Báo cáo khoa học: Experimental and steady-state analysis of the GAL regulatory system in Kluyveromyces lactis docx

Experimental and steady-state analysis of the GAL
regulatory system in Kluyveromyces lactis
Venkat R. Pannala, Sharad Bhartiya and Kareenhalli V. Venkatesh
Department of Chemical Engineering, Indian Institute of Technology, Bombay, Mumbai, India

Keywords
galactose; GAL system; Kluyveromyces
lactis; Saccharomyces cerevisiae; steadystate model
Correspondence
S. Bhartiya ⁄ K. V. Venkatesh, Department of
Chemical Engineering, Indian Institute of
Technology, Bombay, Powai, Mumbai400076, India
Fax: 91 22 25726895
Tel: 91 22 25767225
E-mail:
(Received 10 December 2009, revised
7 May 2010, accepted 12 May 2010)
doi:10.1111/j.1742-4658.2010.07708.x

The galactose uptake mechanism in yeast is a well-studied regulatory network. The regulatory players in the galactose regulatory mechanism (GAL
system) are conserved in Saccharomyces cerevisiae and Kluyveromyces
lactis, but the molecular mechanisms that occur as a result of the molecular
interactions between them are different. The key differences in the GAL
system of K. lactis relative to that of S. cerevisiae are: (a) the autoregulation of KlGAL4; (b) the dual role of KlGal1p as a metabolizing enzyme as
well as a galactose-sensing protein; (c) the shuttling of KlGal1p between
nucleus and cytoplasm; and (d) the nuclear confinement of KlGal80p.
A steady-state model was used to elucidate the roles of these molecular
mechanisms in the transcriptional response of the GAL system. The steadystate results were validated experimentally using measurements of b-galactosidase to represent the expression for genes having two binding sites. The
results showed that the autoregulation of the synthesis of activator
KlGal4p is responsible for the leaky expression of GAL genes, even at high
glucose concentrations. Furthermore, GAL gene expression in K. lactis

shows low expression levels because of the limiting function of the bifunctional protein KlGal1p towards the induction process in order to cope with
the need for the metabolism of lactose ⁄ galactose. The steady-state model of
the GAL system of K. lactis provides an opportunity to compare with the
design prevailing in S. cerevisiae. The comparison indicates that the existence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevisiae as a result of genome duplication has resulted in a system which
metabolizes galactose efficiently.

Introduction
Galactose metabolism in microorganisms occurs
through a well-conserved metabolic pathway which is
tightly regulated. For example, both Saccharomyces
cerevisiae and Kluyveromyces lactis utilize galactose as
an alternative carbon and energy source in the absence
of glucose in the environment. The uptake of galactose
is governed by the well-known Leloir pathway using
enzymes produced via the GAL switch [1]. When galactose is the sole carbon source, the induction and transcription of GAL genes occur via the interplay between

three regulatory proteins, namely Gal4p, Gal80p and
Gal3p ⁄ Gal1p [2–5]. The activator protein (Gal4p)
binds to the upstream activator sequence (UASG) of
each gene for transcription to proceed. The transcription process is inhibited by a repressor protein Gal80p
which binds to the C-terminal activation domain of
Gal4p. However, in the presence of galactose, this
repression is relieved by the inducer protein Gal3p ⁄
Gal1p. In contrast, glucose represses the ability of
galactose to activate the GAL system by multiple

Abbreviations
NINR, noninducing, nonrepressing; UAS, upstream activator sequence; URS, upstream repressor sequence; YPD, yeast–peptone–dextrose.

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V. R. Pannala et al.

mechanisms, and thus terminates the activation of
GAL genes [6–8]. Although the regulatory players are
conserved in various organisms, the molecular mechanisms that occur as a result of the interactions between
them are different. For example, in K. lactis, the synthesis of the transcriptional activator protein KlGal4p
is autoregulated, but its expression is inhibited by glucose [9–11], whereas, in S. cerevisiae, ScGal4p synthesis
is not autoregulated, but its gene expression and activity are repressed and inhibited by glucose [10,12].
Although the GAL system of S. cerevisiae has been
well characterized, a similar degree of quantification
for the GAL system of K. lactis is absent in the
literature.
The GAL system in K. lactis contains two regulatory genes (LAC9 or KlGAL4 and KlGAL80), a
bifunctional gene KlGAL1 and four structural genes
(LAC12, LAC4, KlGAL7 and KlGAL10). The GAL
switch is found in three regulatory states in response
to the availability of various carbon sources. In the
presence of a noninducing, nonrepressing (NINR)
medium, such as glycerol or raffinose, the GAL switch
is in a noninduced state. Under such a condition,
KlGal4p activity is inhibited by the binding of
KlGal80p protein to the C-terminal activation domain
of KlGal4p. In this state, the GAL genes are poised
for induction as they are not subjected to carbon

catabolite repression. In the presence of lactose ⁄ galactose medium, the GAL switch is in an induced state.
The enzyme permease (Lac12p) transports lactose ⁄
galactose into the cytoplasm, which, in combination
with ATP, activate the protein KlGal1p. The protein
KlGal1p, being bifunctional, has both inducer and
galactokinase activity. The activated KlGal1p then
shuttles into the nucleus and interacts with the repressor protein KlGal80p to form a stable tetrameric complex (KlGal1p–KlGal80p2–KlGal1p), thereby relieving
the inhibition of KlGal80p on KlGal4p [13]. Further,
the regulatory proteins KlGal4p, KlGal80p and
KlGal1p are under the GAL promoter, and thus their
synthesis is dependent on the status of the GAL
switch, which in turn is a function of the concentrations of these regulatory proteins. This autocatalytic
effect caused by the feedbacks of the regulatory proteins on the switching of the GAL genes is termed
‘autoregulation’. Although KlGal4p and KlGal1p,
as activators, constitute positive feedback loops,
KlGal80p, as an inhibitor, imparts a negative feedback. Autoregulation, as a molecular mechanism, is
known to yield system level properties, such as signal
amplification and ultrasensitivity [14]. In the presence
of glucose, the GAL switch is in a repressed state. In
S. cerevisiae, glucose represses GAL genes via a
2988

specific repressor protein Mig1p, which binds to the
upstream repressor sequences (URSG) present in GAL
genes [7]. However, in the case of K. lactis, the repression of KLGAL4 is independent of Mig1p, as KlGAL4
has no URSG in its promoter for Mig1p, but glucose
indirectly represses the GAL system by a Mig1p binding site in the KlGAL1 gene [8,15]. Although KlGal4p
has no Mig1p binding site for its gene promoter, its
activity is inhibited directly in the presence of glucose.
It has been shown experimentally that glucose affects

