Verlag des Forschungszentrums Jülich
JUEL-3519 Biosynthesis of Glycine as a Precursor of Riboflavin in Ashbya gossypii The filamentous
fungus Ashbya gossypii is a vitamin B2 overproducer. Enhancement of riboflavin
production is known to be achieved by supplementation of the medium with glycine, a
precursor of vitamin B2 (riboflavin). The present work describes the
characterization and deregulation of glycine biosynthetic pathways in A. gossypii leading
to an improved riboflavin production. In the A. gossypii wild type strain ATCC 10895
supplementation with 80 mM glycine lead to an increase in riboflavin production by at
least 100 % whereas the growth remained unchanged. Nevertheless only 5 % of the added
glycine were consumed during the course of cultivation, which suggested a poor uptake of
the amino acid. On unsupplemented media glycine concentration even increased from 1.9 mM
to 3 mM during cultivation. Consequently the effect of glycine on riboflavin production
can be attributed to a small net uptake as well as to an inhibition of glycine efflux due
to the high extracellular glycine concentration. The glycine biosynthetic enzymes serine
hydroxymethyltransferase, threonine aldolase and glutamate glyoxylate aminotransferase
were detected in crude extracts of A. gossypii with maximum specific activities of 6, 5
and 26 mU/mg protein, respectively. Sucrose density gradient centrifugation of A. gossypii
organelles showed, that glutamate glyoxylate aminotransferase occurs in the mitochondria
of the fungus, thus it is not colocated with isocitrate lyase - the main supplier of
glyoxylate - in the peroxisomes. Glycine formation starting from serine and threonine
could also be demonstrated in vivo using 13C labelling experiments. Likewise
the formation of serine from threonine, which probably proceeds via glycine and therefore
means an unwanted loss of glycine, was shown in vivo. When 70 mM aminomethylphosphonic
acid (AMPS) were added to the culture medium riboflavin production of A. gossypii was
completely inhibited. Screening on AMPS resistance of riboflavin production lead to the
isolation of the strain A.g. AMPS-NM-01. It showed a riboflavin production of 40 mg/g
mycelial dry weight (mdw) even in the absence of glycine, which was significantly higher
than in the wild type strain (5 mg/g mdw) under the same conditions but resembled wild
type riboflavin production in the presence of 80 mM of glycine (30 mg/g mdw). Increased
riboflavin production without glycine supplementation suggested a better intracellular
availability of glycine in the mutant strain. Nevertheless, even in this case riboflavin
production could be increased by glycine supplementation to 95 mg/g mdw. In comparison to
the wild type strain serine hydroxy-methyltransferase specific activity was significantly
reduced from 3 to 1.5 mU/mg protein in the strain A.g. AMPS-NM-01. Therefore the increased
riboflavin production of this strain can be explained by a better intracellular
availability of glycine conditioned by a reduced loss of glycine for the formation of
serine. Using heterologous complementation of a Saccharomyces cerevisiae mutant
auxotrophic for glycine a GLY1 homologous gene with unknown function was isolated from A.
gossypii. Characterization of the corresponding enzymatic activity showed that the
isolated gene as well as the GLY1 gene from S. cerevisiae encode a threonine aldolase. In
contrast to S. cerevisiae the GLY1 knock-out mutant of A. gossypii was not auxotrophic for
glycine, which demonstrated that threonine aldolase plays only a minor role during glycine
biosynthesis of A. gossypii. GLY1 was overexpressed in A. gossypii under the control of
the TEF-promotor and -terminator using the expression vector pAG203. In crude extracts of
A.g. pAG203GLY1 50 mU/mg protein of threonine aldolase specific activity were detected
indicating a tenfold overexpression in comparison to the wild type. When 50 mM threonine
was fed to A.g. pAG203GLY1 an increase in riboflavin production from 2 to 16 mg/g mdw was
determined, an increase never reached with glycine because of its worse uptake. Threonine
was found to be taken up efficiently by this strain. Its conversion to glycine was
confirmed by a striking efflux of glycine into the medium. Extracellular glycine
correspondingly increased from 2 to 44 mM. The requirement to feed threonine in addition
to threonine aldolase overexpression demonstrated a limitation in threonine biosynthesis,
which was confirmed by feeding experiments with threonine precursors.
Monschau, Nicole
Untersuchungen zur Biosynthese von Glycin als Vorstufe von Riboflavin in Ashbya gossypii
115 S., 1998
Neuerscheinungen
Schriften des Forschungszentrums Jülich
Ihre Ansprechperson
Heike Lexis
+49 2461 61-5367
zb-publikation@fz-juelich.de