product. We formerly described methods for the analysis of this
xanthophyll and its precursor carotenoids [5, 6]. Moreover, we
have identified all the genes and enzymes involved in this biosynthetic pathway and the molecular mechanisms of its regulation are
currently under investigation. The mRNA levels of the structural
genes increase in the presence of light [7] or in the absence of
nitrogen [8], but they are kept under low levels under standard
growth conditions. The main control of carotenogenesis is done by
a negative regulator, CarS, whose mutation results in the accumulation of large amounts of mRNA for the structural genes of the
pathway and a high carotenoid biosynthetic activity. The gene carS
codes for a protein of the RING finger (RF) family, with sequence
similarity with protein CrgA of M. circinelloides, whose mutation
results in a similar carotenoid overproducing pattern in this fungus
[9, 10]. The degree of similarity between the CarS and CrgA
proteins is not very high, as expected for two taxonomically distant
fungi, but it covers the most relevant CrgA domains, which include
two amino-terminal RF domains, and a LON protease domain
[11]. At least one of the RF domains of CrgA is essential for its
regulatory function in carotenogenesis, and this seems to be also
the case of CarS, as indicates the occurrence of mutations in a RF
domain in two independent carotenoid overproducing carS
mutants [12]. In other proteins, RF finger domains are typical of
ubiquitin ligases (E3) which together with ubiquitin conjugating
enzymes (E2) mediate ubiquitination of target proteins [13]. This
labeling process frequently is a signal for protein degradation but
also is a mode of regulation. This suggests that CarS might function
as an E3 ubiquitin ligase.
The biological properties of neurosporaxanthin are unknown,
and its potential applications in food or feed industries remain to be
investigated. The development of new techniques to control the
expression of genes related to carotenoid metabolism are a
promising approach to modulate carotenoid production of fungi.
A method of inducible gene expression that allows transcription to
be reversibly turned on or off is known as Tetracycline-Controlled
Transcriptional Activation. This method is based on a negative
regulatory mechanism governing resistance to tetracycline in
Gram-negative bacteria. The regulatory circuit in Escherichia coli
consists of the repression of the Tn10 tetracycline resistance operon
by the binding of the TetR repressor protein to its operator
sequence, TetO [14]. Because of its higher stability, the tetracycline
analog doxycycline is usually used as the inducer signal under
laboratory conditions. In the presence of doxycycline, the antibiotic
binds TetR, provoking its dissociation from TetO and allowing the
transcription of the operon. TetR was adapted for its use in eukaryotic cells with the opposite regulatory role: it was fused to a transcriptional activation domain from the herpes simplex virus protein
16 (VP16) converting it into a hybrid transactivator, known as rtTA
344
Julia Marente et al.
xanthophyll and its precursor carotenoids [5, 6]. Moreover, we
have identified all the genes and enzymes involved in this biosynthetic pathway and the molecular mechanisms of its regulation are
currently under investigation. The mRNA levels of the structural
genes increase in the presence of light [7] or in the absence of
nitrogen [8], but they are kept under low levels under standard
growth conditions. The main control of carotenogenesis is done by
a negative regulator, CarS, whose mutation results in the accumulation of large amounts of mRNA for the structural genes of the
pathway and a high carotenoid biosynthetic activity. The gene carS
codes for a protein of the RING finger (RF) family, with sequence
similarity with protein CrgA of M. circinelloides, whose mutation
results in a similar carotenoid overproducing pattern in this fungus
[9, 10]. The degree of similarity between the CarS and CrgA
proteins is not very high, as expected for two taxonomically distant
fungi, but it covers the most relevant CrgA domains, which include
two amino-terminal RF domains, and a LON protease domain
[11]. At least one of the RF domains of CrgA is essential for its
regulatory function in carotenogenesis, and this seems to be also
the case of CarS, as indicates the occurrence of mutations in a RF
domain in two independent carotenoid overproducing carS
mutants [12]. In other proteins, RF finger domains are typical of
ubiquitin ligases (E3) which together with ubiquitin conjugating
enzymes (E2) mediate ubiquitination of target proteins [13]. This
labeling process frequently is a signal for protein degradation but
also is a mode of regulation. This suggests that CarS might function
as an E3 ubiquitin ligase.
The biological properties of neurosporaxanthin are unknown,
and its potential applications in food or feed industries remain to be
investigated. The development of new techniques to control the
expression of genes related to carotenoid metabolism are a
promising approach to modulate carotenoid production of fungi.
A method of inducible gene expression that allows transcription to
be reversibly turned on or off is known as Tetracycline-Controlled
Transcriptional Activation. This method is based on a negative
regulatory mechanism governing resistance to tetracycline in
Gram-negative bacteria. The regulatory circuit in Escherichia coli
consists of the repression of the Tn10 tetracycline resistance operon
by the binding of the TetR repressor protein to its operator
sequence, TetO [14]. Because of its higher stability, the tetracycline
analog doxycycline is usually used as the inducer signal under
laboratory conditions. In the presence of doxycycline, the antibiotic
binds TetR, provoking its dissociation from TetO and allowing the
transcription of the operon. TetR was adapted for its use in eukaryotic cells with the opposite regulatory role: it was fused to a transcriptional activation domain from the herpes simplex virus protein
16 (VP16) converting it into a hybrid transactivator, known as rtTA
344
Julia Marente et al.