the ability of KlGAL4 to activate the transcription of
GAL genes [16,17]. The activator Gal4p in yeast contains at least three inhibitory domains in its central
region between the activator domains, which become
active in the presence of glucose, but, however, are
independent of the repressor Mig1p [10].
In all of the above three states, the concentration of
the activator KlGal4p plays a vital role in the induction mechanism of the GAL system. The KlGAL4 gene
contains a UASG in its own promoter for the binding
of KlGal4p, resulting in an autoregulatory circuit
which causes a two- to five-fold increase in KlGal4p
concentration in the presence of lactose ⁄ galactose. This
increase is essential for the maximal growth rate on
lactose and has probably evolved to give the organism
a selective advantage in its natural habitat [9]. However, to maintain the repressed state of KlGAL4controlled genes in a glucose-containing medium, the
KlGal4p concentration must be held below a certain
threshold concentration [11]. Although experimental
studies of the K. lactis GAL system have determined
the regulatory components, uncertainties exist in the
way in which these components interact with each
other and their compartmentalization. Until recently,
it was believed that the K. lactis GAL system operated
in a similar manner to the S. cerevisiae GAL system,
where the repressor ScGal80p shuttles between the
nucleus and the cytoplasm [4,18]. However, it was later
shown that, in K. lactis, it is the bifunctional protein
KlGal1p that shuttles between the nucleus and the
cytoplasm [13]. The key differences in the GAL systems
of K. lactis and S. cerevisiae are as follows: (a) the
autoregulation of transcriptional activator KlGal4p;
(b) the dual role of KlGal1p as a metabolizing enzyme

as well as a galactose-sensing protein; (c) the shuttling
of KlGal1p between nucleus and cytoplasm; (d) the
nuclear confinement of KlGal80p; and (e) the fact that
KlGAL4 is the only gene in the GAL system with one
binding site, with the remaining genes having two
binding sites.
Although S. cerevisiae and K. lactis utilize similar
molecular components in the GAL network, the architecture in the organisms differs substantially. Further-

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V. R. Pannala et al.

more, the parameter values also play a role in the
performance of the GAL system in the two yeasts. It
should be noted that K. lactis utilizes the GAL network to metabolize mainly lactose, whereas S. cerevisiae uses it to metabolize melibiose and galactose.
This evolutionary fact also plays a role in the performance of the two networks. Given the above differences in the two GAL networks, it is of interest to
compare the steady-state performances of the networks in S. cerevisiae and K. lactis in response to
galactose and glucose.
We used a steady-state modeling approach to quantify the underlying molecular mechanism for the GAL
system of K. lactis and to obtain a systems’ level
understanding of its behavior. The steady-state model
for the GAL system of K. lactis was validated experimentally by obtaining steady-state protein expression
levels in a wild-type strain and in a mutant strain lacking gene KlGAL80. The steady-state model was then
used to delineate the importance of the autoregulation
of regulatory proteins and parametric sensitivity. Subsequently, we considered a KlGAL80 mutant strain of
K. lactis to determine the importance of the autoregulation of activator KlGal4p and glucose repression.
The K. lactis GAL system model developed in this
work has been validated experimentally. As the systems’ level properties, such as ultrasensitivity and

memory, arising out of the various molecular mechanisms in S. cerevisiae have been well elucidated [19], it
was of interest to compare the steady-state performance of K. lactis with that of S. cerevisiae. Such a
comparison yields the significance of the various
molecular interactions in the two networks with similar
molecular components. The results showed that the
autoregulation of the activator protein Gal4p in
K. lactis is responsible for the leaky expression of GAL
genes, even at high glucose concentration. The comparison indicates that the existence of a protein Gal3p
in S. cerevisiae, dedicated for the sensing of galactose,
arising as a result of genome duplication has resulted
in a system which metabolizes galactose efficiently. We
begin by describing the key features of the model
developed for the wild-type strain of K. lactis, the
detailed equations for which are provided in Supporting information.

Model development
All molecular interactions in the K. lactis GAL system
that have been included in the steady-state model are
shown schematically in Fig. 1. D1 in Fig. 1 represents
the gene LAC9 ⁄ KlGAL4, with one binding site in its
promoter for KlGal4p, whereas the other genes

GAL system in K. lactis

(LAC12, LAC4, KlGAL7, KlGAL10 and KlGAL1),
which have two or more binding sites, are shown as
D2. The activator KlGal4p dimerizes with a dissociation constant K1 and subsequently binds to the operator site of the gene KlGAL4 (D1) with a dissociation
constant Kd:
ẵKlGal4p ỵ ẵKlGal4p  ẵKlGal4p2

1ị

ẵD1 ỵ ẵKlGal4p2  ẵD1 KlGal4p2 Š

ð2Þ

K1

Kd

For genes with two binding sites (D2), dimer Gal4p
binds to the first site with a dissociation constant of
Kd, followed by binding to the second site with a dissociation constant of Kd ⁄ m, where the factor m (>1)
quantifies the cooperative effect of binding of KlGal4p
to the second binding site [3]:
ẵD2 ỵ ẵKlGal4p2 Â ẵD2 KlGal4p2
Kd

3ị

ẵD2 KlGal4p2 ỵ ẵKlGal4p2 Â
Kd
m

4ị

ẵD2 KlGal4p2 KlGal4p2 Š
In the absence of galactose, the repressor protein
KlGal80p dimerizes with a dissociation constant K2
and binds with DNA-bound KlGal4p to inhibit

the transcriptional process. For example, KlGal80p2
interaction with D1–KlGal4p2 can be written as
follows:
ẵKlGal80p2 ỵ ẵD1 KlGal4p2 Â
K3

5ị

ẵD1 KlGal4p2 À KlGal80p2 Š
Similarly, the remaining interactions of KlGal80p2
with DNA–KlGal4p2 complexes can be written (see
Supporting information for details).
In the presence of galactose and ATP, the inducer
KlGal1p is activated, which is ultimately responsible
for relieving the repression of the GAL system by
KlGal80p. The activation of the inducer KlGal1p can
be quantified using a steady-state saturation function
given by [18]:


Gal
6ị
ẵKlGal1p ẳ ẵKlGal1pt
t
Ks ỵ Gal
where ẵKlGal1p represents total activated KlGal1p
t
and [KlGal1p]t represents total KlGal1p concentration.
Ks represents the half-saturation constant for the activation of KlGal1p by galactose (Gal). The activated

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V. R. Pannala et al.

A
0
4

KlGal4p2

0
80

KlGal80p2

4

KlGal4p

12

80

KlGal80p

Lac12p

1

KlGal1p
Galactose

KlGal1p-Galint

Galactose
12
1
ATP

K
K4
Cytoplasm

4

4
K4

80

K1
K3

K2

4

80

Kd
K3

80

GAL genes

UASG
0
4

D1

4
GAL genes

UASG

B

Nucleus

D2

Galactose
12

1
ATP

K
4

4
K1
Cytoplasm

4

80

Kd
GAL genes

Nucleus

UASG
0
4

D1

4
GAL genes

UASG

D2

Fig. 1. (A) Schematic diagram showing the molecular interactions in a Kluyveromyces lactis wild-type strain. (B) GAL system in a K. lactis
strain lacking GAL80. Here, Ki (i = 1–4) represents the dissociation constant for the respective interactions, K represents the distribution
coefficient for KlGal1p shuttling and Kd represents the binding of KlGal4p protein to the DNA. ‘m’ represents the degree of cooperativity. D1
and D2 represent genes with one and two binding sites, respectively.

KlGal1p shuttles into the nucleus with a distribution
coefficient K (see Fig. 1) and is defined as the ratio of
activated KlGal1p in the cytoplasm to the nucleus:
Kẳ

ẵKlGal1p
c
ẵKlGal1p
n

ẵKlGal1p ỵ ẵKlGal80p  ẵKlGal1p KlGal80p
n
n
K4

8ị

7ị

The monomeric form of activated KlGal1p in the
nucleus interacts with the monomeric form of the
2990

repressor KlGal80p with a dissociation constant of K4
as shown in Fig. 1.

