Book/Dissertation / PhD Thesis

FZJ-2017-01017

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Novel genetic tools for production strain development of $Corynebacterium$ $glutamicum$

Hochheim, J. (Corresponding author)FZJ*

2017
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-212-2

Please use a persistent id in citations: http://hdl.handle.net/2128/14002  urn:nbn:de:0001-2017032839

Abstract: The development of new genetic tools plays a key role in the establishment of new and/or improved microbial production strains. Since $\textit{Corynebacterium glutamicum}$ belongs to the most important microorganisms in white biotechnology, the major task of this work was to establish new genome editing tools for this organism. To this end, the following results were obtained:(1) dsDNA recombineering was established as a genome editing method using the RecET recombination system originating from the prophage Rac from $\textit{Escherichia coli}$. Whereas single point mutations could be introduced with high frequencies, full-length genes werenot integrated at the correct genomic target location. In-depth analyses revealed a correlation of DNA substrate length with DNA recombineering efficiency, allowing for only very low efficiencies of 9.8 x 10$^{2}$ recombinant cells per 10$^{10}$ cells with fragments larger than 700 nt. With 5’-phosphorylated DNA fragments as template, dsDNA recombination efficiencies were enhanced by a factor of 10, whereas 5’-phosphorothioation decreased recombination efficiencies by a factor of 5.(2) The CRISPR-Cas9 system was established in $\textit{C. glutamicum}$ allowing for the separation of different genotypes. In a mixture of the two $\textit{C. glutamicum}$ strains ATCC13032 and ATCC13032 with a point mutation resulting in MurE-G81E, all wild-type cells could be eliminated using an appropriate sgRNA. Since it was observed that the presence of Cas9 protein reduced living cell count, Cas9 toxicity was studied in more detail by analyzing various plasmid constructs and Cas9 variants. Exchanging the start codon of $\textit{cas9}$ from ATG to TTG led to highly reduced unspecific activity, whereas CRISPR-Cas9 targeting remained functional.(3) Furthermore, pCLTON2, one of the expression plasmids used for $\textit{C. glutamicum}$, was modified to establish a temperature-sensitive origin of replication. Whereas this plasmid was stably maintained during cultivation at 25 °C, cultivation at 34 °C led to immediate plasmid loss, rendering pCl2-OriTS a valuable tool for genome editing of this bacterium.(4) With the aim to quantify the CRISPR-Cas9 genome editing efficiency, several test systems were evaluated. In the course of these experiments, the ß-galactosidase assay was identified as the most suitable test system for $\textit{C. glutamicum}$, since recombinant cells canbe distinguished from non-edited cells by visual differentiation in an easy time-efficient manner.(5) The established β-galactosidase assay allowed for simulating the combination of genome editing by recombineering and targeted elimination of non-edited cells by CRISPR-Cas9. Depending on the plasmid carrying $\textit{cas9}$, at best an enrichment factor of 5000 of the desired mutant was obtained. Assuming that dsDNA recombineering enables to obtain 1 recombinant per 100,000 cells means that theoretically only a low number of approximately 20 clones must be assayed to retrieve the desired mutant.(6) Site-saturation mutagenesis of the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) at position G81 allowed for the exchange of all amino acids possible in $\textit{C. glutamicum}$. The characterization of the mutant strains revealed a systemic effect on L-lysine accumulation: Increased L-lysine accumulation is coupled to a reduced growth rate, which is most likely a cell response to limited mesodiaminopimelate availability, which is required for both L-lysine and cell wall synthesis. In summary, two new genome editing techniques, dsDNA recombineering and CRISPR-Cas9 targeting, were established for $\textit{Corynebacterium glutamicum}$ enabling integration/deletion of small DNA fragments and separation of different genotypes.

Note: Abgabedatum Dissertation: 25.11.2016 Biotgechnologie 1
Note: Universität Düsseldorf, Diss., 2017

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Contributing Institute(s):

1. Biotechnologie (IBG-1)

Research Program(s):

1. 581 - Biotechnology (POF3-581) (POF3-581)

Appears in the scientific report 2017

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