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| Home > Publications database > Strain and tool development for the production of industrially relevant compounds with $\textit{Corynebacterium glutamicum}$ |
| Book/Dissertation / PhD Thesis | FZJ-2020-04679 |
2021
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
ISBN: 978-3-95806-522-2
Please use a persistent id in citations: http://hdl.handle.net/2128/27136 urn:nbn:de:0001-2021051022
Abstract: $\textit{Corynebacterium glutamicum}$ is one of the most important model organisms in white biotechnology for the industrial production of numerous amino acids, most notably L-glutamate and L-lysine, but also of other metabolites and proteins. In order to further expand possible applications of this organism for the conversion of renewable feedstocks into industrially relevant compounds, two different approaches of strain engineering of $\textit{C. glutamicum}$ were addressed in the present thesis. In the first part of this work, a new expression system based on a chromosomally encoded T7 RNA polymerase and a plasmid-based T7 promoter was established and characterized in $\textit{C. glutamicum}$. For this purpose, the T7 RNA polymerase (gene 1) was integrated into the prophage-free strain $\textit{C. glutamicum}$ MB001 under the control of the IPTG-inducible lacUV5 promoter, resulting in strain MB001(DE3). In addition, the expression plasmids pMKEx1 and pMKEx2 were constructed into which a target gen can be cloned under control of the T7 promoter and, if necessary, can be fused to a polyhistidine-tag or a Strep-tag. The T7 expression system was evaluated with the reporter gene $\textit{eyfp}$ and compared to the well-established pEKEx2 system, which is based on the $\textit{tac}$ promotor. The basal expression of the T7 system was lower than that of the pEKEx2 system. The specific fluorescence of eYFP increased 450-fold after maximal induction of the T7 system with 250 μM IPTG and was 3.5-fold higher than the specific eYFP fluorescence obtained after maximal $\textit{eyfp}$ expression with the pEKEx2 system. Furthermore, it was shown that proteins such as pyruvate kinase or secretory GFP proteins can also be successfully produced in higher amounts with the T7 system than with the pEKEx2 system. In the second part of this work, a genetically encoded biosensor for the cytoplasmic lysine concentration was used to screen for mutated variants of pyruvate carboxylase (PCx), which enabled an improved Llysine production. PCx catalyzes the ATP-dependent carboxylation from pyruvate to oxaloacetate and plays an important role as anaplerotic enzyme. It replenishes the tricarboxylic acid cycle during growth on sugars when intermediates have been removed from the cycle by branching metabolic pathways. This reaction is for example of great importance for L-lysine production and it has already been shown that overexpression of the $\textit{pyc}$ gene as well as the mutation P458S in PCx lead to an increased L-lysine yield in $\textit{C. glutamicum}$. In order to find further advantageous mutations for lysine production, a plasmidbased library with mutated $\textit{pyc}$ variants was generated in this work using error-prone PCR. Out of this library, two PCx variants could be isolated with the help of the lysine biosensor pSenLys-Spec and FACS-based high-throughput screening, which showed an increased lysine formation compared to the strain with wild-type PCx. The variants PCx$^{T343A, I1012S}$ and PCx$^{T132A}$ enabled an increase of the lysine titer by 9% and 19%, respectively, after plasmid-based overexpression of the respective genes in the strain DM1868Δ$\textit{pyc}$/pSenLys-Spec. When the mutations were introduced one by one into the genome of the lysine-producing strain DM1868, the variant PCx$^{T132A}$ produced 7% more lysine and PCx$^{T343A}$ 15% more lysine compared to strain DM1868 encoding wild-type PCx. In previous studies, PCx activity of $\textit{C. glutamicum}$ could only be measured with permeabilized cells. For a more detailed characterization, conditions were established that enabled the measurement of PCx activity in cell-free extracts and the isolation of the enzyme in active form. For purified PCx, K$_{m}$ values of 3.8 mM for pyruvate, 0.6 mM for ATP, and 13.3 mM for bicarbonate were determined. ADP and aspartate inhibited PCx activity with $\textit{K}_{i}$ values of 1.5 mM and 9.3 mM, respectively. Initial characterization of the variants PCx$^{T132A}$ and PCx$^{T343A}$ revealed that their activity was not inhibited up to aspartate concentrations of approx. 7.5 mM. The $\textit{K}_{i}$ values of 13.2 mM for PCx$^{T132A}$ and 10.8 mM for PCx$^{T343A}$ were higher than for wild-type PCx and provide an explanation for the increased lysine production obtained with these variants in $\textit{C. glutamicum}$.
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