The monomeric form of the activated KlGal1p is
known to interact with KlGal80p2 with a positive cooperativity, resulting in a reduction in the dissociation
constant by two (i.e. K4 ⁄ 2) [13]:

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V. R. Pannala et al.

GAL system in K. lactis

ẵKlGal1p ỵ ẵKlGal80p2 Â ẵKlGal1p KlGal80p2 9ị
n
n
K4
2

Furthermore, two monomers of activated KlGal1p
can also interact with the dimer KlGal80p to form
a heterotetrameric complex ½KlGal1pà À KlGal80p2 À
n
KlGal1pà Š with a negative cooperativity, which results
n
in an increase in the dissociation constant by two (i.e.
2K4) [13]:
ẵKlGal1p KlGal80p2 ỵ ẵKlGal1pn Â

n
2K4

ẵKlGal1p
n

KlGal80p2

KlGal1p
n

10ị

The net result of all of these interactions relieves the
inhibition of repression on activator KlGal4p, which
allows the transcription to proceed. The complete
detailed equations for all interactions are given in Supporting information.
Based on the mechanisms shown above, we can
obtain the fractional protein expressions for genes with
one binding site and two binding sites by applying
equilibrium and mass balance equations. Thus, we
define the fractional transcriptional expressions f1 and
f2 as the ratio of mRNA that is transcribed in response
to an input stimulus to the maximum capacity of
mRNA that could be transcribed by the system for
genes with one and two binding sites, respectively. The
fractional transcriptional expressions for genes with
one binding site (D1) and two binding sites (D2) are
given as follows:
f1 ẳ

f2 ẳ

ẵD1 KlGal4p2
D1t

ẵD2 KlGal4p2 ỵ ẵD2 KlGal4p2 À KlGal4p2 Š
D2t

ð11Þ

ð12Þ

where D1t and D2t are the total operator concentrations of genes with one and two binding sites,
respectively. As shown in Fig. 1, [D1–KlGal4p2],
[D2–KlGal4p2] and [D2–KlGal4p2–KlGal4p2] represent
the concentrations of the complexes formed as a result
of the interactions between the genes (D1 and D2) and
KlGal4p2. It should be noted that, in the definition of
f2 [Eqn (12)], it is assumed that the transcriptional
capacity of a D2–KlGal4p2 complex is equal to that of
a D2–KlGal4p2–KlGal4p2 complex. However, the cooperativity in binding to the second site [parameter m
in Eqn (4)] ensures that the complex D2–KlGal4p2–
KlGal4p2 dominates the gene expression quantified by
f2. The fractional protein expression fip, that is the
ratio of protein Pi synthesized for a given transcriptional expression to the maximum expression possible

Pmax, is related to the fractional transcriptional expression as follows [18,20]:
fip ¼

Pi
¼ fin ;
Pmax

for i ẳ 1; 2

13ị

where n is the co-response coefficient and is defined as
the ratio of the log-fold change in protein expression
to the log-fold change in mRNA expression [21]. In
prokaryotes, the typical value of n is close to unity,
indicating that the translational process is quite efficient. It has been shown through microarray experiments that n has a value in the range 0.5–0.75 for
protein expression from genes in S. cerevisiae [22]. Specifically, the GAL genes in S. cerevisiae show an average co-response coefficient of around 0.7 [18]. In this
work, we have assumed a value of 0.7 as the corresponding coefficient in K. lactis. As the gene KlGAL4
with one binding site is autoregulated; the total
KlGal4p concentration (KlGal4pt) is therefore a function of f1p. Further, the autoregulation of KlGAL4
makes it imperative that a basal amount of KlGal4pt0,
necessary to activate the switch from a completely
repressed state (i.e. f1p = 0), exists. Thus, the total
KlGal4pt concentration is dependent on f1p and is
modeled as follows [14]:


14ị
ẵKlGal4pt ẳ ẵKlGal4pt0 1 ỵ f1p q
where q represents the fold-change in [KlGal4pt] from
a noninduced state to a completely induced state corresponding to f1p = 1. Experiments have indicated a
two- to five-fold change in KlGal4p concentration on
induction [9], and we have assumed a value of five for

the parameter q. [KlGal4pt0] represents the basal
KlGal4pt concentration in the noninduced state.
As KlGal80p and KlGal1p are autoregulated with
two binding sites for KlGal4p, their individual total
concentrations can be related to the status of the
switch through f2p [see Eqn (13)] as given below:
½KlGal1pŠt ẳ f2p ẵKlGal1pmax and ẵKlGal80pt
ẳ f2p ẵKlGal80pmax

15ị

The model equations are obtained assuming that
all molecular interactions (as shown in Fig. 1) are at
equilibrium and using total molar balances for the
components together with the constraint imposed by
Eqn (15). All component concentrations are based on
a cell volume of 23 fL [13]. The model consists of 23
concentrations of various complexes, together with
the two transcriptional expressions (f1 and f2) and
two corresponding protein expressions (f1p and f2p).

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V. R. Pannala et al.

Table 1. Parameter values used in the steady-state model.

Parametera

Kluyveromyces
lactis

Source

Kd
K1
K2
K3
K4
K
Ki
Ks
D1t
D2t
m
[KlGal4p]t0
[KlGal4p]t
KlGal80max
KlGal1pmax
n
q
gg

0.62 nM
730 nM

0.1 nM
0.5 nM
83 nM
8
1.13 mM
9 mM
0.072 nM
0.43 nM
10
6.95 nM
32.6 nM
170–340 nM
11000 nM
0.7
5
1.3

Fitted to data
Fitted to data
[13]
[13]
[13]
Fitted to data
Fitted to data
Fitted to data
Calculated
Calculated
Fitted to data
[13]b
Calculated

[13]b
[13]
Assumed [20]
Assumed [9]
Fitted to data

Saccharomyces
cerevisiae
0.2 nM
100 nM
0.1 nM
0.05 nM
0.063 nM
0.4
0.4 mM
1 mM
0.071
0.166
30
5.47 nM
1000 nM
5000 nM
0.5

a

The parameter values reported were based on the K. lactis cell
volume (23 fL). b The parameter values reported in the reference
were based on the K. lactis nucleus volume (2 fL). c The reported
parameter values are from [18,20].

These 27 variables are determined by 27 algebraic
equations (see detailed model development in Supporting information). The mass balance equations are
then solved by the ‘fsolve’ routine of MATLABª to
obtain the response of the GAL system as the fractional protein expression of genes with one (f1p) and
two (f2p) binding sites. Experiments were performed
on glucose and galactose as substrates to measure the
fractional b-galactosidase expression from the gene
LAC4 to quantify (f2p) and validate the developed
model. The equilibrium dissociation constants, cooperativity factor (m) and half-saturation constants
were obtained by fitting the steady-state protein
expressions measured experimentally at various
steady-state glucose and galactose concentrations.
Parameter optimization was performed using the optimization toolbox of MATLAB 7.5 of Math Works
Inc., MA, USA. The parameter values are summarized in Table 1.

