Journal of General Microbiology (1989), 135, 2407-241 1. Printed in Great Britain
2407
Controlling the Growth Rate of Succhuromyces cerevisiue Cells Using the
Glucose Analogue D-Glucosamhe
By E A M O N M. M c G O L D R I C K t A N D A L A N E . WHEALS*
Microbiology Group, School of Biological Sciences, University of Bath, Bath BA2 7A Y, UK
(Received 19 December 1988; revised 8 June 1989; accepted 15 June 1989)
By using competition between glucose and its analogue D-glucosamine, we have produced a
system in which it is possible to vary the steady-state growth rate of populations of
Saccharomyces cerevisiae cells without otherwise altering the composition of the medium or
significantly affecting catabolite repression. We demonstrate that D-glucosamine inhibits the
accumulation of glucose derived label and the phosphorylation of glucose by hexokinase
(EC 2.7.1.1).
INTRODUCTION
Being able to control the growth rate of populations of cells at constant temperature is a useful
facility both for industrial purposes and as an investigative tool for studies on cell growth and
division. Several methods are in wide use (Pirt, 1975).
Batch culture methods rely on altering the overall nutritional composition of the medium or by
changing the carbon source. This method of control suffers from the drawback that general
cellular metabolism may change and cells may be relieved of carbon catabolite repression on
non-fermentable carbon sources (Magasanik, 1961; Polakis & Bartley, 1965).
The chemostat supplies carbon sources, such as glucose, at rates that become limiting for
growth. Although the supply can be adjusted to ensure continued population growth, the
metabolic profile of cells growing with significantly reduced growth rates is typical of cells that
are not catabolite repressed (Beck & von Meyenburg, 1968).
Diffusion capsules (Pirt, 1971) reduce growth by allowing a limited supply of substrate to
diffuse into the medium along a concentration gradient. However, since the requirement for
substrate increases with the exponentially growing population, the supply/demand curve is
constantly changing, making steady growth difficult to maintain.
It has been demonstrated that certain non-metabolizable glucose analogues are capable of
inhibiting the growth of yeast via effects on fermentation (Woodward & Hudson, 1953). We
report here a method of modulating the continuous exponential growth of Saccharomyces
cerevisiae in batch culture using the competition between glucose and its analogue
D-glucosamine to limit the metabolism of glucose without significantly altering the repression
status of the cell. The effect of glucosamine on the uptake and initial metabolism of glucose were
also examined.
METHODS
Strains, media and culture conditions. S . cerevisiae strain SR665-1 (MATa met2 tyrl cyh2 cdc39 g a l l ) was
obtained from S. Reed (Research Institute of Scripps Clinic, La Jolla, CA, USA), and strain D273-lla
(Mata adel his1 trp2) from L. Johnston (National Institute for Medical Research, Mill Hill, UK). Complex media
(YEP) contained 1% (w/v) yeast extract, 2% (w/v) mycological peptone and carbon source as indicated. Agar (2%,
w/v) was used to solidify the media. For liquid cultures, cells were shaken in conical flasks filled to less than 30%
total volume. All growth experiments were done at 23 "C unless stated.
t Present address: Department
0001-5303 0 1989 SGM
of Biochemistry, University of Leeds, Leeds LS2 9JT, UK.
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2408
E. M .
MCGOLDRICK A N D
A.
E. WHEALS
Invertuse assay. Samples (1 ml) were incubated at 3 1 "C for 10 min. Sucrose (0.5%, w/v) was added to start the
reaction which was stopped by addition of 1-5ml dinitrosalicylic acid reagent (Miller, 1959) after an appropriate
time. Colour was developed by boiling for 15 min, the absorbance read at 640 nm and compared with a standard
curve prepared with glucose.
Hexokinase assay. Cells were grown to 1 x lo7 cells ml-I in YEP plus 2% (w/v) glucose at 23 "C. They were
centrifuged, washed twice in sterile distilled water and resuspended in 0.1 M-potassium phosphate buffer (pH 7.0)
containing 10 mwmagnesium chloride. A sample (0.7 ml) of this slurry was mixed with sufficient (450-500 pg)
glass beads to give a thick paste in a 1.5 ml Eppendorf tube. The tube was vortexed three times for 20 s with
periods on ice between each treatment. The supernatant was centrifuged from the tubes by puncturing the base
and standing the tube inside a second decapped 1.5 ml tube. This 'piggyback' arrangement was centrifuged in a
50 ml tube. The crude cell-free extract (1 ml) was passed through a Sephadex G50 column, and eluted with 0.1
M-potassium phosphate buffer (pH 7.0) containing 10 mM-magneSiUm chloride. Hexokinase was then assayed
using the method of Lob0 & Maitra (1977). Protein content was determined using the Bio-Rad protein assay.
Glucose uptake. Cells were grown to 1 X lo7 cells ml-I in YEP plus 2% (w/v) glucose at 23 "C, harvested by
centrifugation (6000 r.p.m. for 5 min in a Sorvall RC5B centrifuge, GSA rotor), washed twice in distilled water
and resuspended at 2 x lo8 cells ml-' in 20 mM-Tris/HCl buffer (pH 6.7). Cell suspension (2.5 ml) was added to
Universal bottles containing appropriate concentrations of D-[ l-14C]glucose(220 MBq mmol-l ; Amersham) and
glucosamine, made to a final volume of 2.5 ml with Tris/HCl buffer. This solution was equilibrated at 30 "C prior
to addition of cells. Samples (1 ml) taken at 20 s intervals were transferred to a 0.45 pm Millipore filter and washed
with 5 ml Tris/HCl buffer. Filters were transferred to 7.5 ml Optiphase Safe (Fisons) scintillation fluid and
counted in an LKB Rackbeta 1217 liquid scintillation spectrometer.
Cell number. This was measured using an Electrozone Celloscope electronic particle counter.
RESULTS
D-Glucosamine is not metabolized at an appreciable rate and does not support growth of S .
cerevisiae (Burger & Hejmova, 1961). Moreover, glucosamine prevents growth of yeast on
glycerol or ethanol (Furst & Michels, 1977). We have extended these observations by failing to
observe growth of S . cerevisiae D273-1 l a on YEP containing 1% (w/v) glucosamine and any of
the following substrates added at 2 % (w/v); raffinose, cellobiose, mannitol, sorbitol, sodium
succinate, sodium acetate, lactic acid, sucrose, galactose or glycerol. Concentrations of
glucosamine greater than 0.1 % completely inhibited growth on 2% (w/v) sodium pyruvate but
allowed, with reductions in rate, continued growth on 0.05 % (w/v) glucose.
The relationship between glucose and glucosamine with respect to effects on growth rate was
further quantified by growing cells at 23 "C in YEP medium supplemented with various
concentrations of glucose and glucosamine (Fig. 1). It was found that for a fixed concentration of
glucose, the glucose :glucosamine ratio would extend the population doubling time in a
predictable manner, and that this effect was greatest at low concentrations of glucose. These
results suggested that a glucose/glucosamine competition may have been reducing the flux of
glucose available for growth.
D-Glucosamine is capable of producing a repressed state similar to glucose repression (Furst &
Michels, 1977) and has been used in the selection of carbon catabolite repression resistant
mutants (Michels & Romanski, 1980). Invertase (EC 3.2.1 .26) catalyses the hydrolysis of
sucrose to fructose and glucose and is subject to glucose repression (Dodyk & Rothstein, 1984). It
has frequently been used as an indicator of the repression status of cells (Matsumoto et al., 1983;
Bailey & Woodward, 1984). Table 1 depicts the relationship between carbon source, growth rate
and invertase activity. Although the addition of 1% (w/v) glucosamine significantly reduces the
growth rate, the average invertase activity in these cells is more typical of the fast-growing,
repressed, glucose-grown cells than of derepressed cells metabolizing pyruvate.
Glucose accumulation and the effect of glucosamine upon it were examined further. In the
absence of glucosamine intact cells were incubated with radiolabelled glucose, and its
intracellular accumulation followed over 20 s intervals. A Lineweaver-Burk (1934) plot of
glucose accumulation against extracellular glucose concentration gave apparent V and K ,
values of 21 nmol per lo8 cells mind' and 1 mM respectively. Increasing concentrations of
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Controlling yeast growth rate
1
2
3
4
5
D-GlUCOSamine concn (%, w/v)
Fig. 1 . Effect of varying the glucose :glucosamine ratio on the growth rate of S. cereuisiue SR665-1.
Cells were grown at 23 "C on YEP medium with various concentrations of glucose and glucosamine.
0 , 0.5% glucose; A, 1.0% glucose; 0,
2.0% glucose. Data are the means of at least two separate
experiments.
Table 1. Growth rate and invertase activity of S. cerevisiae grown on various carbon sources
S. cerevisiae SR665-1 was grown at 23 "C on YEP plus various carbon sources. Invertase activity (mean
f SE of four separate experiments) was determined as described in Methods.
Carbon source
(%, wlv)
Population
doubling time (min)
Invertase activity
[pmol glucose formed
(pg protein)-' min-'1
Glucose (2)
Glucose (2) glucosamine (1)
Sodium pyruvate (2)
120
420
210
0.063 f 0.01
0.16 f 0.01
0.71 f 0.06
+
glucosamine had increasingly inhibitory effects on glucose accumulation (Fig. 2) but the data
failed to fit standard inhibitor kinetic plots. It is possible that glucosamine affects the various
elements of the multi-event glucose uptake system differently.
Since facilitated diffusion is proposed to be involved in glucose uptake, hexokinase activity
plays an essential role in uptake by maintaining the necessary concentration gradient. As an
element of uptake, hexokinase can be examined independently of membrane transport by using
partially purified cell-free extracts. A Lineweaver-Burk plot of hexokinase activity relative to
glucose concentration gives V and K , values of hexokinase for glucose as 4.4 pmol NADPH
formed mg-' min-l and 0.19 mM respectively. The ability of D-glucosamine to inhibit
hexokinase activity was assayed at glucose concentrations around the K , value. In this case the
results generated fit the Dixon plot characteristic of competitive inhibitors (Dixon, 1953) (Fig.
3). This plot gave a Kivalue for the effect of D-glucosamine on the ability of hexokinase to
phosphorylate glucose of 1.7 mM.
DISCUSSION
The results demonstrate that glucosamine can reduce the growth rate of yeast cells
metabolizing glucose. Glucosamine can act as a competive inhibitor of hexokinase in vitro and
inhibit the accumulation of labelled glucose in vivo; but the complex nature of the glucose uptake
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E . M . M C G O L D R I C K A N D A . E . WHEALS
0
10
20
30
40
50
D-Glucosamine concn (mM)
L
-2
'
I
I
1
I
1
I
0
1
D-Glucosamine concn (mM)
-1
Fig. 2
J
2
Fig. 3
Fig. 2. Effect of glucosamine on the accumulation of labelled glucose by intact cells of S.cerevisiue
SR665- 1 . The incubation solution contained various concentrations of labelled glucose and
glucosamine. Accumulation was deemed to begin upon addition of cells. Inhibition is expressed relative
to the maximum observed accumulation of 16.7 nmol glucose per lo8 cells min-l which is assigned a
value of 100%. 0 , 1 7.5 mM-glucose ; A, 1 2.5 mM-glucose ; 0 , 10.0 mM-glucose ; , 7.5 mM-glucose.
Data are the means of at least two replicates.
Fig. 3. Inhibition of hexokinase by D-glucosamine. Hexokinase was assayed in partially purified cell
free extracts of S. cerevisiue SR665-1 in the presence of various concentrations of glucose and
glucosamine. 0 , 0.05 mM-glucose ; A, 0.10 mM-glUCOSe; 0 , 0.20 mM-glucose.
system, in which transport may be linked to phosphorylation, makes it difficult to state, from
these data, the point(s) at which glucosamine disrupts glucose transport. However, the role of
hexokinase in the establishment of catabolite repression in yeast has been demonstrated (Entian
et ai., 1984) and glucosamine induced catabolite repression is well-documented (Furst &
Michels, 1977 ; Hockney & Freeman, 1980). Consequently, glucosamine must cross the
membrane to bind with intracellular hexokinase in vivo and may use the glucose transport system
to gain entry. If so, glucosamine is likely to inhibit both transport and phosphorylation of
glucose.
There are two systems for the transport of glucose into yeast cells (Bisson & Fraenkel, 1983a;
Lang & Cirillo, 1987). The low affinity uptake system (apparent K , 20 mM) is mediated by
constitutive carrier-mediated facilitated diffusion. The high-affinity system (apparent K , 2 mM)
depends on any one of three kinases (hexokinase I and I1 and glucokinase) and is regulated by
catabolite repression and inactivation (Bisson, 1988). There is a reciprocal relationship between
the levels of high- and low-affinity processes (Ramos et al., 1988). Transport of a nonmetabolizable glucose analogue, 6-deoxy-~-glucose,is kinase-dependent in the same way as
glucose suggesting that the role of the kinases is not merely through phosphorylation and
metabolism of the sugar itself. The glucose analogue is transported with biphasic kinetics with
high and low K , components (Bisson & Fraenkel, 1983b).
The uptake results reported here referred to the intracellular accumulation of glucose-derived
label and were therefore a measure of the total system rather than its individual components.
The non-standard inhibition kinetics shown in Fig. 2 suggested that both high- and low-affinity
components were being affected. At the higher glucose concentrations (17.5 mM) the lowaffinity system was being used and was subject to standard competitive inhibition from
D-glucosamine. However, the reduced effect of the inhibitor at lower glucose concentrations
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Controlling yeast growth rate
241 1
(7.5 mM) suggested that the high-affinity system was now becoming more important and this
system had a lower affinity for the inhibitor. Indeed, the ability to study hexokinase independent
of transport allowed stronger conclusions to be reached. The K , and Ki values of 0.19 mM and
1-7 mM were sufficiently similar to support the observation that glucosamine was acting as a
competitive inhibitor of hexokinase.
Glucosapine provides the same trigger for repression as glucose. However, since catabolism
proceeds no further, its energetic contribution is zero, there is a reduction in the energy available
from glycolysis and a consequential reduction in growth rate. Although certain strains of yeast
show a resistance to glucosamine, the principle of competition by a non-metabolizable
catabolite repressor can be extended to alternative glucose analogues (Woodward et al., 1953).
The controlled reduction of growth rate allowed by this system may bring the convenience of the
shake flask to experiments that previously demanded a glucose-limiting chemostat.
Furthermore, it will now be possible to vary the growth rate over a wide range without altering
the catabolite-repression status of the cells or the composition of the medium.
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