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.
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.
%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}}
}