This title appears in the Scientific Report :
2013
Please use the identifier:
http://hdl.handle.net/2128/5485 in citations.
Increasing the NADPH supply for whole-cell biotransformation and development of a novel biosensor
Increasing the NADPH supply for whole-cell biotransformation and development of a novel biosensor
In the first part of this work, the pentose phosphate pathway (PPP) was investigated as a source of NADPH in reductive whole-cell biotransformation using $\textit{Escherichia coli}$ and $\textit{Corynebacterium glutamicum}$ as hosts and glucose as reductant. The reduction of methyl acetoacetate to t...
Saved in:
Personal Name(s): | Siedler, Solvey (Corresponding author) |
---|---|
Contributing Institute: |
Biotechnologie; IBG-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH, Zentralbibliothek, Verlag
2013
|
Physical Description: |
127 S. |
Dissertation Note: |
Heinrich-Heine-Universität Düsseldorf, Diss., 2013 |
ISBN: |
978-3-89336-900-3 |
Document Type: |
Dissertation / PhD Thesis |
Research Program: |
ohne Topic |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / health
66 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
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520 | |a In the first part of this work, the pentose phosphate pathway (PPP) was investigated as a source of NADPH in reductive whole-cell biotransformation using $\textit{Escherichia coli}$ and $\textit{Corynebacterium glutamicum}$ as hosts and glucose as reductant. The reduction of methyl acetoacetate to the chiral (R)-methyl hydroxybutyrate (MHB) served as a model reaction for NADPH-dependent reactions and was catalyzed by an alcohol dehydrogenase (ADH) from $\textit{Lactobacillus brevis}$. Partial cyclization of the PPP in $\textit{E. coli}$ and $\textit{C. glutamicum}$ was achieved by deletion of the phosphofructokinase gene $\textit{pfkA}$, which prevents fructose 6-phosphate catabolism in the glycolytic pathway. The $\textit{pfkA}$-deficient mutants carrying the $\textit{L. brevis}$ ADH showed a doubled MHB-per-glucose ratio compared to the parent strains. In $\textit{E. coli}$ the partial PPP cyclization in the Δ$\textit{pfkA}$ mutant was proven by $^{13}$C-flux analysis, which showed a negative net flux through the phosphoglucose isomerase reaction. Furthermore, the flux through pyruvate kinase was found to be absent in the Δ$\textit{pfkA}$ mutant, indicating that a low phosphoenolpyruvate (PEP) concentration limited glucose uptake via the phosphotransferase system (PTS). PTS-independent glucose uptake and phosphorylation via the glucose facilitator and glucose kinase from $\textit{Zymomonas mobilis}$ enhanced the specific MHB productivity by 21% in the $\textit{E. coli ΔpfkA}$ mutant. Deletion of glyceraldehyde 3-phosphate dehydrogenase ($\textit{gapA}$) theoretically results in a completely cyclized PPP and a ratio of 12 mol NADPH per mol glucose 6-phosphate. A $\textit{C. glutamicum ΔgapA}$ mutant showed a ratio of 7.9 mol MHB per mol glucose, which is the highest one reported so far. Formation of the by-product glycerol presumably was responsible for not achieving a higher ratio. In the second part of this work, a biosensor was developed which is capable of detecting a lowered intracellular NADPH/NADP+ ratio and trigger the synthesis of an autofluorescent protein. DNA microarray analysis of $\textit{E. coli}$ during biotransformation showed an upregulation of $\textit{soxS}$ transcription after MAA addition, suggesting that the SoxR regulator known to upregulate soxS expression is activated by a lowered NADPH/NADP$^{+}$ ratio. Subsequently, the $\textit{soxS}$ promoter was fused on a plasmid with the gene encoding yellow fluorescent protein (eYFP). $\textit{E. coli}$ transformed with this plasmid showed fluorescence when MAA was added to the culture. The final fluorescence [...] | ||
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