[15]. The binding of rtTA to an eukaryotic promoter allows transcriptional activation, but this is prevented by its interaction with
doxycycline. This regulatory circuit, known as Tet-off, was changed
to a Tet-on system by further modifications in the rtTA protein, so
that it is only able to recognize the target promoter sequence when it
is bound to doxycycline (reviewed in Ref. [16]). In addition, the
promoter sequence was improved by the addition of several TetO
binding elements (tetO), resulting in an optimized doxycyclineresponsive promoter [17, 18]. Therefore, the use of the Tet-on
promoter system allows the control of transcription of any target
gene through the addition of doxycycline to the medium (Fig. 1).
We show that the Tet-on system may be used to modulate
carotenoid biosynthesis in fungi through the controlled expression
of a key regulatory gene. To check the efficiency of this methodology, we have constructed a Tet-on regulated version of carS and we
have introduced it in a carS mutant by transformation. We show
that carotenoid production is repressed by the addition of doxycycline to the medium through the activation of the negative regulator CarS. This method may be particularly useful for the controlled
expression of genes for bottleneck steps in the carotenoid pathways.
2 Materials
2.1 Construction of
Plasmids pPO3 and
pPO5
1. Plasmid pLR14 [12], containing gene carS from Fusarium
fujikuroi.
2. Plasmid pGEM
® -T easy (Promega) for PCR fragment cloning.
3. Plasmid pVG3 [19].
4. Primers carS-PmeI-1F 5
0 -CCAGTTTAAACGTACTATACG
CACGCAAT-3
0 and carS-PmeI-1R: 5
0 -CGAGTTTAAACGTA
Fig. 1 Control of transcription of carS gene by doxycycline using Tet-on system. The Tet-on system consists in
two modules: (a) Transactivator module: Transactivator rtTA2 is constitutively expressed under the control of
Aspergillus glyceralhehyde-3P dehydrogenase promoter (PgpdA). (b) Expression module: A gene of interest is
expressed under the doxycycline-dependent Tet-on promoter. In our case, this gene is carS, that encodes a
down-regulator of carotenoid gene expression. In the presence of Dox (represented by orange circles), rtTA
activates the Tet-on promoter and the gene carS is transcribed. In the absence of Dox, rtTa is not active and
low transcription is expected. Dox: doxycycline
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
345
doxycycline. This regulatory circuit, known as Tet-off, was changed
to a Tet-on system by further modifications in the rtTA protein, so
that it is only able to recognize the target promoter sequence when it
is bound to doxycycline (reviewed in Ref. [16]). In addition, the
promoter sequence was improved by the addition of several TetO
binding elements (tetO), resulting in an optimized doxycyclineresponsive promoter [17, 18]. Therefore, the use of the Tet-on
promoter system allows the control of transcription of any target
gene through the addition of doxycycline to the medium (Fig. 1).
We show that the Tet-on system may be used to modulate
carotenoid biosynthesis in fungi through the controlled expression
of a key regulatory gene. To check the efficiency of this methodology, we have constructed a Tet-on regulated version of carS and we
have introduced it in a carS mutant by transformation. We show
that carotenoid production is repressed by the addition of doxycycline to the medium through the activation of the negative regulator CarS. This method may be particularly useful for the controlled
expression of genes for bottleneck steps in the carotenoid pathways.
2 Materials
2.1 Construction of
Plasmids pPO3 and
pPO5
1. Plasmid pLR14 [12], containing gene carS from Fusarium
fujikuroi.
2. Plasmid pGEM
® -T easy (Promega) for PCR fragment cloning.
3. Plasmid pVG3 [19].
4. Primers carS-PmeI-1F 5
0 -CCAGTTTAAACGTACTATACG
CACGCAAT-3
0 and carS-PmeI-1R: 5
0 -CGAGTTTAAACGTA
Fig. 1 Control of transcription of carS gene by doxycycline using Tet-on system. The Tet-on system consists in
two modules: (a) Transactivator module: Transactivator rtTA2 is constitutively expressed under the control of
Aspergillus glyceralhehyde-3P dehydrogenase promoter (PgpdA). (b) Expression module: A gene of interest is
expressed under the doxycycline-dependent Tet-on promoter. In our case, this gene is carS, that encodes a
down-regulator of carotenoid gene expression. In the presence of Dox (represented by orange circles), rtTA
activates the Tet-on promoter and the gene carS is transcribed. In the absence of Dox, rtTa is not active and
low transcription is expected. Dox: doxycycline
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
345