326939800011}}, url = {https://doi.org/10.1111/jam.12333}, usage-count-last-180-days = {{0}}, usage-count-since-2013 = {{20}}, volume = {{115}}, web-of-science-categories = {{Biotechnology \& Applied Microbiology; Microbiology}}, year = {{2013}} }