Results
Steady-state model response for the wild-type
strain of K. lactis
Experiments were performed at different galactose
concentrations with glycerol as the background
2992

medium and the steady-state b-galactosidase activity
was measured. It should be noted that the b-galactosidase activity represents the protein expression from
a GAL gene with two binding sites for KlGal4p, and
its measurement was used to quantify f2p. The
dynamic profile of b-galactosidase expression for
three different galactose concentrations is shown in
Fig. 2A. The activity of b-galactosidase reached a

steady value after approximately 12 h. The steadystate value was obtained by averaging over the last
three time points from the individual fed-batch experiment. The steady-state values of the b-galactosidase
activity of cells grown at different galactose concentrations (0.002–0.44 m) are shown by squares in
Fig. 2B. These steady-state points represent the
means of three independent experiments at each
galactose concentration. The steady-state b-galactosidase activities are represented by the fractional
protein expressions by normalizing with a maximum
b-galactosidase activity observed in a mutant
K. lactis strain lacking KlGAL80. The steady-state
model was simulated to validate the protein expression profiles with respect to galactose. The full line
in Fig. 2B shows the simulated fractional expression
of proteins for genes with two binding sites (f2p)
which are also responsible for the synthesis of
b-galactosidase. The steady-state experimental data
were used to estimate the model parameters, as indicated in Table 1 (see model development section in
Supporting information for details). The fitted binding constants were of a similar order of magnitude
as those reported for Gal4p binding to GAL genes in
S. cerevisiae [20]. It should be noted that the model
is able to predict the experimental steady-state
response (full line in Fig. 2B). The broken line
depicts the model prediction of fractional protein
expression corresponding to genes with one binding
site. It is clear from Fig. 2B that genes with one
binding site show a leaky expression of 9% of the
maximum, even in the absence of galactose. However, the protein expression corresponding to genes
with two binding sites is tightly regulated by the
GAL switch, with basal expression levels of only 2%.
Furthermore, as shown in Fig. 2B, the maximum
expression in the wild-type strain in the presence of
high galactose concentration is only 37% and 35%

for one and two binding sites, respectively, relative
to the maximum possible expression when D2 is
completely bound by KlGal4p2 [see Eqn (12)]. This
maximum value can be achieved by a strain lacking
repressor KlGal80p. The steady-state GAL response
curves for one and two binding site genes (see
Fig. 2B) can be represented by the Hill equation:

FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS


V. R. Pannala et al.

GAL system in K. lactis

B 0.4
Fractional protein
expression

0.3
0.25
0.2
0.15
0.1
0.05
0

C

0

5

10
15
Time (h)

20

D

Concnetration (nM)

106
104
102
100

10–2
10−5

10−3
10−1
Galactose (M)

101

0.3
0.2

ηH = 1.12
ηH = 1.25

0.1
0
10−4

25

KlGAl4pt (nM)

Fractional betagal
expression

A

10−3

10−2 10−1 100
Galactose (M)

101

20
18
16

ηH = 1.12

14

12
10
10−5

10−3
10−1
Galactose (M)

101

Fig. 2. (A) Time course of fractional b-galactosidase expression in a typical fed-batch experiment to obtain steady-state expression values
for f2p in a Kluyveromyces lactis wild-type strain. Diamonds, squares and circles represent experiments with galactose concentrations of
0.022, 0.077 and 0.16 M, respectively. (B) Steady-state fractional protein expression with varying galactose concentrations for the K. lactis
wild-type strain. The full line represents the predicted fractional protein expression for genes with two binding sites (f2p), and the broken line
represents the expression levels for genes with one binding site (f1p). Experimental data for the expression of genes with two binding sites
(f2p) are shown by filled squares. (C) Model predictions of total KlGal80p, KlGal1p and nuclear activated KlGal1p concentrations with varying
galactose concentration in a K. lactis wild-type strain. The full line represents total KlGal1p, the dotted line represents total KlGal80p and the
broken line represents activated KlGal1p in the nucleus. (D) Model prediction of total KlGal4p concentration with varying galactose concentrations in a K. lactis wild-type strain.

f1p ẳ

f2p ẳ

Galị1:12
Galị1:12 ỵ0:07ị1:12
Galị1:25
Galị1:25 ỵ0:1ị1:25

!
0:375

16ị

0:35

ð17Þ

!

The values of the Hill coefficients are close to unity
for genes with one and two binding sites, indicating a
typical Michaelis–Menten response. It should be
noted that the half-saturation constants were 0.07
and 0.1 m for genes with one and two binding sites,
respectively.
Figure 2C shows the variation of total KlGal1p (full
line), activated KlGal1p in the nucleus (broken line)
and total KlGal80p (dotted line) at different galactose
concentrations. It is observed that total KlGal1p
changes from 777 to 11 000 nm when the medium
changes from NINR to a high galactose concentration.
Of the total KlGal1p, 0.11% exists in the activated
state at low galactose concentrations, whereas 99% of

total KlGal1p is activated at high galactose concentrations. Thus, it should be noted that, although KlGalpt
shows a 14-fold change in its concentration on maximal induction, the corresponding fold change in activated KlGal1p is very high (approximately 13 000).
Similarly, KlGal80p changes from 24 to 342 nm on
induction. The variation in these total regulatory protein concentrations is caused by autoregulation. The
basal level of KlGal80p protein was sufficient for the
system to exist in a repressed state in the absence of

galactose. Activated KlGal1p in the nucleus would be
absent in NINR medium and its concentration corresponds to 1190 nm in the maximally induced state,
representing a 10th of the total KlGal1p concentration.
Thus, the ratio of activated KlGal1p in the nucleus to
total KlGal80p is 3.5, which is in the range of the
three- to six-fold ratio observed in Anders et al. [13].
Furthermore, in the K. lactis GAL system, the synthesis of activator protein KlGal4p is also autoregulated
by having one binding site in its gene promoter region.
As a result, the total KlGal4p concentration changes
from 10.0 to 20 nm (see Fig. 2D), which is necessary

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2994

Fractional protein expression (f2p)

A

1

0.8

0.6

0.4

0.2

0
10−4

B
Fractional protein expression (f2p)

Fig. 3. (A) Effect of the distribution coefficient K on the GAL
switch: fractional protein expression of genes with two binding
sites (f2p) for three different K values. Broken line, K = 4; full line,
nominal K = 8; dotted line, K = 16. (B) Effect of autoregulation of
KlGal4p: comparison of model prediction between the wild-type
(full line) and the strain lacking autoregulation of KlGal4p (broken
line). The constitutive expression of KlGal4p was maintained at its
basal level of 6.95 nM. (C) Fractional protein expression for two
binding site genes for the following conditions. (a) KlGAL80 alone
was not autoregulated (dashed–dotted line) and expressed constitutively to its maximum value. The regulated bifunctional protein
KlGal1p was not sufficient to interact with excessive repressor,
leading to the complete repression of the GAL system. (b) Both
KlGAL80 and KlGAL1 were autoregulated, representing the
wild-type strain (solid line). (c) Both KlGAL80 and KlGAL1 were not
autoregulated (dotted line) and constitutively expressed to the
maximum expression achieved under induced conditions. As both
regulatory proteins were in excess and the KlGal1p concentration in
the nucleus was three- to six-fold higher than the repressor

KlGal80p concentration, the switch is able to function normally,
yielding protein expression levels similar to those of the wild-type.
(d) KlGAL1 was not autoregulated and was constitutively expressed
to its maximum concentration (broken line). In this case, the autoregulation of the repressor KlGAL80 results in a low KlGal80p concentration, leading to the activation of the switch at low galactose
concentrations.

the system response shows a two-fold reduction in
expression levels (broken line). This reduction in gene
expression is a result of insufficient concentration of
the activator, as autoregulation of KlGAL4 in the
wild-type increases the availability of KlGal4p by twoto five-fold [9,15]. However, when KlGAL4 is constitu-

