This title appears in the Scientific Report :
2010
Systemische Analyse des Citratzyklus in $\textit{Corynebacterium glutamicum}$
Systemische Analyse des Citratzyklus in $\textit{Corynebacterium glutamicum}$
A more holistic view on L-lysine formation is offered by systems biology approaches making use of genome-scale in silico analyses together with metabolic engineering. A genome-scale metabolic model for $\textit{C. glutamicum}$ has recently been generated (Kjeldsen 2009) and optimized. Its analyses,...
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Personal Name(s): | van Ooyen, Jan (Corresponding author) |
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Contributing Institute: |
Biotechnologie 1; IBT-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2010
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Physical Description: |
117 S. |
Dissertation Note: |
Universität Düsseldorf, Diss., 2010 |
ISBN: |
978-3-89336-731-3 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Biotechnologie |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health
41 |
Subject (ZB): | |
Publikationsportal JuSER |
A more holistic view on L-lysine formation is offered by systems biology approaches making use of genome-scale in silico analyses together with metabolic engineering. A genome-scale metabolic model for $\textit{C. glutamicum}$ has recently been generated (Kjeldsen 2009) and optimized. Its analyses, as well as literature data, pointed at citrate synthase (CS) as a promising target to further increase L-lysine yields. Aim of this work was to clarify if a correlation between flux over CS and L-lysine yield as seen in flux balance analysises could be proven experimentally. This work shows that the CS gene $\textit{gltA}$ of $\textit{C. glutamicum}$ is encoded by two monocistronic transcripts with their transcript initiation sites located 121 bp and 357 bp upstream of the translational start site. Northern blot analyses revealed that the short transcript prevails during growth on acetate, whereas the long transcript is dominant during growth on glucose. Further Experiments revealed that the transcriptional regulators RamA, RamB and the global regulator GlxR are involved in the regulation of $\textit{gltA}$ expression in a complex scenario. Based on this knowledge the entire upstream region of $\textit{gltA}$ was replaced with significantly reduced transcription activity of a $\textit{dapA}$-promoter collection. As a result nine mutants were obtained, exhibiting a gradual decrease in the CS specific activity from 32 %, respectively 6 % of the original CS activity. These strains were assayed for their L-lysine formation. Already the reduction of the CS activity to 32 % yielded a significant increase in L-lysine yield from 0.16 to 0.20 g g$^{-1}$. With a further decrease in the CS activity the yield is further increasing up to 0.32 g g$^{-1}$ at the lowest CS activity of 6 %. At the same time the growth rate was gradually decreased, showing a correlation between CS activity and L-lysine yield accompanied by a decreased growth rate. Thus, CS can be used as a switch between lysine production on the one hand and biomass formation on the other hand. Using the L-lysine producer DM1933 and lowering its specific CS activity to 10 %, a L-lysine yield of 0.5 g g$^{-1}$ could be obtained, being the highest yield reported so far. Global gene expression analyses revealed amongst others that with reduced CS activity genes controlled by RamA and RamB were altered in their expression. Furthermore, quantifications of intracellular metabolite concentrations revealed an increase of glycolytic intermediates, as well as aspartate and aspartate-derived amino acids, at reduced CS activity, whereas citrate, 2-oxoglutarate and proline concentrations decreased. Extracellular rates of the strains during batch fermentation were determined in bioreactors, showing that the specific rates of glucose uptake and CO$_{2}$ production were not affected by reduced CS activity, whereas growth rate was reduced and the L-lysine formation rate nearly doubled. Space time yield obtained were thus up to 35% higher than in the parental strain. Taken together the data show that influencing CS is a very powerful instrument to increase L-lysine, and probably other aspartate derived aminoacids, although systemic consequences are not fully understood. |