YEAST
VOL.
7: 119-125 (1991)
Production of the STA2-Encoded Glucoamylase in Saccharomyces cerevisiae is Subject to Feed-back Control N. I. SUNTSOV, S. V. KUCHIN, M. A. NEYSTAT, S. V. MASHKO A N D S. V. BENEVOLENSKY Institute of Genetics and Selection of Industrial Microorganisms. Moscow 113545, USSR
Received 14 February 1990; revised I6 June 1990
Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified: repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold difference in the rate of G A production) can be displayed by either glucose-limited or glycerol- plus ethanolconsuming cultures: the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated G A . It is proposed that the feed-back control involved in G A production can be attenuated by starch which can thus ‘induce’ higher rates of G A production compared to the basal mode. K E Y WOKDS -
Saccharomyces cerevisiae; yeast; starch; secretion; extracellular glucoamylase; feed-back control.
INTRODUCTION
conditions (Yamashita and Fukui, 1984a; Inui et al., 1989). Diastatic strains of Saccharomyces cerevisiae are The effect of different carbon sources on GA capable of producing extracellular glucoamylase expression has been examined revealing that the (GA) (1,4-a-~-glucanglucohydrolase, EC 3.2.1.3) highest levels of GA activity and GA mRNA are and can, therefore, utilize exogenous starch. accumulated by cultures grown in rich medium The yeast extracellular GA is a heavily glycosy- supplemented with starch (Inui el al., 1989) or lated protein with an average molecular weight of glycerol plus ethanol (Pretorius er al., 1986). All about 300000, data reported so far suggesting a sugars tested (including GA-hydrolysable maltose) variety of forms of this enzyme with respect to its reduce GA expression (Pretorius el al., 1986). In this report, we have examined the production structure and enzymatic properties, which areapparently dependent on a particular Saccharomyces of GA by a Saccharomyces strain in different growth strain and on growth conditions (Yamashita et a f . , conditions, identifying three distinct GA produc1984, 1985; Modena et al., 1986; Kleinman et al., tion modes: induced, basal and repressed. We also present evidence supporting the involvement of 1988). Three identical unlinked genes, STA1, STA2 and feed-back control in GA production. We propose STA3, are known to be the structural genes for GA, that the induced mode demonstrated by earlyeach conferring upon the cell the ability to produce logarithmic cultures in starch-containing media, is GA (Tamaki, 1978). GA production directed by the related to the feed-back control-relieved state. STAI-STA3 genes can beconstitutively repressed in a dominant and epistatic fashion by the allele(s) MATERIALS AND METHODS STAlO (or I N H I ) present in most S. cerevisiae strains (Polaina and Wiggs, 1983; Yamashita and Strains We used Saccharornyces cerevisiae strains 249 Fukui, 1984b). STAIO has been shown to affect GA expression at the transcriptional level (Pardo et al., (STA2 stalO), 501 (sta2-1 stal0) and 509 (sla2-3 1986). Heterozygosity at MAT (in diploids) and stalO) from our collection. Strains 501 and 509 I N H I inhibit GA expression at the transcriptional are isogenic to 249 except that they carry two or post-transcriptional levels depending on growth independent sta2 mutations (Kuchin et al., 1990). 0749-503X~91:020119-07$05.00 0 1991 by John Wiley & Sons Ltd
120 Media
YP medium (also referred to as rich medium) containing 0.5% yeast extract and 1 .O% bacto-peptone (Difco) was supplemented with: 2.0% glucose (YPD), 2.0% galactose (YPGal), 2.0% maltose (YPMal), 2.0% sucrose (YPSuc), 3.0% glycerol plus 1.5% ethanol (YPGE), 1.0,2.0 or 4.0% soluble starch (YPSI, YPS2 and YPS4, respectively), 2.0% glucose plus 1.0% starch (YPDS). Growth conditions
Batch cultures were grown in shake flasks on an orbital shaker (250 rpm) at 28°C with an initial cell titre of 5.0 x lo5 cells/ml, starting with a 24-h preculture grown on plates containing YPD medium solidified withl.570 agar. Chemostat cultures were run using a 350-ml fermenter (BioFlo Model C30, New Brunswick Scientific Co.) at 28°C and pH 5.4.The dissolved oxygen tension was maintained above 40% air saturation at an agitation of 300 rpm. After an increase in dilution rate, the time taken for six vessel volumechanges was allowed in order to reach a new steady state. To inoculate fermenter cultures, a 24-h batch culture of strain 249 grown in YPD was used. Determination of cell titres
Cell titres were determined microscopically using a cell counting chamber. Enzyme assays
GA activity wasquantified by determining the rate ofreleaseofglucosefromamylose(Serva;0.5%, wjv, final concentration in 100 mM phosphate buffer, pH 5.4, at 37°C. Glucose content was measured either enzymatically using the glucose oxidase-peroxidase method (Berezin et al., 1977), or by determining the content of reducing sugars as described by Jamieson et ul. (1969). Invertase (IN) activity was determined according to Jamieson et al. (1 969). The GA and IN units are expressed, respectively, as nmol and pmol of glucose released/min. Proportionality of the measured GA and IN activities to the corresponding enzyme amounts was occasionally confirmed by monitoring these activities in successive dilutions of culture supernatants. Probes were diluted with distilled water.
