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
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 galac-
tose is the sole carbon source, the induction and tran-
scription 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 (UAS
G
)of
each gene for transcription to proceed. The transcrip-
tion 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
Keywords
galactose; GAL system; Kluyveromyces
lactis; Saccharomyces cerevisiae; steady-
state model

the GAL system of K. lactis provides an opportunity to compare with the
design prevailing in S. cerevisiae. The comparison indicates that the exis-
tence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevi-
siae as a result of genome duplication has resulted in a system which
metabolizes galactose efficiently.
Abbreviations
NINR, noninducing, nonrepressing; UAS, upstream activator sequence; URS, upstream repressor sequence; YPD, yeast–peptone–dextrose.
FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2987
mechanisms, and thus terminates the activation of
GAL genes [6–8]. Although the regulatory players are
conserved in various organisms, the molecular mecha-
nisms that occur as a result of the interactions between
them are different. For example, in K. lactis, the syn-
thesis of the transcriptional activator protein KlGal4p
is autoregulated, but its expression is inhibited by glu-
cose [9–11], whereas, in S. cerevisiae, ScGal4p synthesis
is not autoregulated, but its gene expression and activ-
ity 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 regula-
tory 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

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
specific repressor protein Mig1p, which binds to the
upstream repressor sequences (URS
G
) present in GAL
genes [7]. However, in the case of K. lactis, the repres-
sion of KLGAL4 is independent of Mig1p, as KlGAL4
has no URS
G
in its promoter for Mig1p, but glucose
indirectly represses the GAL system by a Mig1p bind-
ing 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 con-
tains 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 induc-
tion mechanism of the GAL system. The KlGAL4 gene
contains a UAS
G
in its own promoter for the binding
of KlGal4p, resulting in an autoregulatory circuit

molecular components in the GAL network, the archi-
tecture in the organisms differs substantially. Further-
GAL system in K. lactis V. R. Pannala et al.
2988 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 net-
work to metabolize mainly lactose, whereas S. cerevi-
siae uses it to metabolize melibiose and galactose.
This evolutionary fact also plays a role in the perfor-
mance of the two networks. Given the above differ-
ences in the two GAL networks, it is of interest to
compare the steady-state performances of the net-
works in S. cerevisiae and K. lactis in response to
galactose and glucose.
We used a steady-state modeling approach to quan-
tify 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 experi-
mentally by obtaining steady-state protein expression
levels in a wild-type strain and in a mutant strain lack-
ing gene KlGAL80. The steady-state model was then
used to delineate the importance of the autoregulation
of regulatory proteins and parametric sensitivity. Sub-
sequently, we considered a KlGAL80 mutant strain of
K. lactis to determine the importance of the autoregu-
lation of activator KlGal4p and glucose repression.
The K. lactis GAL system model developed in this
work has been validated experimentally. As the sys-

and subsequently binds to the opera-
tor site of the gene KlGAL4 (D1) with a dissociation
constant K
d
:
½KlGal4pþ½KlGal4p ¢
K
1
½KlGal4p
2
ð1Þ
½D1þ½KlGal4p
2
 ¢
K
d
½D1 À KlGal4p
2
ð2Þ
For genes with two binding sites (D2), dimer Gal4p
binds to the first site with a dissociation constant of
K
d
, followed by binding to the second site with a dis-
sociation constant of K
d
⁄ m, where the factor m (>1)
quantifies the cooperative effect of binding of KlGal4p
to the second binding site [3]:
½D2þ½KlGal4p

follows:
½KlGal80p
2
þ½D1 À KlGal4p
2
 ¢
K
3
½D1 À KlGal4p
2
À KlGal80p
2

ð5Þ
Similarly, the remaining interactions of KlGal80p
2
with DNA–KlGal4p
2
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]:
½KlGal1p
Ã
t
¼½KlGal1p

