Verlag des Forschungszentrums Jülich
JUEL-4130
Netzer, Roman
Untersuchungen zur Glykolyse und zum L-Serin-Stoffwechsel in Corynebacterium glutamicum
IV, 141 S., 2004
Abstract
The amino acid L-serine is a central metabolite in the metabolism and of increasing
importance for the industrial use. The aim of this work was to investigate if L-serine
can be produced biotech nologically by Corynebacterium glutamicum and to study the
influence of L-serine degradation and the provision of glycolytic precursors.
To improve the availability of glycolytic precursors for the L-serine biosynthesis, a
pyruvate kinase and 3-phosphoglycerate mutase deletion mutant was constructed . It
could be shown that pyruvate kinase is essential for growth both on the non PTS
(phosphotransferase system) sugar ribose and on the gluconeogenetic substrates
acetate and citrate. Global gene expression analyses and enzyme assays with a
suppressor mutant showed, that the growth of the pyruvate kinase mutant on acetate
and citrate could be restored by overexpression of the mez gene encoding the malic
enzyme. Further it was shown, that the pyruvate kinase mutant accumulates
glycolytic precursors for L-serine biosynthesis.
To inactivate the 3-phosphoglycerate mutase, the functional expressed gene was
identified by sequence analysis and growth experiments. A pgm deletion mutant was
only able to grow in presence of both a glycolytic and a gluconeogenetic substrate .
Thereby, the non-PTS sugar maltose has been metabolised preferentially to glucose
that is taken up via a PTS system . It has been shown that the mutant accumulates
the unphosphorylated L-serine precursor glycerate .
To investigate the L-serine degradation the L-serine dehydratase encoding gene was
identified in the C. glutamicum genome by sequence comparison . The subsequent
deletion of the sdaA gene resulted in a 1 .8-fold decreased L-serine degradation rate
in comparison to the wild type but the overexpression of sdaA led to a 2-fold
increased degradation rate. Interestingly, the overexpression of sdaA enabled
C. glutamicum to grow on L-serine.
The deletion of the sdaA gene in a C. glutamicum strain overexpressing the L-serine
biosynthesis genes revealed a 960-fold gain of the L-serine accumulation . After an
additional deletion of either the 3-phosphoglycerate mutase or pyruvate kinase
encoding gene, a further increase of the L-serine formation was found. Further it was
shown that the deletion of sdaA and the simultaneous reduction of the serine
hydroxymethyltransferase activity, another L-serine converting enzyme, in a strain
overexpressing the L-serine biosynthesis genes, led to a tremendous increase of
L-serine accumulation up to 100 mM.
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