N. I. SUKTSOV E r AL.
Table I . Effect of the carbon source on the stationaryphase GA activity level in the culture medium Cell titre
Medium
cells/ml
( x lo8)
GA unitsiml
GA units Per 10* cells
30 22
3.1
~~
YPD YPGal YPMal YPSUC YPSI YPS2 YPS4 YPGE
17 7.1 12 5.0 3.1 5.2 8.0
6.9
25 15 160 I80 250 100
1.8 2.1 3.0 52 35 31
14
Ultrafiltration
Ultrafiltration of cell-free culture supernatants was carried out using Millipore equipment; the membrane used was PTTK (cut-off 30 000).
RESULTS Effect of the carbon source on glucoamylase production
The accumulation of GA was examined in culture supernatants of strain 249 grown in rich medium supplemented with the following carbon sources: glucose, galactose, maltose, sucrose, starch and glycerol plus ethanol. Cells were grown until the stationary phase. Culture samples were then collected and the corresponding cell titres counted. The cell-free culture supernatants were examined for GA activity. The levels of enzyme activity (Table 1 ) suggest that the carbon sources tested can be placed in threecategories: (i)stdrch (the highest levels ofGA accumulation); (ii) glucose, galactose, maltose and sucrose (the lowest levels of GA accumulation); (iii) glycerol plus ethanol (an intermediate level of GA accumulation). The results reveal a more apparent difference if related to the corresponding cell titres. Chemostat cultivation
The increased production of GA displayed by cultures grown on starch could be due to the effect of starch per se. On the other hand, this may be a response to glucose limitation. The two possibilities can in principle be distinguished in glucose-limited chemostat culture. We ran chemostat cultures in
121
PRODUCTION OF THE STAIENCODED GLYCOAMYLASE
Tablc 2. G A and IN production rates in chemostat cultures
PR (enzyme unitsih per lo8cells) ~~
Medium D ( h YPD YPGE YPD YPDS
’)
0.10 0.20
~
~~
~
GA
IN
1.5k0.2 I .8 5 0.2 2.8 f0.3 2.2 5 0 . 2
90k 10 34k 7 160k20
n.d.
Standard errors were estimated from triplicate determinations; nd.. not determined.
three media (YPD, YPGE and YPDS) at two dilution rates (0.10 and 0.20 h-I) assaying for cell titres and activities of GA and IN (as a control). The results were expressed in terms of production rate (PR), i.e. the enzyme activity produced by an average cell per unit time (equation I):
PR = ( E x D)iT
(1)
where E and Tare the steady-state enzyme activity level and cell titre, respectively; D, the dilution rate. As suggested by the results (Table 2), glucose limitation does not stimulate GA induction per se under conditions used. Indeed, at D=O.lO h-I, the PR values for YPGE and YPD (1.8 and 1.5 GA units/h per 10’ cells, respectively) cannot be considered as different within the error range. Furthermore, we observed no induction for YPDS compared to YPD (2.2 versus 2.8 GA units/h per lo8 cells; D = 0.20 h-I). although starch was abundant in the cell environment as indicated by iodine staining. The results also suggest that the rates of both GA and IN production are proportional to D ;the percell-cycle amounts of secreted GA and I N thus appear to remain relatively constant despite the decrease in the doubling time from 10 to 5 hours. I t should be noted that the chemostat cultures would be washed out at a D of less than 0.30 h-’; therefore. it was impossible to achieve significantly higher specific growth rates, which, as we reasoned, might be a restrictive condition with respect to the induction of higher rates of G A production. Dynamics ofgrowth and G A accumulation using diflerent carbon sources To elucidate the conditions in which starch could induce the production of increased quantities of GA, we studied the dynamics of growth and GA
accumulation in logarithmic cultures of strain 249 grown in YPSI, YPS2, YPS4, YPGE and YPD media (Figure I). To obtain a more detailed pattern of the growth-phase modulation of GA production, we interpreted the dynamics data in terms of per-cell PR, as was the case with chemostat cultures. In this case, however, a more complicated mathematic approach would be required since parameters of a periodic culture are subject to a continuous change. A formal equivalent of equation 1 can be presented as follows (equation 2): 1 dE(t) PR(t) = x T(t) dr where E(t)and T(t)are, respectively, the level of GA activity and the cell titre at time t. Equation 2 can be readily applied to inspecting the PR function throughout the logarithmic phase because E(t) and T(t)increaseexponentiallywith time. The log(E)and log(7J points were thus easily fitted by linear regression. The resulting functions, log(E(1 ) ) and log(T(t)),weresubsequentlypotentiated and applied to equation 2. The PR functions thus obtained (Figure 2) reveal dependence on the carbon source used: PR(t) for YPD (not plotted) represents the repressed mode of GA production and is vanishingly close to zero on the scale used; the PR curve for YPGE and the bunch of curves for YPSI-YPS4 are considerably gapped yielding an approximately seven-fold difference in PR in the early-exponential phase. We have termed the latter two production modes ‘basal’ (for YPGE) and ‘induced’ (for YPS 1-YPS4). Notably, the basal PR(r) functionis relativelyconstant throughout the exponential phase. In contrast, the induced PR values, although initially high, tend to decrease exponentially towards the basal PR value, which is ultimately reached in the lateexponential phase, establishing a pattern resembling that found in the chemostat experiments. ~
~
Glucoamylase production is subject tofeed-back control Ascanbeseenin Figure2, thePRcurvesforYPSlYPS4 media are closely compacted on the versustime plot; therefore, the synchronous decrease in PR in these media is unlikely to be caused by a gradual exhaustion of starch from the cell environment. Our hypothesis was that it might be the extracellularly accumulated GA that was involved in the inhibition of GA production.