ð7Þ
The monomeric form of activated KlGal1p in the
nucleus interacts with the monomeric form of the
repressor KlGal80p with a dissociation constant of K
4
as shown in Fig. 1.
½KlGal1p
Ã
n
þ½KlGal80p ¢
K
4
½KlGal1p
Ã
n
À KlGal80pð8Þ
The monomeric form of the activated KlGal1p is
known to interact with KlGal80p
2
with a positive co-
operativity, resulting in a reduction in the dissociation
constant by two (i.e. K
4
⁄ 2) [13]:
0
4
0
80
KlGal4p
2

4
80
80
4
4
K
1
D1
G
A
L
g
e
n
e
s
UAS
G
K
d
K
2
K
3
K
3
K
4
Galactose
Nucleus

n
e
s
UAS
G
K
d
Galactose
A
B
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, K
i
(i = 1–4) represents the dissociation constant for the respective interactions, K represents the distribution
coefficient for KlGal1p shuttling and K
d
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.
GAL system in K. lactis V. R. Pannala et al.
2990 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS
½KlGal1p
Ã
n
þ½KlGal80p
2
 ¢
K
4
2
½KlGal1p

¢
2K
4
½KlGal1p
Ã
n
À KlGal80p
2
À KlGal1p
Ã
n

ð10Þ
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 Sup-
porting 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 f
1
and
f
2
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

are the total operator concentra-
tions of genes with one and two binding sites,
respectively. As shown in Fig. 1, [D1–KlGal4p
2
],
[D2–KlGal4p
2
] and [D2–KlGal4p
2
–KlGal4p
2
] represent
the concentrations of the complexes formed as a result
of the interactions between the genes (D1 and D2) and
KlGal4p
2
. It should be noted that, in the definition of
f
2
[Eqn (12)], it is assumed that the transcriptional
capacity of a D2–KlGal4p
2
complex is equal to that of
a D2–KlGal4p
2
–KlGal4p
2
complex. However, the co-
operativity in binding to the second site [parameter m
in Eqn (4)] ensures that the complex D2–KlGal4p

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 effi-
cient. It has been shown through microarray experi-
ments that n has a value in the range 0.5–0.75 for
protein expression from genes in S. cerevisiae [22]. Spe-
cifically, the GAL genes in S. cerevisiae show an aver-
age co-response coefficient of around 0.7 [18]. In this
work, we have assumed a value of 0.7 as the corre-
sponding coefficient in K. lactis. As the gene KlGAL4
with one binding site is autoregulated; the total
KlGal4p concentration (KlGal4p
t
) is therefore a func-
tion of f
1p
. Further, the autoregulation of KlGAL4
makes it imperative that a basal amount of KlGal4p
t0
,
necessary to activate the switch from a completely
repressed state (i.e. f
1p
= 0), exists. Thus, the total
KlGal4p
t
concentration is dependent on f
1p
and is

[see Eqn (13)] as given below:
½KlGal1p
t
¼ f
2p
½KlGal1p
max
and ½KlGal80p
t
¼ f
2p
½KlGal80p
max
ð15Þ
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 (f
1
and f
2
) and
two corresponding protein expressions (f
1p
and f
2p

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
medium and the steady-state b-galactosidase activity
was measured. It should be noted that the b-galacto-
sidase activity represents the protein expression from
a GAL gene with two binding sites for KlGal4p, and
its measurement was used to quantify f
2p
. 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 steady-
state value was obtained by averaging over the last
three time points from the individual fed-batch exper-
iment. The steady-state values of the b-galactosidase
activity of cells grown at different galactose concen-
trations (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-galactosi-
dase 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 expres-
sion profiles with respect to galactose. The full line