C

10−3

10−2
10−1
Galactose (M)

100

101

10−3

10−2
10−1
Galactose (M)

100

101

0.4

0.3

0.2

0.1

0
10−4

Fractional protein expression (f2p)

for the GAL system to express at its protein expression
levels in the induced state.
It is of interest to elicit the influence of the nucleocytoplasmic shuttling of KlGal1p on the behavior of the
switch. The steady-state model is simulated by varying
the value of the distribution coefficient above and
below its nominal value (K = 8). On halving the value
of K from eight to four, a greater amount of the inducer KlGal1p is available in the nucleus, which results
in an ultrasensitive response of protein expression corresponding to genes with two binding sites, together
with a decrease in the threshold value (broken line in
Fig. 3A). The sensitivity as measured by the Hill coefficient is 2.4, a nearly two-fold increase over the wildtype sensitivity. However, doubling the value of the
shuttling constant to 16 shuts off the expression
because of a lack of the inducer in the nucleus (dotted
line in Fig. 3A). Thus, the distribution coefficient is a

key parameter in the operation of the GAL switch.
The steady-state model has been evaluated for regulatory designs of the GAL system. It is of interest to
ascertain the role of autoregulation in the synthesis of
activator protein KlGal4p in K. lactis as the synthesis
of the corresponding activator in S. cerevisiae is not
autoregulated. Figure 3B shows that, on constitutive
expression of KlGal4p at a value corresponding to the
uninduced concentration of KlGal4p in the wild-type,

0.4

0.3

0.2

0.1

0
10−5

10−3
10−1
Galactose (M)

101

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V. R. Pannala et al.

tively expressed at 20 nm, which corresponds to a
KlGal4p concentration at the induced level, the fractional protein expression is similar to that of the wildtype response. Model simulations suggest that, in this
case, the excess KlGal4p binds to the free basal
KlGal80p, and thus the fractional protein expression
remains similar to the wild-type expression (results not
shown). The steady-state model was further simulated
to determine the effect of autoregulation of KlGAL1
and KlGAL80. Figure 3C shows the response when the
synthesis of both regulatory proteins was not autoregulated (dotted line), and they were expressed constitutively at their maximum concentration, which
corresponds to the maximally induced concentration in
the wild-type strain. It should be noted that the
sensitivity of the response of such a mutant strain to
galactose is higher than the sensitivity observed in the
wild-type response (see Fig. 3C, full line). However,
when the synthesis of the repressor KlGAL80 alone is
not autoregulated and is constitutively expressed at
wild-type levels (340 nm), the regulated amount of
KlGal1p is insufficient to interact with the high levels
of KlGal80p in the nucleus, leading to the inhibition
of the GAL switch and thereby reducing expression
levels to zero (broken–dotted line in Fig. 3C). When
KlGAL80 is autoregulated and KlGAL1 is expressed
constitutively, the GAL switch is induced at a lower
galactose concentration and shows wild-type expression levels at a high galactose concentration (broken
line in Fig. 3C). Thus, it is observed that, for the GAL
system to function normally, the autoregulation of
KlGAL80 is essential if KlGAL1 is autoregulated.
Steady-state model response for a K. lactis
mutant strain lacking GAL80

The steady-state model for the wild-type strain can be
validated by predicting the behavior of a mutant strain
lacking the repressor gene KlGAL80. The expression of
GAL genes of such a mutant is independent of galactose concentration. However, glucose represses the
transcriptional activator KlGal4p, thereby inhibiting
the expression of GAL genes. To evaluate the effect of
glucose on GAL gene expression, experiments were
performed in a fed-batch mode operated at different
average glucose concentrations. The fractional protein
expressions were measured as the steady-state b-galactosidase concentration relative to the maximum
b-galactosidase concentration obtained in glycerol
medium. A typical experimental run that aimed to
maintain a constant glucose concentration of 57 ±
4 mm is shown in Fig. 4A. The expression of LAC4,
the gene for b-galactosidase expression with two

GAL system in K. lactis

binding sites for KlGal4p, is also shown in Fig. 4A.
The cells were grown in glycerol medium until the
absorbance at 600 nm (A600) attained a value between
0.8 and 1 before the addition of glucose, where the initial protein expression (i.e. at t = 0) was 48% of the
maximum value. The enzyme profile indicates that
glucose represses protein expression and reaches a
steady-state value of about 28% of the maximum
about 6 h after glucose addition. Similar experiments
were performed to obtain steady-state protein expressions at different average glucose concentrations in the
range 0–57 mm.
In order to predict the response of the mutant strain
lacking KlGal80p, all interactions pertaining to

KlGal80p were eliminated in the wild-type model. This
subsystem is shown in Fig. 1B. Although it is known
that glucose inhibits the synthesis of KlGal4p, the
mechanism of repression is not clearly understood.
Equation (14), which represents the effect of autoregulation on KlGAL4 expression, is modified to reflect the
inhibition by glucose using a Hill equation:
!!
g
Ki g
ẵKlGal4pt ẳ ẵKlGal4pt0 1 ỵ f1p q
g
Ki g ỵ Glcgg
18ị
It should be noted that, in the absence of KlGal80p,
GAL gene expression is negatively dependent only on
glucose. Ki represents the inhibitory constant on glucose and gg represents the Hill coefficient. Equations
(S-39)–(S-52) in Supporting information were solved
to relate the fractional protein expression to varying
glucose concentrations. The steady-state protein
expressions at different glucose concentrations were
obtained and are shown by the squares in Fig. 4B.
The experimental data were used to identify the halfsaturation constant Ki and the Hill coefficient gg in
Eqn (18), which were estimated to be 1.13 mm and
1.3, respectively. Except for Ki and gg, all other model
parameters used to predict the mutant behavior are
identical to those of the wild-type (see Table 1). Figure 4B shows a comparison between the experimental
data and model simulations. It can be seen that the
protein expressions are leaky for GAL genes with one
and two binding sites (18% and 28%, respectively),
even at high glucose concentrations (> 104 lm), thus

indicating partial repression. However, the maximum
expression in the absence of glucose demonstrated that
genes with one binding site could express only 73% of
the maximum, whereas genes with two binding sites
could express completely (see Fig. 4B). This implies
that the concentration of the activator KlGal4pt is
limiting, even in the absence of glucose. The inhibitory

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GAL system in K. lactis

V. R. Pannala et al.

B

Fractional protein
expression/glucose (M) * 101

A

Fractional protein
expression (f1p & f2p)

1
0.6
0.5

0.4
0.3
0.2
0.1
0

0

5

0.6

0.4

ηH = 1.67

ηH = 2.04

0.2

D

1
0.8

0.6

0 0
10

15

102
104
Glucose (μM)

106

1
Fractional protein
expression (f1p & f2p)

Fractional transcriptional
expression (f1 & f2)

C

10
Time (h)

0.8

ηH = 3.19

0.4

ηH = 1.89

0.2
0

10−4 10−3 10−2 10−1 100
KlGal4pt (μM)

101

0.8
0.6

ηH = 2.57
ηH = 1.64

0.4
0.2
0
10−4 10−3 10−2 10−1 100
KlGal4pt (μM)