122
N. I. SUNTSOV ET AL.
B
A
I
i/!I
I
t
t
t
t
L
D
C
a
0
20
40
0
20
40
60
T i m e , hrs Figure 1. Dynamics of growth and accumulation of GA in the culture medium in the logarithmic phase. Cells of strain 249 were grown in shake flasks containing the following media: YPSl (A),YPS2 (.)and YPS4(3)(AandC);YPGE(O)andYPD(.)(BandD). Growth wasmonitored by counting the cell titre (A and B). Cell-free culture supernatants were examined for GA activity (C and D).
To test this, cells of strain 249 were grown in YPGE medium (to ensure the basal GA production mode) up to the mid-exponential phase, and the entire cell-free supernatant was then subjected to ultrafiltration to remove macromolecules greater than 30 OOO. GA and IN activities were found to be completely removed from the culture supernatant by this procedure. The cells were subsequently resuspended in the filtered supernatant and put onto
the shaker for further growth. The break in growth did not exceed 30 min. Except for the ultrafiltration, the same manipulations were applied to the control culture. In both the tested and the control cultures GA and IN activities and cell titres were monitored. It was found that GA and IN activity levels were quickly restored up to the levels exhibited by the control culture, although growth was not affected. Estimations carried out according to equation 2
PRODUCTION OF THE STAPENCODED GLYCOAMYLASE
123 level also reaches that of the control shortly after growth is resumed so that the PR value approaches a maximum of about 46 GA units/h per 10’ cells by the end of the period followed (8 h following the substitution) (Figure 3, curve 3). Similar results have been obtained using another sra2 mutant, 501 (not shown). The PR value for the control culture has been estimated that is relatively constant at about 3.5-2.5 GA units/h per lo8 cells. I t should be outlined that the IN accumulation pattern remains unchanged when GA, but not IN, is missing from the cell environment.
0
10
20
30
Time, hrs Figure 2. G A production rates (the PR functions) in the logarithmic phase. Cells of strain 249 were grown in Y PSI (centre line), YPS2 (dashed line), YPS4 (dotted line) and YPGE (solid line). The curves were obtained by mdthematic treatment of data depicted in Figure I asdescribed in the text. Theconsiderable lag observed during growth in YPGE medium has been omitted to align the period of logarithmic growth in YPGE to those in Y PS I , Y PS2 and Y PS4, so that f = 0 corresponds to a cell titre of 2.6 x ICP cellsiml in all these cultures.
indicate that the rate of GA production increases for at least 8 h following ultrafiltration to reach a value of approximately 15 units/h per lo8 cells (Figure 3, curves 1 and 2), which is close to the PR values characteristic of the induced GA production mode (12-16 unitsih per lo8 cells) found for the early-log starch-grown cultures. However, it was not clear whether it was GA itself whose absence brought about the increase in the rate of GA production. Indeed, arrested on the membrane might be an extracellular regulatory factor universal in its function to both GA and IN (or to other secretory protein also). To clear up this point, we used strain 509, an isogenic sta2 mutant of strain 249, as a donor of GA-free culture supernatant. The donor was grown in parallel with the tested strain under the same conditions. In the midexponential phase the wild-type culture liquid of strain 249 was replaced by the mutant one (instead of the ultrafiltration). In this case, the GA activity
DISCUSSION In this paper, we describe the effect of different growth conditions on the production of GA by a diastatic strain of S. cerevisiae. First, we have classified several carbon sources by their impact on the stationary-phase GA activity levels accumulated in culture supernatants. The carbon sources tested fall into three categories: (i) starch (the highest levels of GA accumulation); (ii) monosaccharides and disaccharides, including GA-hydrolysable maltose (the lowest levels of GA accumulation); (iii) glycerol plus ethanol (an intermediate level of GA accumulation). We have also studied the dynamics of growth and enzyme accumulation in batch cultures grown on several carbon sources. The obtained results were interpreted using a composite function, PR(r), defined as the rate of GA production by an average cell at time t . Comparisons of the log-phase PR curves using several carbon sources have revealed a more dramatic difference between these substrates than might be expected from the initial screening: starch, or some products of its breakdown, appear to induce GA production in the early-exponential phase cells. Such cells display a PR value which is induced approximately seven-fold over the value found for the ethanol-plus glycerol-consuming early-exponential phase culture. We have termed the two GA production modes ‘induced’ and ‘basal’, respectively. However, the PR(r) function for starch-grown cultures exhibits an exponential decrease during the logarithmic phase: the lowest PR value reached in the late-logarithmic phase is close to the basal PR value. In contrast, the basal PR(r) tends to be relatively unchanged throughout the log phase. To find a basis for such a decrease, we have put forward the hypothesis that GA production may be interfered with by extracellularly accumulating GA. To test this, we havecarried out experiments in which
124
N. 1. SUNTSOV E T A L .
0
36
40
44
0
36
40
44
T i m e , hrs
Figure3. Effect ofremovalofextracellularlyaccumulatedproteinon thedynamicsofGAandINaccumulationintheculturemedium. Cells of strain 249 were grown in YPGE medium until the mid-logarithmic phase. At 36 h cells were pelleted by centrifugation and the ccll-free supernatant was ( I ) ultrafiltered to remove macromolecules greater than 30 OOO; (2) kept unfiltered;(3) replaced by the GA-free supernatant of strain 509 grown in parallel under the same conditions. The pelleted cells were subsequently resuspended in each of the three above liquids to resume growth. Culture supernatants were examined for GA and IN activities (B and C, respectively). Cell titres were also determined (A). PR functions were then derived as described in the text.
