Title
Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: Verification and complementation of hypotheses derived from transcriptomics
Author
Wierckx, N.
Ruijssenaars, H.J.
de Winde, J.H.
Schmid, A.
Blank, L.M.
TNO Kwaliteit van Leven
Publication year
2009
Abstract
The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism. © 2009 Elsevier B.V. All rights reserved.
Subject
Biology
Biomedical Research
Metabolic engineering
Metabolic flux analysis
Phenol
Pseudomonas putida S12
Solvent tolerance
Carbon fluxes
Carbon metabolism
Cellular events
Complementation
Encoding genes
Energy productions
Glucose 6 phosphates
In-vitro
Metabolic engineering
Metabolic flux
Metabolic flux analysis
Pentose phosphate pathway
Phenol production
Phosphate production
Phosphoenolpyruvates
Physiological effects
Pseudomonas putida
Pseudomonas putida S12
Pyruvate kinase
Pyruvates
Solvent tolerance
Substrate uptake
TCA cycle
Transcriptional changes
Transcriptome analysis
Transcriptomics
Up-regulation
Encoding (symbols)
Gene encoding
Glucose
Phenols
Solvents
Substrates
Metabolism
bacterial protein
glucose 6 phosphate
gntP protein
gnuK protein
lactate dehydrogenase
malate dehydrogenase
malic acid
oprB 1 protein
oxaloacetic acid
phenol
phosphoenolpyruvate
porin
pykA protein
pyruvate kinase
pyruvic acid
unclassified drug
article
bacterial metabolism
bacterial strain
carbon metabolism
carbon source
complex formation
controlled study
energy metabolism
gene mutation
gluconeogenesis
metabolic engineering
nonhuman
priority journal
Pseudomonas putida
transcription regulation
transcriptomics
upregulation
Pseudomonas putida
To reference this document use:
http://resolver.tudelft.nl/uuid:425c092c-9610-44df-9bd5-e967bf10d0c5
DOI
https://doi.org/10.1016/j.jbiotec.2009.06.023
TNO identifier
241705
ISSN
0168-1656
Source
Journal of Biotechnology, 143 (2), 124-129
Document type
article