Verlag des Forschungszentrums Jülich
JUEL-3995
Schlösser, Thomas
Molekularbiologische Untersuchungen zur Regulation der an der Riboflavinbiosynthese beteiligten Gene bei Ashbya gossypii
98 S., 2002
Ashbya gossypii is a natural overproducer of riboflavin (vitamin 82). Nothing has yet been
published on the regulation of the genes involved in riboflavin biosynthesis. First indications
that a global regulation mechanism is involved in regulating the RIB-genes were obtained by
supplementation experiments with cAMP. The cAMP analogs 8-bromo-cAMP, dibutyryl-
cAMP, and chlorophenylthio-cAMP led to a complete inhibition of riboflavin formation in
concentrations of 5 mM. Supplementation of the medium with cGMP, AMP, and 12-0-
tetradecanoyl-phorbol-13-acetate had no influence on riboflavin formation. Presumably a
protein kinase A (PKA) mediated signal transduction pathway is involved in the regulation of
the riboflavin biosynthesis pathway. RT -P CR analysis showed a strong increase in relative
mRNA concentrations of the genes RIB3, RIB4, and RIB5 after cultivation for 24 h in the
riboflavin production phase in comparison to the growth phase after cultivation for 8 h. An
increase in the mRNA concentration of these genes was observed in comparison to the
constitutively expressed genes TEF or ACT1. The investigation of relative RIB2 and RIB7
mRNA concentrations revealed on I y a weak (RIB2) or no increase (RIB7) in the production
phase, respectively. A reproducible detection of RIB1-mRNA was not possible. The observed
induction of the genes RIB3, RIB4, and RIB5 in the production phase is consistent with the
stoichiometry of riboflavin biosynthesis. The corresponding enzymes 3,4-dihydroxy-2-
butanone 4-phosphate synthase, lumazin synthase, and riboflavin synthase are used twice
for the formation of one riboflavin molecule. Expression of the RIB3 gene was investigated in
more detail by an independent approach. Reporter studies with a RIB3 promoter-lacZfusion
showed on average an 8-fold increase of β-galactosidase specific activity in the production
phase. The increase of specific activity was observed in strains transformed with a reporter
plasmid as weil as in site-directed transformants. For a better resolution of growth and
production phase a fermenter cultivation of the strain AgRIB3placZ-1 was performed. The
increase of relative RIB3 mRNA concentrations and reporter activity in the late growth phase
correlated with the beginning of riboflavin overproduction which was triggered by a decline in
growth rate. Such a regulatory mechanism has never been described for a RIB3 gene
encoding the key enzyme in riboflavin biosynthesis. By gradual deletion of the RIB3 promoter
a regulatory region was found between -250 bp and -200 bp upstream of the initiation
codon. For further research on the regulation mechanism of riboflavin biosynthesis a strategy
for isolating a transcription factor gene was evolved. A genomic library of Ashbya gossypii
was used to mediate expression of an RIB3 promoter-lacZ reporter plasmid in the host
Saccharomyces cerevisiae.
Neuerscheinungen
Schriften des Forschungszentrums Jülich
Ihre Ansprechperson
Heike Lexis
+49 2461 61-5367
zb-publikation@fz-juelich.de