6.3 Natural Yeast Promoters 115Carbon catabolite repression is the set of regulatory mechanisms forcing cells
to preferentially use glucose and fructose over other carbon sources (reviewed in
[69, 70]). For example, the GAL circuit is fully repressed by glucose, even when
galactose is present in the culture medium [51]. Carbon catabolite repression
affects the levels and activity of enzymes involved in energy metabolism. We can
distinguish two main ways of transcription initiation repression by glucose. The
first way is direct, when the presence of glucose triggers the recruitment of tran-
scription repressors to the target promoters. Mig1 represses transcription initia-
tion via Cyc8–Tup1. When glucose is depleted, Mig1 is phosphorylated by Snf1
and is consequently relocalized to the cytoplasm, thus abolishing repression of
the target promoters [69]. A well‐characterized target of Mig1 is GAL1, which
contains the cognate TFBS upstream of its TATA element [54, 55]. The second
way is indirect, when transcription repression is achieved by inactivating tran-
scription activators. For example, Adr1 is inactive in the presence of glucose;
therefore it cannot trigger transcription initiation [71]. A well‐characterized
natural target of this transcription activator is alcohol dehydrogenase 2 (ADH2),
which is repressed when yeast is grown in glucose [72]. When the TFBS recog-
nized by Adr1 is cloned in front of the fermentative alcohol dehydrogenase 1
(ADH1) gene, its expression shows catabolite repression [15]. Carbon catabolite
repression can be exploited to repress genes of interest until glucose is depleted
in the culture medium. Besides the ADH2 promoter [73], that of JEN1, the main
lactate and pyruvate transporter, is also used. The JEN1 promoter was initially
selected to construct biosensors for measuring sugar concentrations, since it
reacts specifically to carbon sources and is insensitive to most types of cell
stresses [74].
6.3.2 Constitutive Promoters
A constitutive promoter displays a relatively constant activity that is not signifi-
cantly altered by stimuli. In most cases, the activity of constitutive promoters is
coupled to the growth rate, which depends on the level of glucose, the preferred
carbon source of yeast [69, 75, 76]. This constant transcription is ensured by a
complex combination of TFBSs.
The most used constitutive promoters belong to genes involved in primary cell
metabolism such as glycolysis and fermentation. The main reason for this selec-
tion is historical; mutations affecting these genes were relatively easily isolated
and characterized. The regulation of the expression of glycolytic and fermen-
tative enzymes takes place mainly at the transcriptional level and correlates
with glucose concentration and growth curve stage [76–80]. The promoters of
these genes share common TFBSs recognized by the regulators Rap1 and Gcr1
[29, 81, 82], which ensure coordinated transcription [83, 84]. The promoters of
phosphoglycerate kinase 1 (PGK1) and glyceraldehyde‐3‐phosphate dehydroge-
nase 3 (TDH3) are among the strongest ones known [77, 80]. The promoter of
the fermentative ADH1 was one of the first used to overexpress a heterologous
protein in yeast [5]. A vector containing this promoter was also used to produce
the human hepatitis B vaccine [7]. Although considered strong, the ADH1
promoter is weaker than those of PGK1 or TDH3 [9, 80].