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:
Table 1. Parameter values used in the steady-state model.
Parameter
a
Kluyveromyces
lactis Source
Saccharomyces
cerevisiae
K
d
0.62 nM Fitted to data 0.2 nM
K
1
730 nM Fitted to data 100 nM
K
2
0.1 nM [13] 0.1 nM
K
3
0.5 nM [13] 0.05 nM
K
4
83 nM [13] 0.063 nM
K 8 Fitted to data 0.4
K
i
1.13 mM Fitted to data 0.4 mM

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].
GAL system in K. lactis V. R. Pannala et al.
2992 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS
f
1p
¼
GalðÞ
1:12
GalðÞ
1:12
þ 0:07ðÞ
1:12
!
 0:375 ð16Þ
f
2p
¼
GalðÞ
1:25
GalðÞ
1:25
þ 0:1ðÞ

sponds 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 synthe-
sis 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
10
−5
10
−3
10
−1
10
1
10
−5
10
−3
10
−1
10
1
10
–2
10
0

0
0.1
0.2
0.3
0.4
Galactose (M)
Fractional protein
expression
0 5 10 15 20 25
0
0.05
0.1
0.15
0.2
0.25
0.3
Time (h)
Fractional betagal
expression
η
H
= 1.12
η
H
= 1.25
η
H
= 1.12
AB
CD

responding 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 coef-
ficient is 2.4, a nearly two-fold increase over the wild-
type 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 regu-
latory 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,
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 two-
to five-fold [9,15]. However, when KlGAL4 is constitu-
10
−4
10
−3
10
−2
10

0.3
0.4
Galactose (M)
Fractional protein expression (f
2p
)
10
−5
10
−3
10
−1
10
1
0
0.1
0.2
0.3
0.4
Galactose (M)
Fractional protein expression (f
2p
)
A
B
C
Fig. 3. (A) Effect of the distribution coefficient K on the GAL
switch: fractional protein expression of genes with two binding
sites (f
2p

type 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 autoreg-
ulated (dotted line), and they were expressed constitu-
tively 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 expres-
sion 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

sions 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 autoregu-
lation on KlGAL4 expression, is modified to reflect the
inhibition by glucose using a Hill equation:
½KlGal4p
t
¼½KlGal4p
t0
 1 þ f
1p
 q Â
K
g
g
i
K
g
g
i
þ Glc
g
g
! !

protein expressions are leaky for GAL genes with one
and two binding sites (18% and 28%, respectively),
even at high glucose concentrations (> 10
4
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 KlGal4p
t
is
limiting, even in the absence of glucose. The inhibitory
V. R. Pannala et al. GAL system in K. lactis
FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2995
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:
f
1p
¼ 0:18 þ
½K
1p

1:67
½K
1p


lacking KlGAL80 was further used to evaluate the
fractional transcriptional (f
i
, i = 1, 2) and protein
expressions (f
ip
, i = 1, 2) for genes with one and two
binding sites at different total KlGal4p (KlGal4p
t
)
concentrations in the absence of glucose. Total
KlGal4p was varied by independently changing
KlGal4p
t0
and setting the glucose concentration to
zero in Eqn (18) [or Eqn (S-49) in Supporting infor-
mation]. Figure 4C shows the fractional transcriptio-
nal expression at various KlGal4p
t
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 ultrasen-
sitive 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 (K
0.5
) were

0.2
0.4
0.6
0.8
1
Fractional transcriptional
expression (f
1
& f
2
)
KlGal4p
t
(μM)
10
−4
10
−3
10
−2
10
−1
10
0
10
1
0
0.2
0.4
0.6

2p
)
η
H
= 3.19
η
H
= 1.89
η
H
= 1.64
η
H
= 2.57
η
H
= 2.04
η
H
= 1.67
AB
CD
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 m
M (full line) and precultured on glycerol (30 gÆL
–1
) for 12–16 h until
A
600
= 0.8–1.0 was achieved. Triangles represent glucose concentration, circles represent fractional protein expression as measured by

concen-
trations. 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 pro-
tein expressions were less sensitive relative to the frac-
tional 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 KlGal4p
t
concentration
was in excess of 32.6 nm. This was approximately 1.8-
fold higher than the experimental observation that the
KlGal4p
t
concentration in the induced cells in a wild-
type strain was 17.4 nm [13]. The expression of genes
with two binding sites (f
2p
) at various glucose concen-
trations 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 repres-
sion of the GAL genes by the repressor KlGal80p in
the wild-type strain. In summary, the steady-state anal-
ysis demonstrated that GAL gene expression was sensi-
tive but leaky in response to repression by glucose,
largely as a result of the regulated expression of

response of the KlGAL80 mutant when KlGAL4 is
constitutively expressed at 32 nm. This structural alter-
ation results in complete repression at high glucose
concentration, as is observed in S. cerevisiae. A com-
parison 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. ce-
revisiae GAL system not only exhibits a higher expres-
sion, 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 intro-
duced (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 perfor-

GAL system, whilst excluding the roles of other regula-
tory proteins. Despite the equivalent role of the tran-
scriptional 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 coeffi-
cients of 1.3 and 2.1 for genes with one and two bind-
ing 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, illustrat-
ing 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
Fig. 5. (A) Steady-state response of genes with two binding sites

10
−2
10
−1
10
−5
10
−4
10
−3
10
−2
10
−1
0
0.2
0.4
0.6
0.8
1
Glucose (M)
Fractional
protein expression (f
2p
)
10
−5
10
−3
10

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 con-
stant K
i
which has a value of 1 mm for genes with two
binding sites for S. cerevisiae, whereas the correspond-
ing 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 aut-
oregulatory 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 repres-
sion, even at a high concentration of glucose, and a
requirement for greater amounts of transcriptional
activator. Thus, it can be hypothesized that S. cerevisi-
ae has evolved to eliminate the autoregulatory mecha-
nism in Gal4p synthesis, resulting in a complete
repression of GAL genes by glucose using a lower con-
centration 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 cooper-
ativity during the binding of the transcriptional activa-
tor to the two binding site genes in the S. cerevisiae
GAL system. Thus, the repression by glucose is stron-
ger in S. cerevisiae.
The wild-type response of the GAL system of both

S. cerevisiae has evolved to give a system that is not
constrained in the availability of the inducer
(ScGal3p). Another important difference in the regula-
tory 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 regu-
lated by genes with one binding site. As autoregulation
by genes with two binding sites results in tighter regu-
lation, 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, respec-
tively, 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

from the nucleus to the cytoplasm, the amount of
KlGal1p available for metabolism would be deter-
mined 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. cerevisi-
ae to yield an optimal response to the efficient metabo-
lism 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 microcen-
trifuge tubes. The cells were precultured in yeast–peptone–
dextrose (YPD) medium (yeast extract, 10 gÆL
)1
; peptone,
20 gÆL
)1
; dextrose, 20 gÆL
)1
) and streaked out onto agar

tion similar to that of the preculture medium until A
600
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 concen-
tration of glucose ⁄ galactose in the flask. For studies on the
K. lactis GAL80 mutant strain, different average steady-
state glucose concentrations (0–57 mm) were maintained
(±10%) in the flask using two standard glucose solutions
with concentrations 10–50-fold of the required concentra-
tion. 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 maxi-
mum protein expression was noted for a medium lacking
glucose. The data are provided as the fraction of the maxi-
mum 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
Table 2. Comparison of the concentrations of the regulatory proteins in Saccharomyces cerevisiae and Kluyveromyces lactis in three regula-
tory states based on the availability of the carbon source. All concentrations are in n
M.
Repressed NINR Induced
S. cerevisiae K. lactis S. cerevisiae K. lactis S. cerevisiae K. lactis
Gal4p
t

measurement which were stored in breaking buffer immedi-
ately 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 sys-
tem 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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should be addressed to the authors.
GAL system in K. lactis V. R. Pannala et al.
3002 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS