This title appears in the Scientific Report :
2014
Please use the identifier:
http://hdl.handle.net/2128/5695 in citations.
L-Cystein-Bildung mit $\textit{Corynebacterium glutamicum}$ und optische Sensoren zur zellulären Metabolitanalyse
L-Cystein-Bildung mit $\textit{Corynebacterium glutamicum}$ und optische Sensoren zur zellulären Metabolitanalyse
L-Cysteine is an important amino acid and is in addition to L-methionine the most abundantsulfur containing molecule inside the cell. It provides reduced sulfur for the synthesis of avariety of other sulfur containing compounds. Besides its importance for microbial growththere is also a growing inte...
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Personal Name(s): | Hoffmann, Kristina (Corresponding author) |
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Contributing Institute: |
Biotechnologie; IBG-1 |
Published in: | 2014 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2014
|
Physical Description: |
VI, 81 S. |
Dissertation Note: |
Heinrich-Heine-Universität Düsseldorf, Diss., 2013 |
ISBN: |
978-3-89336-939-3 |
Document Type: |
Dissertation / PhD Thesis |
Research Program: |
ohne Topic |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health
69 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
L-Cysteine is an important amino acid and is in addition to L-methionine the most abundantsulfur containing molecule inside the cell. It provides reduced sulfur for the synthesis of avariety of other sulfur containing compounds. Besides its importance for microbial growththere is also a growing interest for L-cysteine in the food, pharmaceutical and cosmetics industries and therefore a need for the production of L-cysteine by fermentation exists. Consequently, the main objective of this study was to construct a L-cysteine producer based on an efficient $\textit{Corynebacterium glutamicum}$ L-serine producing strain. Furthermore the development of metabolite sensors for intracellular detection of sulfur availability should be achieved. The following results were obtained:(1) The L-serine producer Ser4 contains the plasmid pser$\textit{ACB}$. To facilitate further genetic work concerning L-cysteine formation with this strain, the production of L-serine without a plasmid was desirable. The chromosomal integration of the gene $\textit{serA}^{fbr}$,coding for a feedback-resistant 3-phosphoglycerate dehydrogenase, led already to slight L-serine formation. Studies on a promoter exchange of $\textit{serA}^{fbr}$ in combination with plasmid based expression of the genes $\textit{serC}$ and $\textit{serB}$ revealed that strong expression of $\textit{serA}^{fbr}$ together with that of $\textit{serC}$ and $\textit{serB}$ is necessary to obtain high L-serine production.(2) In contrast to the expression of genes encoding heterologous feedback-resistant serine acetyltransferases the increased expression of native $\textit{cysE}$ from $\textit{C. glutamicum}$ led to improvement of L-cysteine formation from 0.95 ± 0.35 mM to 5.6 ± 0.8 mM in the L-serine producer. In combination with expression of $\textit{cysK}$, coding for the $\textit{O-acetylserine}$ sulfhydrylase, 7.3 ± 0.7 mM L-cysteine accumulated in the culture supernatant. The deletion of genes encoding cysteine desulfurase AecD to prevent L-cysteine degradation and homoserine actyltransferase MetX to disable L-methionine synthesis as well as the expression of the operon $\textit{fpr2-cysIXHDNYZ}$ necessary for assimilatory sulfate reduction had a moderate effect on L-cysteine formation. (3) To construct metabolite sensors for the detection of the sulfur acceptor molecules O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) promoter regions of genes NCgl1289 and $\textit{cysI}$ including CysR-binding sites were fused to the reporter gene $\textit{eyfp}$. Additionally, the respective sensor cassettes contained the gene of transcriptional regulator CysR, which activates the genes for the assimilatory sulfate reduction. It could be shown that intracellular elevated levels of OAS and OAH led to increased specific fluorescence at the single cell level and that cultures cultivated under limiting sulfur conditions showed increased fluorescence.(4) To analyze the impact of the transcriptional regulator CysR on L-cysteine formation the gene $\textit{cysR}$ was expressed under control of the native promoter as well as P$_{tac}$. It could be shown, that increased expression of CysR does not necessarily improve the L-cysteineformation. The results indicate the delicate regulation of sulfur metabolism in $\textit{C. glutamicum}$, in which the three transcriptional regulators CysR, McbR and SsuR are involved. In summary, L-cysteine formation with $\textit{C. glutamicum}$ could be improved up to 7.3 ± 0.7 mM, but due to the complex regulation of sulfur metabolism it proved to be difficult to achieve high production. Furthermore, the metabolite sensors for O-acetyl-L-serine and O-acetyl-L-homoserine facilitate $\textit{in vivo}$ studies at the single-cell level to detect intracellular levels of the respective metabolites. Therefore, they represent an important tool for the analysis of cellular sulfur metabolism. |