101

Fig. 4. (A) Time course of fractional b-galactosidase expression in a mutant strain lacking KlGAL80. A typical fed-batch operation aimed at
maintaining an average steady-state glucose concentration of 57 mM (full line) and precultured on glycerol (30 gỈL–1) for 12–16 h until
A600 = 0.8–1.0 was achieved. Triangles represent glucose concentration, circles represent fractional protein expression as measured by
b-galactosidase expression and broken lines represent glucose concentrations (within ±10%). (B) Steady-state response of Kluyveromyces lactis mutant strain lacking GAL80. Comparison of the experimental data with the model prediction for different average steady-state
glucose concentrations. Full and broken lines represent model predictions for genes with one (f1p) and two (f2p) binding sites, respectively,
and circles with error bars represent the experimental data of fractional b-galactosidase expression. (C) Model predictions for fractional transcriptional expression of genes with one (full line, f1) and two (broken line, f2) binding sites for varying KlGal4pt concentration in a K. lactis
mutant strain lacking KlGAL80. (D) Model predictions for fractional protein expression of genes with one (full line, f1p) and two (broken line,
f2p) binding sites for varying KlGal4pt concentration in a K. lactis mutant strain lacking KlGAL80.

effect of glucose on the profiles of protein expression
for one and two binding sites was quantified using a

Hill equation, accommodating the leaky expression, as
follows:
!
ẵK1p 1:67
0:55
19ị
f1p ẳ 0:18 ỵ
ẵK1p 1:67 ỵ ẵGlc1:67

f2p ẳ 0:28 ỵ

ẵK2p 2
ẵK2p 2 ỵ ½GlcŠ2

!
 0:7

ð20Þ

where K1p and K2p are the half-saturation constants
for genes with one and two binding sites, whose values
were estimated to be 0.82 and 1.33 mm, respectively.
The values of the Hill coefficients indicate that the
inhibitory response is ultrasensitive.
The steady-state model of the K. lactis mutant strain
lacking KlGAL80 was further used to evaluate the
fractional transcriptional (fi, i = 1, 2) and protein
2996

expressions (fip, i = 1, 2) for genes with one and two

binding sites at different total KlGal4p (KlGal4pt)
concentrations in the absence of glucose. Total
KlGal4p was varied by independently changing
KlGal4pt0 and setting the glucose concentration to
zero in Eqn (18) [or Eqn (S-49) in Supporting information]. Figure 4C shows the fractional transcriptional expression at various KlGal4pt concentrations
obtained by the solution of Eqns (S-39)–(S-52) in
Supporting information. It can be observed from
Fig. 4C that the transcriptional responses were ultrasensitive for genes with both one and two binding sites,
with Hill coefficients of 1.89 and 3.19, respectively. The
genes with two binding sites were more sensitive than
those with one binding site as a result of the effect of
cooperativity. The half-saturation constants (K0.5) were
determined to be 0.024 and 0.012 lm for genes with
one and two binding sites, respectively. This implied that
the expression of genes with one binding site required
a larger amount of total KlGal4p concentration,

FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS


V. R. Pannala et al.

indicating amplification as a result of cooperativity for
genes with two binding sites.
Figure 4D shows the simulation results for the fractional protein expressions at various KlGa4pt concentrations. The Hill coefficients for genes with one and
two binding sites were evaluated to be 1.64 and 2.57,
respectively. It should be noted that the fractional protein expressions were less sensitive relative to the fractional transcriptional expressions for both gene types
because of the inefficiencies in mRNA translation into
protein. The genes with two binding sites were
expressed completely when the KlGal4pt concentration

was in excess of 32.6 nm. This was approximately 1.8fold higher than the experimental observation that the
KlGal4pt concentration in the induced cells in a wildtype strain was 17.4 nm [13]. The expression of genes
with two binding sites (f2p) at various glucose concentrations was also compared for the wild-type and
mutant strain lacking Gal80p (Fig. S1, see Supporting
information). The fractional protein expression in the
wild-type was lower by three-fold at all concentrations
of glucose. This was a result of the additional repression of the GAL genes by the repressor KlGal80p in
the wild-type strain. In summary, the steady-state analysis demonstrated that GAL gene expression was sensitive but leaky in response to repression by glucose,
largely as a result of the regulated expression of
KlGAL4.
Comparative study with the GAL system of
S. cerevisiae
A similar model development for the GAL system of
S. cerevisiae has been reported in the literature [18]. As
the two species of yeast are related evolutionarily, with
similar regulatory components, it is of interest to compare the steady-state performances of the GAL systems
of the two organisms. The steady-state modeling strategy discussed above was used to compare the performance of the GAL systems in both mutant (lacking
gene GAL80) and wild-type strains of S. cerevisiae and
K. lactis. Figure 5A compares the steady-state protein
expression levels for the two binding site genes for the
GAL80-lacking mutant strains of S. cerevisiae (broken
line) and K. lactis (full line) at different glucose concentrations. The steady-state response for the two
mutant strains shows that, at varying glucose concentration, the two binding site genes of S. cerevisiae were
completely repressed (see Fig. 5A, broken line). However, the K. lactis GAL system shows a leaky response,
with about 25% expression in a high glucose concentration medium (see Fig. 5A, full line). Nevertheless,
the half-saturation constants in both cases are approxi-

GAL system in K. lactis

mately equal. As the difference in the two mutant

strains is primarily in the autoregulation of KlGAL4, it
is of interest to evaluate the performance of the K. lactis GAL switch when the autoregulation of KlGAL4 is
eliminated, thereby mimicking the GAL system of
S. cerevisiae. The broken line in Fig. 5B shows the
response of the KlGAL80 mutant when KlGAL4 is
constitutively expressed at 32 nm. This structural alteration results in complete repression at high glucose
concentration, as is observed in S. cerevisiae. A comparison of the induction of the GAL system in the
wild-type strains of K. lactis and S. cerevisiae is shown
in Fig. 5C. It is evident that the response of the S. cerevisiae GAL system not only exhibits a higher expression, but is more sensitive to the concentration
of galactose, with half-saturation constants of 1 and
100 mm for S. cerevisiae and K. lactis GAL responses,
respectively. The S. cerevisiae GAL system shows a
2.3-fold higher expression under maximal induction.
The comparison between the two systems presented
above considers both species of yeast as they have
evolved, characterized by their distinct structural
motifs and parameters. To eliminate the influence of
specific parameters and to focus attention only on the
structural elements of regulation, we re-engineered a
given species of yeast in silico so that its regulatory
structure mimicked that of the other species whilst
retaining the parameters of the former. The in silico
re-engineering study indicated that the leaky behavior
in response to glucose inhibition was also obtained in
S. cerevisiae when autoregulation of Gal4p was introduced (Fig. S2A, see Supporting information). This
implies that the leaky phenotype is a characteristic of
the structural motif and not the parameters. Both
re-engineered systems showed that nucleocytoplasmic
shuttling is a key mechanism determining the performance in response to galactose (Fig. S2B,C, see
Supporting information).

Discussion
The GAL systems of K. lactis and S. cerevisiae employ
common proteins to effect the regulation of galactose
uptake, indicating their evolutionary relationship.
However, the regulatory schemes in the two yeasts are
markedly different. Although S. cerevisiae has been
well characterized, quantitative studies of the GAL system in K. lactis are relatively sparse. Thus, the steadystate model of the GAL system of K. lactis presented
in this work provides an opportunity to compare the
K. lactis regulatory design of the GAL system with that
of S. cerevisiae. Experimental comparisons between
related organisms have been reported in the literature.

