Verlag des Forschungszentrums Jülich
JUEL-4284
Diesveld, Ramon
Nutzung heterologer Transporter zur Steigerung der L-Threonin-Bildung mit Corynebacterium glutamicum
V, 114 S., 2008
Abstract
Transporters take part in the most essential processes of the cell due to their function in
substrate and product transport. In biotechnological applications export is often the limiting
step. The main objective of this thesis was to analyze transport processes in
Corynebacterium glutamicum and to overcome export limitations in L-threonine production
by the use of heterologous transporters.
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 glyA and 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 Escherichia coli the two
transporters RhtA and RhtC could effectively catalyze the export of L-threonine in
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 C. glutamicum DM1800 pEC-T18 homfbr thrB thrE pEKEx2 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.
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