G A was removed from the cell environment either by ultrafiltration (elimination of G A and other macromolecules greater than 30000) or by culture liquid substitutions using GA-non-producing sta2 mutants (isogenic to the tested STA2 strain) as donors of GA-free culture supernatant. It must be noted that, although the ‘basal’ carbon source (ethanol plus glycerol) was used in these experiments, the maximum PR value observed during the resulting secretion outburst was at least as high as theinduced PRvaluefound for theearly-exponential phase, starch-grown cultures. As suggested by the ultrafiltrationexperiments, IN may also besubject to feed-back control, since its production is enhanced following ultrafiltration. This is consistent with evidence reported earlier (Iurkevich. 1974). The observation that the IN accumulation pattern remains unchanged when GA, but not IN, is missing from the cell environment, suggests specificity of the G A feed-back control. Thus, we propose that the inducing role of starch may consist in attenuating the response of the cells to the presence of GA in the cell environment. The mechanism ofsuch attenuation is not known. It can be assumed that starch competes with the cell surface for G A molecules. In another line of speculation. starch could interact with the cell surface
In a report by Pretorius et al. (1986), it was pointed out that, unlike in rich medium, the presence of starch in synthetic complete medium was sufficient for the induction of considerably higher levels of G A mRNA and G A activity than in the use of glycerol plus ethanol. The attenuation model would then suggest that the G A feed-back response occurs at the level of G A mRNA production. This seems to find support in our current experiments. At the same time, the translational level could also be affected, as was believed in the case of yeast I N (Iurkevich and Galcheva-Gargova, 1975). In conclusion, we would like to note that feedback control-defective cells could be utilized for the production of increased quantities of yeast-secreted enzymes.
making the latter inaccessible or insensitive to GA.
Uspekhisovremennoi hiologii ( U S .R.R.)77,360-368.
REFERENCES Bcrezin, I. V., Rabinovich, M . L. and Sinitsyn, A. P. (1977). Study of applicability of quantitative kinetic spectrophotometric method for glucose determination. Biokhimiya (U.S.S.R.)42, 1631-1637. Inui. M., Fukui, S. and Yamashita, I . (1989). Genetic controls of STAl gene expression in yeast. Agric. Biol. Chcm. 53,741-748. lurkevich, V. V. (1974). Biosynthesis of secretory enzymes can be regulated by their presence in the medium.
125 lurkcvich, V. V. and Galcheva-Gargova, Z. 1. (1975). Localization of the effect of P-fructosidase biosynthesis rcgulation in yeast by the level of active enzyme of the medium. Dokludv Akudemii Nuuk SSSR ( I ' . S . S . R . j 225.97 I - 973. Jamicson. A. D., Pruitt, K. P. and Caldwell, R. S. (1969). An improved amylase assay. J . Dent. Res. 48,483--487. Kleinrnan. M . K . , Wilkinson, A. E., Wright. I. P.. Evans. I . H. and Bevan. E. A. (1988). Purification and properties of an extracellular glucoamylasc from a diastatic strain of .%cchurom)~cr.s cc~revisiue.Biochem. J . 249, 163 170. Kuchin, S. V., Neystat, M. A.. Mashko, S. V., Gerasimenko. 0.G . and Benevolensky, S. V. (1990).A mutational analysis of the starch utilization system in the yeast .Sacchoroni~w.s cerevisiue. Molekuliurnuya hio1ogij.u. mikrohiologiju i viruso1ogi)u ( C'.S.S. R.J , 5 5 . '7--29. Modena. D.. Vanoni. M., Englard, S. and Marmur, J . ( 1986). Biochemical and immunological chardcterization of the S T A k n c o d e d extracellular glucoamylasc from Succharom,rces diustuticus. Arch. Biochem. Biophj'.Y.248, 138-150. Pardo. J. M.. Polaina. J . and Jimenez. A. (1986). Cloning of the STA? and SGA genes encoding glucoamylases in yeasts and regulation of their expression by the STA 10 gene of Sacc.haromyrs cerevisiue. Nuclric Acids Res. 14.4701- 4718.
Polaina, J. and Wiggs, M. Y. (1983). STAIO: a gene involved in the control of starch utilization by Succhuromyces. Curr. Genet. 7, 109--112. Pretorius. I . S., Modena, D., Vanoni. M.. Englard. S. and Marmur, J. (1986). Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Succhuromyces species. Mol. Cell. B i d . 6, 3034-3041. Tamaki, H . (1978). Genetic studies of ability to ferment starch in Succhuromyces: gene polymorphism. Mol. Gen. Genet. 164,205-209. Yamashita, 1. and Fukui, S. (1984a). Mating signals control expression of both starch fermentation penes and a novel flocculation gene FLOR in the yeast Succhuromj.ces. Agric. Biol. Chem. 47, 2889.-2896. Yamashita, I . and Fukui. S. (1984b). Genetic background of glucoamylase production in the yeast Succharom y e s . Agric. B i d . Chem. 48, 137-. I4 1 . Yamashita, I., Hatano, T. and Fukui, S. (1984). Subunit structure of glucoam ylase of Succhuromyx~diusruticus. AKric. Biol. Chem. 48, 16 1 I - I6 16. Yamashita. I.. Nakamura. M. and Fukui, S. (1985). Diversity of molecular structures in the yeast extracellular glucoamylases. J . G m . Appl. Microhiul. 31, 339 401.
VOL.
7: 119-125 (1991)
Production of the STA2-Encoded Glucoamylase in Saccharomyces cerevisiae is Subject to Feed-back Control N. I. SUNTSOV, S. V. KUCHIN, M. A. NEYSTAT, S. V. MASHKO A N D S. V. BENEVOLENSKY Institute of Genetics and Selection of Industrial Microorganisms. Moscow 113545, USSR
Received 14 February 1990; revised I6 June 1990
Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified: repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold difference in the rate of G A production) can be displayed by either glucose-limited or glycerol- plus ethanolconsuming cultures: the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated G A . It is proposed that the feed-back control involved in G A production can be attenuated by starch which can thus ‘induce’ higher rates of G A production compared to the basal mode. K E Y WOKDS -
Saccharomyces cerevisiae; yeast; starch; secretion; extracellular glucoamylase; feed-back control.
INTRODUCTION
conditions (Yamashita and Fukui, 1984a; Inui et al., 1989). Diastatic strains of Saccharomyces cerevisiae are The effect of different carbon sources on GA capable of producing extracellular glucoamylase expression has been examined revealing that the (GA) (1,4-a-~-glucanglucohydrolase, EC 3.2.1.3) highest levels of GA activity and GA mRNA are and can, therefore, utilize exogenous starch. accumulated by cultures grown in rich medium The yeast extracellular GA is a heavily glycosy- supplemented with starch (Inui el al., 1989) or lated protein with an average molecular weight of glycerol plus ethanol (Pretorius er al., 1986). All about 300000, data reported so far suggesting a sugars tested (including GA-hydrolysable maltose) variety of forms of this enzyme with respect to its reduce GA expression (Pretorius el al., 1986). In this report, we have examined the production structure and enzymatic properties, which areapparently dependent on a particular Saccharomyces of GA by a Saccharomyces strain in different growth strain and on growth conditions (Yamashita et a f . , conditions, identifying three distinct GA produc1984, 1985; Modena et al., 1986; Kleinman et al., tion modes: induced, basal and repressed. We also present evidence supporting the involvement of 1988). Three identical unlinked genes, STA1, STA2 and feed-back control in GA production. We propose STA3, are known to be the structural genes for GA, that the induced mode demonstrated by earlyeach conferring upon the cell the ability to produce logarithmic cultures in starch-containing media, is GA (Tamaki, 1978). GA production directed by the related to the feed-back control-relieved state. STAI-STA3 genes can beconstitutively repressed in a dominant and epistatic fashion by the allele(s) MATERIALS AND METHODS STAlO (or I N H I ) present in most S. cerevisiae strains (Polaina and Wiggs, 1983; Yamashita and Strains We used Saccharornyces cerevisiae strains 249 Fukui, 1984b). STAIO has been shown to affect GA expression at the transcriptional level (Pardo et al., (STA2 stalO), 501 (sta2-1 stal0) and 509 (sla2-3 1986). Heterozygosity at MAT (in diploids) and stalO) from our collection. Strains 501 and 509 I N H I inhibit GA expression at the transcriptional are isogenic to 249 except that they carry two or post-transcriptional levels depending on growth independent sta2 mutations (Kuchin et al., 1990). 0749-503X~91:020119-07$05.00 0 1991 by John Wiley & Sons Ltd
120 Media
YP medium (also referred to as rich medium) containing 0.5% yeast extract and 1 .O% bacto-peptone (Difco) was supplemented with: 2.0% glucose (YPD), 2.0% galactose (YPGal), 2.0% maltose (YPMal), 2.0% sucrose (YPSuc), 3.0% glycerol plus 1.5% ethanol (YPGE), 1.0,2.0 or 4.0% soluble starch (YPSI, YPS2 and YPS4, respectively), 2.0% glucose plus 1.0% starch (YPDS). Growth conditions
Batch cultures were grown in shake flasks on an orbital shaker (250 rpm) at 28°C with an initial cell titre of 5.0 x lo5 cells/ml, starting with a 24-h preculture grown on plates containing YPD medium solidified withl.570 agar. Chemostat cultures were run using a 350-ml fermenter (BioFlo Model C30, New Brunswick Scientific Co.) at 28°C and pH 5.4.The dissolved oxygen tension was maintained above 40% air saturation at an agitation of 300 rpm. After an increase in dilution rate, the time taken for six vessel volumechanges was allowed in order to reach a new steady state. To inoculate fermenter cultures, a 24-h batch culture of strain 249 grown in YPD was used. Determination of cell titres
Cell titres were determined microscopically using a cell counting chamber. Enzyme assays
GA activity wasquantified by determining the rate ofreleaseofglucosefromamylose(Serva;0.5%, wjv, final concentration in 100 mM phosphate buffer, pH 5.4, at 37°C. Glucose content was measured either enzymatically using the glucose oxidase-peroxidase method (Berezin et al., 1977), or by determining the content of reducing sugars as described by Jamieson et ul. (1969). Invertase (IN) activity was determined according to Jamieson et al. (1 969). The GA and IN units are expressed, respectively, as nmol and pmol of glucose released/min. Proportionality of the measured GA and IN activities to the corresponding enzyme amounts was occasionally confirmed by monitoring these activities in successive dilutions of culture supernatants. Probes were diluted with distilled water.
N. I. SUKTSOV E r AL.
Table I . Effect of the carbon source on the stationaryphase GA activity level in the culture medium Cell titre
Medium
cells/ml
( x lo8)
GA unitsiml
GA units Per 10* cells
30 22
3.1
~~
YPD YPGal YPMal YPSUC YPSI YPS2 YPS4 YPGE
17 7.1 12 5.0 3.1 5.2 8.0
6.9
25 15 160 I80 250 100
1.8 2.1 3.0 52 35 31
14
Ultrafiltration
Ultrafiltration of cell-free culture supernatants was carried out using Millipore equipment; the membrane used was PTTK (cut-off 30 000).
RESULTS Effect of the carbon source on glucoamylase production
The accumulation of GA was examined in culture supernatants of strain 249 grown in rich medium supplemented with the following carbon sources: glucose, galactose, maltose, sucrose, starch and glycerol plus ethanol. Cells were grown until the stationary phase. Culture samples were then collected and the corresponding cell titres counted. The cell-free culture supernatants were examined for GA activity. The levels of enzyme activity (Table 1 ) suggest that the carbon sources tested can be placed in threecategories: (i)stdrch (the highest levels ofGA accumulation); (ii) glucose, galactose, maltose and sucrose (the lowest levels of GA accumulation); (iii) glycerol plus ethanol (an intermediate level of GA accumulation). The results reveal a more apparent difference if related to the corresponding cell titres. Chemostat cultivation
The increased production of GA displayed by cultures grown on starch could be due to the effect of starch per se. On the other hand, this may be a response to glucose limitation. The two possibilities can in principle be distinguished in glucose-limited chemostat culture. We ran chemostat cultures in
121
PRODUCTION OF THE STAIENCODED GLYCOAMYLASE
Tablc 2. G A and IN production rates in chemostat cultures
PR (enzyme unitsih per lo8cells) ~~
Medium D ( h YPD YPGE YPD YPDS
’)
0.10 0.20
~
~~
~
GA
IN
1.5k0.2 I .8 5 0.2 2.8 f0.3 2.2 5 0 . 2
90k 10 34k 7 160k20
n.d.
Standard errors were estimated from triplicate determinations; nd.. not determined.
three media (YPD, YPGE and YPDS) at two dilution rates (0.10 and 0.20 h-I) assaying for cell titres and activities of GA and IN (as a control). The results were expressed in terms of production rate (PR), i.e. the enzyme activity produced by an average cell per unit time (equation I):
PR = ( E x D)iT
(1)
where E and Tare the steady-state enzyme activity level and cell titre, respectively; D, the dilution rate. As suggested by the results (Table 2), glucose limitation does not stimulate GA induction per se under conditions used. Indeed, at D=O.lO h-I, the PR values for YPGE and YPD (1.8 and 1.5 GA units/h per 10’ cells, respectively) cannot be considered as different within the error range. Furthermore, we observed no induction for YPDS compared to YPD (2.2 versus 2.8 GA units/h per lo8 cells; D = 0.20 h-I). although starch was abundant in the cell environment as indicated by iodine staining. The results also suggest that the rates of both GA and IN production are proportional to D ;the percell-cycle amounts of secreted GA and I N thus appear to remain relatively constant despite the decrease in the doubling time from 10 to 5 hours. I t should be noted that the chemostat cultures would be washed out at a D of less than 0.30 h-’; therefore. it was impossible to achieve significantly higher specific growth rates, which, as we reasoned, might be a restrictive condition with respect to the induction of higher rates of G A production. Dynamics ofgrowth and G A accumulation using diflerent carbon sources To elucidate the conditions in which starch could induce the production of increased quantities of GA, we studied the dynamics of growth and GA
accumulation in logarithmic cultures of strain 249 grown in YPSI, YPS2, YPS4, YPGE and YPD media (Figure I). To obtain a more detailed pattern of the growth-phase modulation of GA production, we interpreted the dynamics data in terms of per-cell PR, as was the case with chemostat cultures. In this case, however, a more complicated mathematic approach would be required since parameters of a periodic culture are subject to a continuous change. A formal equivalent of equation 1 can be presented as follows (equation 2): 1 dE(t) PR(t) = x T(t) dr where E(t)and T(t)are, respectively, the level of GA activity and the cell titre at time t. Equation 2 can be readily applied to inspecting the PR function throughout the logarithmic phase because E(t) and T(t)increaseexponentiallywith time. The log(E)and log(7J points were thus easily fitted by linear regression. The resulting functions, log(E(1 ) ) and log(T(t)),weresubsequentlypotentiated and applied to equation 2. The PR functions thus obtained (Figure 2) reveal dependence on the carbon source used: PR(t) for YPD (not plotted) represents the repressed mode of GA production and is vanishingly close to zero on the scale used; the PR curve for YPGE and the bunch of curves for YPSI-YPS4 are considerably gapped yielding an approximately seven-fold difference in PR in the early-exponential phase. We have termed the latter two production modes ‘basal’ (for YPGE) and ‘induced’ (for YPS 1-YPS4). Notably, the basal PR(r) functionis relativelyconstant throughout the exponential phase. In contrast, the induced PR values, although initially high, tend to decrease exponentially towards the basal PR value, which is ultimately reached in the lateexponential phase, establishing a pattern resembling that found in the chemostat experiments. ~
~
Glucoamylase production is subject tofeed-back control Ascanbeseenin Figure2, thePRcurvesforYPSlYPS4 media are closely compacted on the versustime plot; therefore, the synchronous decrease in PR in these media is unlikely to be caused by a gradual exhaustion of starch from the cell environment. Our hypothesis was that it might be the extracellularly accumulated GA that was involved in the inhibition of GA production.
122
N. I. SUNTSOV ET AL.
B
A
I
i/!I
I
t
t
t
t
L
D
C
a
0
20
40
0
20
40
60
T i m e , hrs Figure 1. Dynamics of growth and accumulation of GA in the culture medium in the logarithmic phase. Cells of strain 249 were grown in shake flasks containing the following media: YPSl (A),YPS2 (.)and YPS4(3)(AandC);YPGE(O)andYPD(.)(BandD). Growth wasmonitored by counting the cell titre (A and B). Cell-free culture supernatants were examined for GA activity (C and D).
To test this, cells of strain 249 were grown in YPGE medium (to ensure the basal GA production mode) up to the mid-exponential phase, and the entire cell-free supernatant was then subjected to ultrafiltration to remove macromolecules greater than 30 OOO. GA and IN activities were found to be completely removed from the culture supernatant by this procedure. The cells were subsequently resuspended in the filtered supernatant and put onto
the shaker for further growth. The break in growth did not exceed 30 min. Except for the ultrafiltration, the same manipulations were applied to the control culture. In both the tested and the control cultures GA and IN activities and cell titres were monitored. It was found that GA and IN activity levels were quickly restored up to the levels exhibited by the control culture, although growth was not affected. Estimations carried out according to equation 2
PRODUCTION OF THE STAPENCODED GLYCOAMYLASE
123 level also reaches that of the control shortly after growth is resumed so that the PR value approaches a maximum of about 46 GA units/h per 10’ cells by the end of the period followed (8 h following the substitution) (Figure 3, curve 3). Similar results have been obtained using another sra2 mutant, 501 (not shown). The PR value for the control culture has been estimated that is relatively constant at about 3.5-2.5 GA units/h per lo8 cells. I t should be outlined that the IN accumulation pattern remains unchanged when GA, but not IN, is missing from the cell environment.
0
10
20
30
Time, hrs Figure 2. G A production rates (the PR functions) in the logarithmic phase. Cells of strain 249 were grown in Y PSI (centre line), YPS2 (dashed line), YPS4 (dotted line) and YPGE (solid line). The curves were obtained by mdthematic treatment of data depicted in Figure I asdescribed in the text. Theconsiderable lag observed during growth in YPGE medium has been omitted to align the period of logarithmic growth in YPGE to those in Y PS I , Y PS2 and Y PS4, so that f = 0 corresponds to a cell titre of 2.6 x ICP cellsiml in all these cultures.
indicate that the rate of GA production increases for at least 8 h following ultrafiltration to reach a value of approximately 15 units/h per lo8 cells (Figure 3, curves 1 and 2), which is close to the PR values characteristic of the induced GA production mode (12-16 unitsih per lo8 cells) found for the early-log starch-grown cultures. However, it was not clear whether it was GA itself whose absence brought about the increase in the rate of GA production. Indeed, arrested on the membrane might be an extracellular regulatory factor universal in its function to both GA and IN (or to other secretory protein also). To clear up this point, we used strain 509, an isogenic sta2 mutant of strain 249, as a donor of GA-free culture supernatant. The donor was grown in parallel with the tested strain under the same conditions. In the midexponential phase the wild-type culture liquid of strain 249 was replaced by the mutant one (instead of the ultrafiltration). In this case, the GA activity
DISCUSSION In this paper, we describe the effect of different growth conditions on the production of GA by a diastatic strain of S. cerevisiae. First, we have classified several carbon sources by their impact on the stationary-phase GA activity levels accumulated in culture supernatants. The carbon sources tested fall into three categories: (i) starch (the highest levels of GA accumulation); (ii) monosaccharides and disaccharides, including GA-hydrolysable maltose (the lowest levels of GA accumulation); (iii) glycerol plus ethanol (an intermediate level of GA accumulation). We have also studied the dynamics of growth and enzyme accumulation in batch cultures grown on several carbon sources. The obtained results were interpreted using a composite function, PR(r), defined as the rate of GA production by an average cell at time t . Comparisons of the log-phase PR curves using several carbon sources have revealed a more dramatic difference between these substrates than might be expected from the initial screening: starch, or some products of its breakdown, appear to induce GA production in the early-exponential phase cells. Such cells display a PR value which is induced approximately seven-fold over the value found for the ethanol-plus glycerol-consuming early-exponential phase culture. We have termed the two GA production modes ‘induced’ and ‘basal’, respectively. However, the PR(r) function for starch-grown cultures exhibits an exponential decrease during the logarithmic phase: the lowest PR value reached in the late-logarithmic phase is close to the basal PR value. In contrast, the basal PR(r) tends to be relatively unchanged throughout the log phase. To find a basis for such a decrease, we have put forward the hypothesis that GA production may be interfered with by extracellularly accumulating GA. To test this, we havecarried out experiments in which
124
N. 1. SUNTSOV E T A L .
0
36
40
44
0
36
40
44
T i m e , hrs
Figure3. Effect ofremovalofextracellularlyaccumulatedproteinon thedynamicsofGAandINaccumulationintheculturemedium. Cells of strain 249 were grown in YPGE medium until the mid-logarithmic phase. At 36 h cells were pelleted by centrifugation and the ccll-free supernatant was ( I ) ultrafiltered to remove macromolecules greater than 30 OOO; (2) kept unfiltered;(3) replaced by the GA-free supernatant of strain 509 grown in parallel under the same conditions. The pelleted cells were subsequently resuspended in each of the three above liquids to resume growth. Culture supernatants were examined for GA and IN activities (B and C, respectively). Cell titres were also determined (A). PR functions were then derived as described in the text.
G A was removed from the cell environment either by ultrafiltration (elimination of G A and other macromolecules greater than 30000) or by culture liquid substitutions using GA-non-producing sta2 mutants (isogenic to the tested STA2 strain) as donors of GA-free culture supernatant. It must be noted that, although the ‘basal’ carbon source (ethanol plus glycerol) was used in these experiments, the maximum PR value observed during the resulting secretion outburst was at least as high as theinduced PRvaluefound for theearly-exponential phase, starch-grown cultures. As suggested by the ultrafiltrationexperiments, IN may also besubject to feed-back control, since its production is enhanced following ultrafiltration. This is consistent with evidence reported earlier (Iurkevich. 1974). The observation that the IN accumulation pattern remains unchanged when GA, but not IN, is missing from the cell environment, suggests specificity of the G A feed-back control. Thus, we propose that the inducing role of starch may consist in attenuating the response of the cells to the presence of GA in the cell environment. The mechanism ofsuch attenuation is not known. It can be assumed that starch competes with the cell surface for G A molecules. In another line of speculation. starch could interact with the cell surface
In a report by Pretorius et al. (1986), it was pointed out that, unlike in rich medium, the presence of starch in synthetic complete medium was sufficient for the induction of considerably higher levels of G A mRNA and G A activity than in the use of glycerol plus ethanol. The attenuation model would then suggest that the G A feed-back response occurs at the level of G A mRNA production. This seems to find support in our current experiments. At the same time, the translational level could also be affected, as was believed in the case of yeast I N (Iurkevich and Galcheva-Gargova, 1975). In conclusion, we would like to note that feedback control-defective cells could be utilized for the production of increased quantities of yeast-secreted enzymes.
making the latter inaccessible or insensitive to GA.
Uspekhisovremennoi hiologii ( U S .R.R.)77,360-368.
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125 lurkcvich, V. V. and Galcheva-Gargova, Z. 1. (1975). Localization of the effect of P-fructosidase biosynthesis rcgulation in yeast by the level of active enzyme of the medium. Dokludv Akudemii Nuuk SSSR ( I ' . S . S . R . j 225.97 I - 973. Jamicson. A. D., Pruitt, K. P. and Caldwell, R. S. (1969). An improved amylase assay. J . Dent. Res. 48,483--487. Kleinrnan. M . K . , Wilkinson, A. E., Wright. I. P.. Evans. I . H. and Bevan. E. A. (1988). Purification and properties of an extracellular glucoamylasc from a diastatic strain of .%cchurom)~cr.s cc~revisiue.Biochem. J . 249, 163 170. Kuchin, S. V., Neystat, M. A.. Mashko, S. V., Gerasimenko. 0.G . and Benevolensky, S. V. (1990).A mutational analysis of the starch utilization system in the yeast .Sacchoroni~w.s cerevisiue. Molekuliurnuya hio1ogij.u. mikrohiologiju i viruso1ogi)u ( C'.S.S. R.J , 5 5 . '7--29. Modena. D.. Vanoni. M., Englard, S. and Marmur, J . ( 1986). Biochemical and immunological chardcterization of the S T A k n c o d e d extracellular glucoamylasc from Succharom,rces diustuticus. Arch. Biochem. Biophj'.Y.248, 138-150. Pardo. J. M.. Polaina. J . and Jimenez. A. (1986). Cloning of the STA? and SGA genes encoding glucoamylases in yeasts and regulation of their expression by the STA 10 gene of Sacc.haromyrs cerevisiue. Nuclric Acids Res. 14.4701- 4718.
Polaina, J. and Wiggs, M. Y. (1983). STAIO: a gene involved in the control of starch utilization by Succhuromyces. Curr. Genet. 7, 109--112. Pretorius. I . S., Modena, D., Vanoni. M.. Englard. S. and Marmur, J. (1986). Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Succhuromyces species. Mol. Cell. B i d . 6, 3034-3041. Tamaki, H . (1978). Genetic studies of ability to ferment starch in Succhuromyces: gene polymorphism. Mol. Gen. Genet. 164,205-209. Yamashita, 1. and Fukui, S. (1984a). Mating signals control expression of both starch fermentation penes and a novel flocculation gene FLOR in the yeast Succhuromj.ces. Agric. Biol. Chem. 47, 2889.-2896. Yamashita, I . and Fukui. S. (1984b). Genetic background of glucoamylase production in the yeast Succharom y e s . Agric. B i d . Chem. 48, 137-. I4 1 . Yamashita, I., Hatano, T. and Fukui, S. (1984). Subunit structure of glucoam ylase of Succhuromyx~diusruticus. AKric. Biol. Chem. 48, 16 1 I - I6 16. Yamashita. I.. Nakamura. M. and Fukui, S. (1985). Diversity of molecular structures in the yeast extracellular glucoamylases. J . G m . Appl. Microhiul. 31, 339 401.