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GAL system in K. lactis

Fractional
protein expression (f2p)

A

1
0.8
0.6
0.4
0.2

0
10−5

Fractional
protein expression (f2p)

B

Fractional
protein expression (f2p)

10−4

10−3
Glucose (M)

10−2

10−1

1

0.6
0.4
0.2

10−4

10−3
Glucose (M)

10−2

10−1

1
0.8
0.6
0.4
0.2
0
10−5

10−3
10−1
Galactose (M)

101

For example, it has been demonstrated that the tryptophan biosynthetic operon in Gram-positive bacterial
species is regulated differently [23]. Another example is
the regulation of the Pho operon (PhoP ⁄ PhoQ) in
bacterial systems, wherein the regulatory proteins are
homologous, but they regulate different genes in
different species [24,25]. However, none of these studies have compared the quantitative responses from a
2998

Fig. 5. (A) Steady-state response of genes with two binding sites
(f2p) in GAL80 mutant strains of Saccharomyces cerevisiae and
Kluyveromyces lactis. Model predictions are represented by a broken line for S. cerevisiae and a full line for K. lactis. Steady-state

experimental data are shown by circles and squares for S. cerevisiae and K. lactis, respectively. (B) Model prediction of the steadystate response (f2p) of a mutant K. lactis strain lacking KlGAL80 in
the absence of autoregulation of KlGAL4 (broken line). The full line
represents the model prediction for a mutant strain lacking
KlGAL80 with the synthesis of the transcriptional activator KlGal4p
autoregulated. Squares represent the experimental fractional protein expression. (C) Steady-state response of two binding site
genes (f2p) for wild-type strains of S. cerevisiae and K. lactis. The
broken line and circles represent the S. cerevisiae wild-type strain
response and the full line and squares represent the K. lactis wildtype strain response. Model predictions are represented by the broken line for S. cerevisiae and the full line for K. lactis. Steady-state
experimental data are shown by circles and squares for S. cerevisiae and K. lactis, respectively.

0.8

0
10−5

C

V. R. Pannala et al.

system with varied network topology, but using similar
regulatory proteins.
A mutation corresponding to the deletion of the
GAL80 gene results in a subsystem that focuses attention on the role of activator Gal4p (or KlGal4p) in the
GAL system, whilst excluding the roles of other regulatory proteins. Despite the equivalent role of the transcriptional activator in the two organisms, ScGal4p
and KlGal4p share similarity in nuclear localization,
DNA binding and transcriptional activation only [26].
The difference between the two activators lies in the
fact that, although ScGAL4 is constitutively expressed
in the absence of glucose, the expression of KlGAL4 is
transcriptionally regulated and results in a different

overall system behavior of the GAL system in K. lactis
[11,12]. In both yeasts, it has been shown that glucose
affects the ability of the activator (KlGAL4 or LAC9)
to activate transcription of the GAL genes [16,17]. It
has been shown that, in S. cerevisiae, in the absence of
the ScGal80p protein, the gene expression levels
depend critically on the concentration of ScGal4p [20].
A steady-state analysis for S. cerevisiae indicated that
the expression of GAL genes shows a steep response
with respect to ScGal4p concentration, with Hill coefficients of 1.3 and 2.1 for genes with one and two binding sites, respectively [20]. However, K. lactis has Hill
coefficients of 1.64 and 2.57 for genes with one and
two binding sites (see Fig. 4D), respectively, illustrating that the autoregulatory mechanism prevalent in
K. lactis makes GAL gene expression more sensitive to
total activator concentration relative to that observed
in S. cerevisiae. However, the steady-state expression
profiles of the genes with one and two binding sites in
the S. cerevisiae GAL system demonstrated complete

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V. R. Pannala et al.

repression at a glucose concentration of 10 mm [20],
whereas, for K. lactis (see Fig. 2B), leaky expression of
GAL genes was observed even at 100 mm glucose. This
is also reflected in the inhibitory half-saturation constant Ki which has a value of 1 mm for genes with two
binding sites for S. cerevisiae, whereas the corresponding value for K. lactis is 1.3 mm, a 1.3-fold increase.
Furthermore, in the case of K. lactis, the maximum
total KlGal4p concentration in the KlGAL80 mutant

strain was estimated to be 32.6 nm, a six-fold increase
relative to that observed in S. cerevisiae. Thus, the autoregulatory mechanism for the synthesis of KlGal4p in
K. lactis yielded a sensitive response, as observed by
the Hill coefficient, but at the expense of leaky repression, even at a high concentration of glucose, and a
requirement for greater amounts of transcriptional
activator. Thus, it can be hypothesized that S. cerevisiae has evolved to eliminate the autoregulatory mechanism in Gal4p synthesis, resulting in a complete
repression of GAL genes by glucose using a lower concentration of the transcriptional activator Gal4p. The
lower requirement of Gal4p for the expression of genes
with two binding sites is a result of the strong cooperativity during the binding of the transcriptional activator to the two binding site genes in the S. cerevisiae
GAL system. Thus, the repression by glucose is stronger in S. cerevisiae.
The wild-type response of the GAL system of both
yeasts is based on a complex regulatory design. In
K. lactis, Gal3p is absent and KlGal1p plays a dual
role of induction as well as metabolism of galactose to
galactose-1-phosphate. However, ScGal3p is a homologous protein derived through genome duplication of
ScGal1p, and is exclusively a regulatory protein. Furthermore, unlike in S. cerevisiae, where Gal80p shuttles
from the nucleus to the cytoplasm on induction, in
K. lactis KlGal1p is a shuttling protein. A steady-state
analysis shows that translocation imparts ultrasensitivity to galactose in both K. lactis and S. cerevisiae. In
K. lactis, as KlGal1p is required in the cytoplasm to
metabolize galactose, the shuttling of KlGal1p to the
nucleus is limiting, resulting in a relatively lower induction of the two binding site genes of about 35%, relative to an expression level of 82% observed in
S. cerevisiae, relative to the respective GAL80 mutant
strains. It appears that, in the design of K. lactis, the
nucleocytoplasmic shuttling of KlGal1p limits the performance of the GAL system. As KlGal1p plays a dual
role, as both inducer and galactokinase, our analysis
indicates that only 10% of total KlGal1p translocates
into the nucleus to act as inducer, and the majority of
KlGal1p is available for galactose metabolism in the
cytoplasm. The limiting concentration of KlGal1p in

