Nutzung heterologer Transporter zur Steigerung der L-Threonin-Bildung mit $\textit{Corynebacterium glutamicum}$

Although the amino acids L-aspartate, L-serine and L-threonine are co-consumed together with glucose, transcriptome analysis did not reveal any distinct indication of increased expression of the amino acid importers involved in the uptake. However, a hitherto unknown transcriptional regulation of the genes $\textit{glyA}$ and $\textit{serA}$ encoding for serine hydroxymethyltransferase and 3-phosphoglycerate dehydrogenase by L-serine was discovered. Of the four analyzed transporters RhtA, RhtB, RhtC and YeaS from $\textit{Escherichia coli}$ the two transporters RhtA and RhtC could effectively catalyze the export of L-threonine in $\textit{C. glutamicum}$. Whereas the use of peptides without expression of RhtC led to the accumulation of up to 140 mM of cell-internal L-threonine, the cell-internal concentration was reduced to a maximum of 10 mM due to RhtC-mediated export. Under these conditions a maximal export rate of 8.9 nmol min$^{-1}$ mg TG$^{-1}$ was determined for RhtC and of 5.8 nmol min$^{-1}$ mg TG$^{-1}$ for RhtA. Moreover, it was shown that RhtA also exports L-serine in addition to L-threonine. In addition to the export, increased L-threonine accumulations were achieved in the strain background of $\textit{C. glutamicum}$ DM1800 pEC-T18 hom$^{fbr}$ $\textit{thrB thrE}$ pEKEx2 $\textit{rhtC}$. Inactivation of the threonine dehydratase resulted in a rise of 20%, increased transcription of the pyruvate carboxylase by 5% and reduced citrate synthase activity by 15%. The maximal export rate obtained was 13.3 nmol min$^{-1}$ mg TG$^{-1}$ and the maximal accumulation of L-threonine was 66 mM with a yield of 0.3 mol L-threonine per mol glucose. This is a 900% increase compared to the strain present at the beginning of this thesis. Due to the product spectrum and the internal concentration it can therefore be assumed that by using the heterologous transporter RhtC the export of L-threonine is no longer limiting