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: venks@che.iitb.ac.in (Received 10 December 2009, revised 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2987 GAL system in K lactis 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- FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 D1 in Fig 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 2ị 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  ẵD2 KlGal4p2 Kd 3ị ẵD2 KlGal4p2 ỵ ẵKlGal4p2  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 D1KlGal4p2 can be written as follows: ẵKlGal80p2 ỵ ẵD1 KlGal4p2  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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2989 GAL system in K lactis V R Pannala et al A KlGal4p2 80 KlGal80p2 KlGal4p 12 80 KlGal80p Lac12p KlGal1p Galactose KlGal1p-Galint Galactose 12 ATP K K4 Cytoplasm 4 K4 80 K1 K3 K2 80 Kd K3 80 GAL genes UASG D1 GAL genes UASG B Nucleus D2 Galactose 12 ATP K 4 K1 Cytoplasm 80 Kd GAL genes Nucleus UASG D1 GAL genes UASG D2 Fig (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 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]: 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 ẵKlGal1p ỵ ẵKlGal80p2  ẵKlGal1p KlGal80p2 9ị n n K4 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  n 2K4 ẵKlGal1p n KlGal80p2 KlGal1p Š 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 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; 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]: À Á 14ị ẵKlGal4pt ẳ ẵKlGal4pt0 ỵ f1p q where q represents the fold-change in [KlGal4pt] from a noninduced state to a completely induced state corresponding to f1p = 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: ẵKlGal1pt ẳ f2p ẵKlGal1pmax and ẵKlGal80pt ẳ f2p ẵKlGal80pmax 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 (f1 and f2) and two corresponding protein expressions (f1p and f2p) FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2991 GAL system in K lactis V R Pannala et al Table 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 1.13 mM mM 0.072 nM 0.43 nM 10 6.95 nM 32.6 nM 170–340 nM 11000 nM 0.7 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 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 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 (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 C 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 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 (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 ¼ f2p ¼ 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2993 GAL system in K lactis V R Pannala et al 2994 Fractional protein expression (f2p) A 0.8 0.6 0.4 0.2 10−4 B Fractional protein expression (f2p) Fig (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 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 10−5 10−3 10−1 Galactose (M) 101 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 ± 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 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 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 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2995 GAL system in K lactis V R Pannala et al B Fractional protein expression/glucose (M) * 101 A Fractional protein expression (f1p & f2p) 0.6 0.5 0.4 0.3 0.2 0.1 0 0.6 0.4 ηH = 1.67 ηH = 2.04 0.2 D 0.8 0.6 0 10 15 102 104 Glucose (μM) 106 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 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 10−4 10−3 10−2 10−1 100 KlGal4pt (μM) 101 Fig (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: ! ½K1p Š1:67  0:55 19ị f1p ẳ 0:18 ỵ ẵK1p 1:67 ỵ ẵGlc1:67 f2p ẳ 0:28 ỵ ẵK2p ẵK2p ỵ ẵGlc2 !  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 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2997 GAL system in K lactis Fractional protein expression (f2p) A 0.8 0.6 0.4 0.2 10−5 Fractional protein expression (f2p) B Fractional protein expression (f2p) 10−4 10−3 Glucose (M) 10−2 10−1 0.6 0.4 0.2 10−4 10−3 Glucose (M) 10−2 10−1 0.8 0.6 0.4 0.2 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 (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 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 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 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 FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS 2999 GAL system in K lactis V R Pannala et al Table 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 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 = 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 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Mol Cell Biol 13, 7566–7576 Rose M & Botstein D (1983) Construction and use of gene fusions to lacZ (b-galactosidase) that are expressed in yeast Methods Enzymol 101, 167–180 3002 29 Adams A, Gottschling DE, Kaiser CA & Stearns T (1997) Methods in Yeast Genetics Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 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 Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2987–3002 ª 2010 The Authors Journal compilation ª 2010 FEBS ... 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... absence of autoregulation of Gal4 p in S cerevisiae reduces the burden of maintaining basal Gal4 p Thus, the gene duplication, shuttling of the repressor instead of the inducer and the absence of autoregulation... 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