GAL system in K. lactis

the nucleus results in a low expression of GAL genes
in K. lactis. Should KlGal80p have translocated to the
cytoplasm, a crisp apportioning of KlGal1p for regulatory and metabolism purposes would not be possible.
Thus, the genome duplication event of Gal1p in
S. cerevisiae has evolved to give a system that is not
constrained in the availability of the inducer
(ScGal3p). Another important difference in the regulatory design is the fact that the regulatory proteins
KlGal1p and KlGal80p are autoregulated by genes
with two binding sites, whereas, in S. cerevisiae, the
regulatory proteins ScGal3p and ScGal80p are regulated by genes with one binding site. As autoregulation
by genes with two binding sites results in tighter regulation, all metabolizing proteins in both S. cerevisiae
and K. lactis are regulated by genes with two binding
sites. As KlGal1p is a metabolizing protein in K. lactis,
it is also regulated by two binding site genes. As the
ratio of inducer KlGal1p and repressor KlGal80p must
be finely tuned, it is essential that KlGal80p must also
be regulated by two binding site genes. The analysis
can also be used to compare the basal and induced
concentrations of the regulatory proteins in the two
yeasts. It was observed that the total concentrations of
KlGal4p, KlGal80p and KlGal1p were 10, 24 and
777 nm, respectively, in the noninduced state and 20,
342 and 11 000 nm, respectively, in the induced state.
Similarly, the concentrations of ScGal80p and ScGal3p
in S. cerevisiae were 50 and 250 nm, respectively, in
the noninduced state, and 600 and 3200 nm, respectively, in the induced state. The activator Gal4p in
S. cerevisiae is constitutively expressed with a total

concentration of 5.47 nm [18]. Thus, the basal levels of
the total concentrations of regulatory proteins were
higher in K. lactis (except for KlGa80p) and were
induced only to 100-fold on induction with galactose.
Similarly, the basal levels of the total regulatory protein concentrations were lower in S. cerevisiae relative
to K. lactis, but were induced to 100 to 1000-fold with
galactose [4]. The concentrations of the regulatory proteins for the two strains in repressed (glucose), NINR
(glycerol) and induced (galactose) media are summarized in Table 2. These stoichiometric constraints, in
addition to the regulatory structure, play key roles in
the operation of the switch.
In summary, S. cerevisiae appears to have evolved a
superior design for the uptake of galactose by evolutionary distinct proteins for the induction of GAL
genes (Gal3p) and for the metabolism of galactose
(Gal1p). Clearly, the S. cerevisiae GAL system exhibits
higher expression levels than the K. lactis GAL system
in response to a given galactose concentration. Furthermore, the S. cerevisiae GAL system is turned on at

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V. R. Pannala et al.

Table 2. Comparison of the concentrations of the regulatory proteins in Saccharomyces cerevisiae and Kluyveromyces lactis in three regulatory states based on the availability of the carbon source. All concentrations are in nM.
Repressed
S. cerevisiae
Gal4pt

Gal80pt
Gal3pt
Gal1pt
D1t
D2t

0.002
0.49
2.4
0.0712
0.1

NINR
K. lactis
6.95
25.6
830
0.0712
0.43

S. cerevisiae
5.47
50
250
0.0712
0.16

a lower threshold. Moreover, the leaky expression of
K. lactis at higher glucose concentration indicates the
greater burden that it has to bear relative to S. cerevisiae, which is completely repressed. As, in K. lactis,

KlGal1p plays a dual role, the design requires that a
large quantity of KlGal1p is earmarked for metabolism, with about 10% shuttling into the nucleus for
regulatory function under induced conditions. If, however, the design involved the shuttling of KlGal80p
from the nucleus to the cytoplasm, the amount of
KlGal1p available for metabolism would be determined by the KlGal80p concentration and thus would
affect the metabolism. Furthermore, the autoregulation
of the transcriptional activator KlGal4p results in
leaky expression under both repressive (high glucose)
and noninducing (zero galactose) conditions. The
absence of autoregulation of Gal4p in S. cerevisiae
reduces the burden of maintaining basal Gal4p. Thus,
the gene duplication, shuttling of the repressor instead
of the inducer and the absence of autoregulation of
the transcriptional activator have endowed S. cerevisiae to yield an optimal response to the efficient metabolism of galactose. However, it would be interesting to
evaluate in future the response of the K. lactis GAL
system to lactose, which is the niche for K. lactis.

Materials and methods
Strain
The K. lactis GAL80 mutant and wild-type strains used in
this study were JA6D801 and JA6, respectively [27]. The
strains were stored in 20% glycerol at )20 °C in microcentrifuge tubes. The cells were precultured in yeast–peptone–
dextrose (YPD) medium (yeast extract, 10 gỈL)1; peptone,
20 gỈL)1; dextrose, 20 gỈL)1) and streaked out onto agar
plates. A single colony was picked out from the agar plate
to re-inoculate YPD broth grown in a shake flask until it
reached the exponential phase. Slants were prepared using
cultures grown in the shake flask and stored for experimen-

3000

Induced
K. lactis
10
24
777
0.0712
0.43

S. cerevisiae

K. lactis

5.47
642
3200

20
342

0.0712
0.16

11 · 103
0.0712
0.43

tal use. In each experiment, inoculum was prepared by a
loopful of culture from the slant.

Medium for preculture
A cotton-stoppered Borosil flask (500 mL) containing
100 mL working volume of 10 gỈL)1 yeast extract, 20 gỈL)1
peptone and 20 gỈL)1 dextrose ⁄ 30 gỈL)1 glycerol was used.
The pH was adjusted to 5.5 by the addition of 1 m HCl.
The cells were grown in a shake flask at 30 °C on a rotary
shaker at 240 r.p.m. until A600 reached 1.0–1.5. Subsequently, the experimental flask was inoculated with 10%
(w ⁄ v) cell mass of A600 = 1.

Experimental procedures
Steady-state experiments on glucose and galactose were
carried out independently in a fed-batch mode. Initially,
K. lactis strain was grown in a shake flask with a composition similar to that of the preculture medium until A600
attained a value between 0.8 and 1.0 in a rotary shaker at
30 °C and 240 r.p.m. After this, the experiment was carried
out in a fed-batch mode by the addition of glucose ⁄
galactose at regular intervals whilst measuring the concentration of glucose ⁄ galactose in the flask. For studies on the
K. lactis GAL80 mutant strain, different average steadystate glucose concentrations (0–57 mm) were maintained
(±10%) in the flask using two standard glucose solutions
with concentrations 10–50-fold of the required concentration. The protein concentrations of b-galactosidase were
measured for different average steady-state concentrations
of glucose. All the steady-state experiments were carried
out with glycerol as the background medium, and the maximum protein expression was noted for a medium lacking
glucose. The data are provided as the fraction of the maximum value of b-galactosidase expression. For studies on
the K. lactis wild-type strain, different batch experiments
were performed using different galactose concentrations
(0.002–0.44 m) with glycerol as the background medium,
and the fractional b-galactosidase expression was measured
dynamically. The data obtained from these experiments

FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS


V. R. Pannala et al.

were tabulated as the steady-state fractional protein
expressed at different average steady-state glucose ⁄ galactose
concentrations.

Substrate and enzyme activity measurements
Glucose and galactose were measured using HPLC,
employing a Lachrom L-7490 HPLC system (Mumbai,
India) and a Biorad Aminex HPX-87H column (Mumbai,
India) attached with a guard column in series. b-Galactosidase activity was measured by taking 2A600 cells for each
measurement which were stored in breaking buffer immediately at –20 °C for later extraction. The yeast cells were
lyzed by the addition of glass beads (0.5 mm), and the
activity of b-galactosidase was measured by the method of
crude extracts, as reported by Rose and Botstein [28] and
Adams et al. [29]. All the experiments were carried out in
triplicate and deviations in protein expression data are
shown by error bars in the results. The fluctuations in
the average steady-state concentrations of glucose in the
fed-batch experiments were within acceptable limits.

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Supporting information
The following supplementary material is available:
Fig. S1. Effect of repressor KlGal80p on the GAL system of K. lactis.
Fig. S2. In silico re-engineering of the GAL systems of
S. cerevisiae and K. lactis.
Doc. S1. Detailed steady-state model development for
K. lactis.
This supplementary material can be found in the
online version of this article.
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FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS