Verlag des Forschungszentrums Jülich
JUEL-4093
Schlüpen, Christina
Untersuchungen zur Bedeutung verschiedener Enzyme des Glycin-Stoffwechsels für die Riboflavin-Bildung in Ashbya gossypii
108 S., 2003
The filamentous fungus Ashbya gossypii is an important riboflavin (vitamin B2) overproducer
used in industrial scale. Productivity of the fungus is limited by glycine, a precursor of the de
novo purine biosynthesis . Supplementation of the medium with glycine leads to an increase
in riboflavin production .
The first objective of this thesis was the characterization of the two serine
hydroxymethyltransfe rase (SHMT) isoenzymes, which are involved in glycine metabolism, in
order to improve the glycine supply for the riboflavin production . For a subcellular localization
of both SHMT isoenzymes mutants transformed with HA-fusions were used. By
immunodetection SHMT1 was localized in the mitochondria and SHMT2 in the cytosol .
Disruption of SHM2 resulted in a significant increase of riboflavin overproduction . The SHMT
specific activity decreased about 85 % from 3 mU/mg protein to 0.5 mU/mg protein . After
additional inactivation of SHM1 a remaining SHMT activity of 3 % was detected, which was
shown to be a side activity of threonine aldolase . The enhanced riboflavin overproduction of
SHM2 disruptants was explained by a reduced flux from glycine to serine thus leading to an
elevated pool of the riboflavin precursor glycine . Evidence was obtained by 13C-labeling
experiments . When 13C1-threonine was fed, more than 50 % of the label was detected in C1
of glycine, resulting from threonine aldolase activity . More than 30 % labeling determined in
C1 of serine could be explained by a serine synthesis via SHMT. Knockout of SHM1 had no
detectable effect on serine labeling but disruption of SHM2 led to a decrease in serine (2 -
5 %) and an increase in glycine (59 - 67 %) labeling in position C1. These data indicate the
modified carbon flux. Disruption of SHM2 led to a reduced growth rate in minimal medium .
Supplementation with 1 mM adenine restored wild-type growth, which showed that growth of
AgΔSHM2 is one-carbon limited.
Since overexpression of the threonine aldolase gene had been shown to replace glycine by
threonine limitation of riboflavin synthesis, the second objective of this thesis was a
deregulation of threonine biosynthesis in A. gossypii. By heterologous complementation of a
Saccharomyces cerevisiae mutant showing threonine auxotrophy the AgHOM3 gene
encoding a monofunctional aspartate kinase was rescued . In crude extracts of A. gossypii an
aspartate kinase specific activity of 5 mU/mg protein was detected. A mutant disrupted in
HOM3 lost this enzyme activity and showed homoserine auxotrophy. The riboflavin
production of AgΔHOM3 was significantly increased, when the growth was homoserine
limited . Supplementation of the medium with homoserine restored wild-type growth but at the
same time reduced production of riboflavin below wild-type level . Presumably an enhanced
induction of the RIB genes was the reason for the increased riboflavin production of
AgΔHOM3.
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