Yeast Culture Responses: Galactose Pathway Enzyme Regulation

Archives of
Hicrebiolo!ly
Arch Microbiol (1983) ~35 : 115 - t 19
9 Springer-VerIag1983
Transient responses of yeast continuous cultures to qualitative changes
in the nutrient supply. Induction and repression of the galactose pathway enzymes
M. J. Galindez*, N. Ruiz Ordfiz*, P. Herrero, and F. Moreno
Departamento Interfacultativo de Bioquimica, Facultad de Medicina Universidad de Oviedo, Oviedo, Spain
Abstract. Induction and repression kinetics of alphagalactosidase, galactose uptake system and Leloir pathway
enzymes were studied in chemostat cultures by changing the
medium feed from glucose (11 mM) to glucose and galactose
(11 mM; 17 mM respectively) in the induction experiments;
and from galactose (11 mM) or (111 raM) to galactose plus
glucose (83 mM) in the repression experiments.
Basal levels of alpha-galactosidase and glucose uptake
could be estimated in glucose-limited yeast cells, but it was not
possible to detect any glactose pathway enzyme activity. In
the repression experiments under galactose-limited or galactose-sufficient yeast cells, atpha-galactosidase and galactokinase decayed with Ka = -0.21 h - l = - D ; that is, synthesis of these enzymes ceased (catabolite repression). In
contrast transferase and epimerase activities and galactose
uptake, decreased with Kd values of - 0.33 and - 0.54 h - 1,
showing that these activities were also subject to catabolite
inactivation.
Key words: Induction - Catabolic repression - Galactose
metabolism - Yeast
The bulk of our existing knowledge of transient phenomena
such as enzyme induction and repression comes from observations of batch cultures in which there are supersaturating
concentrations of the carbon sources, although it is assumed
that microbial growth in most natural environments occurs at
subsaturating concentrations of the energy sources (Tempest
and Neijssel 1976). It is well known that the continuous
culture technique is a way to maintain microbial cultures
under nutrient limited growth conditions. This technique has
been widely used for studies in microbial physiology (Tempest
and Neij ssel 1976), usually employing steady state conditions,
but limited information is available about microbial responses to changes in environment studied in chemostat, and
this existent information for the most part concerns bacteria
(Ryu and Mateles 1968; Dean 1972; Ashby and Harrison
1980). However, the observation of transient responses of a
yeast culture growing in steady state to a gradual change in
the level of an inducer or a repressor can render much
* Present address: Escuela Nacional de Ciencias Biol6gicas, Instituto
Polit~cnico Nacional, Mexico
Offprint requests to: F. Moreno
Abbreviations: PPO, 2.5-diphenyloxazole; POPOP, (1,4-bis[2-(5-phen-
yloxazoIyl)lbenzene
information about the physiology and regulatory mechanisms of the lower eukaryotes.
One yeast system which has been both biochemically and
genetically well defined, and which appears well suited for the
study of metabolic regulation in eukaryotic cells, is the
galactose utilizing system of Saccharomyces. The utilization
of galactose by Saccharomyees requires the induction of four
proteins: a specific galactose transport protein and the Leloir
pathway enzymes galactokinase (EC 2.7.1.6), uridyltransferase (EC 2.7.7.12) and UDP-glucose 4-epimerase (EC
5.1.3.2).
Classic genetic analysis has revealed both positive and
negative regulatory genes that coordinately affect the appearance of the Leloir pathway enzymes (Douglas and
Hawthorne 1964, 1966), and there is evidence indicating that
alpha-glactosidase (EC 3.2.1.22) and the galactose pathway
enzymes are genetically co-regulated (Kew and Douglas
1976).
In this communication we report some kinetic data on the
induction of alpha-galactosidase, the galactose uptake system
and the Leloir pathway enzymes, by D-galactose in unsteady
state continuous culture. We also report the decay kinetics of
all five activities in response to a gradual accumulation of
glucose in the culture vessel.
Materials and methods
Materials
Sigma (St. Louis, MO, USA) reagents and enzymes were used
throughout. Yeast Nitrogen Base was obtained from Difco
Lab., Detroit, MI, USA.
Yeast strain and culture conditions
The strain used was Saccharomyces carlsbergensis G-517
(Gillitand 1969), which correspond in the Spanish Collection
of Yeast Cultures (CECT, Departamento de Microbiologia,
Facultad de Biologla, Universidad de Salamanca) to the
number 1317, is haploid type a, auxotrophic for pantothenate, inositol and pyridoxine.
The composition of the basal medium was: 0.02 ~ yeast
extract, 0.067 % yeast nitrogen base. The carbon source was
as indicated below. Cultivations were carried out in a 1.51
chemostat with working volume of 0.971. The temperature
was maintained at 30 ~C, pH was kept at 5.0 _+ 0.2 and the
aeration rate was 3.01. min. the dilution rate (D) in all
experiments was 0.21 _+ 0.01 h-1.
116
When conducting an induction experiment, cells growing
in steady state under glucose limited conditions (medium
reservoir concentration S~ = 11 mM) were subject to a step
change in medium composition by switching the feed line to a
medium containing a mixture of glucose (Sr = 11 mM) and
galactose (S~ = 17 mM), when we return to the original state
by switching the feed line to a medium only containing
glucose (Sr = 11 raM), we can study the effect of removing the
inductor from the culture medium, maintaining constant the
glucose concentration (S~ = 11 mM).
When conducting a repression experiment, the step
change in medium composition was from galactose
(S~ = 11 mM) to a mixture of galactose (S~ = 11 mM) and
glucose (S~ = 83 raM) or from galactose (S~ = 111 raM), cells
growing in galactose-sufficient (say, N-limited) chemostat
culture, to a mixture of galactose (S~ = 111 raM) and glucose
(S~ = 83 mM). Sample collection was initiated immediately
after a step change.
II
,
j
~
,
,
E
,It
100
10
,,.4
I "-A-''-A
O
LU
E
/
0
J
10
tlJ
<
I
<
n
&
s
/foOo_oO~lp-o--o--o
O
FO
<
iJ
9
3.I
/---~]
A~A--A/
<
.A
<
O
O
t
o - - o ~ o / /--- o.o
Sample collection
s t e a d y statc
l/
t
t
II
,
,
t
/
,,,
3
o
F-
2
i
~
tll
,
,
l/
/
,I
E
E
!
B
?
_d
m
"2"'
"'%
I--~--~---~
0
x
0
, r
0
O
O
_1
7
1'
1
2
TIME
Cell-free extracts preparation
Washed cells were resuspended in 50 mM potassium phosphate buffer, pH 7.5, and disrupted in a Vibrogen Cell Mill
(Edmond Biihler Co.) by vibration for 10rain with glass
beads (0.5 mm in diameter). The supernatant obtained after
centrifugation at 10,000 • for 5rain was used as the enzyme source.
[IJ
~0
O
teady state
Samples were withdrawn from the culture effluent stream into
tubes that were submerged in an ice bath. Cells were separated
from the medium by centrifugation at 4,000 x g for 3 min,
twice washed and frozen at - 7 0 ~ until assayed. Supernatants were also freeze-stored for sugar analysis. For the
assay of galactose uptake, samples withdrawn from the
culture effluent were used immediately.
O
3
4
'~ "
(h)
Fig. 1 A, B. Induction kinetics of alpha-galactosidase and galactose
uptake system (A). Transient variation in glucose and galactose levels
during induction (13). Yeast cells growing in steady state at D = 0.21 h - 1
under glucose limited growth were subjected to a gradually increasing
level in D-galactose by changing the nutrient supply from glucose to
glucose plus galactose. In B, the dotted line represents the theoretical
galactose accumulation where it is not consumed by cells according to
the equation ~ = Sr ( 1 - e-Dg, where Sr is the galactose concentration
in the nutrient supply
Analytical methods
Galactokinase was assayed according to Blume and Bentler
(1975). Galactose-l-phosphate uridyltransferase was determined by the Maxwell method (Maxwell et al. 1962) except
that the reaction was started by adding the substrate
galactose-l-phosphate to give 1.5 mM concentration in the
reaction mixture. UDP-glucose 4-epimerase was assayed
according to Moreno et al. (1981). To 100 gl of 10 mM TrisHC1 buffer, pH 8.7, 5 - 25 gl of sample were added and the
reaction was started by the addition of 10 ktl of 5 mM UDPgalactose. The mixture was incubated at 28 ~C for 5 - 25 min.
The reaction was stopped with 25 gl of 0.1 N HC1. The tubes
were immersed in boiling water for 5 min to hydrolize both the
UDP-glucose formed and the unreacted UDP-galactose; then
25 lal of 0.1 N N a O H were added to neutralize the mixture and
the glucose liberated was determined using glucose oxidase.
Alpha-galactosidase was determined using p-nitrophenyl
alpha-D-galactoside as described previously (Moreno et al.
1979). One unit of enzyme activity was defined, for all these
enzymes, as the amount of enzyme that catalyzes the conversion of 1 gmol of substrate to product per min under the
described conditions.
Glucose and galactose were assayed enzymically by using
glucose oxidase and galactose oxidase respectively by methods described elsewhere. The cell density was determined
turbidometrically at 600 nm and converted to dry weight
concentration by use of a calibration curve. For the galactose
uptake assay, cells withdrawn from the chemostat (2 ml) were
centrifuged and washed. To the cell suspension was added
1 ml of basal medium containing 5 mM galactose and I gCi of
D-(1Jr
and incubated 4 rain at 30 ~C. Two milliliter of ice cold water added and the cells collected by filtration
on glass fiber disks (Whatman GF/C), washed twice with ice
cold water. Dried filters were counted in a toluene based
scintillation fluid (PPO 0.5~o; POPOP 0.03~) with a
Beckman liquid scintillation counter.
Results and discussion
Induction experiments
When Saccharomyces carlsbergensis G-517 was cultivated in a
continuous culture under glucose limited conditions, basal
levels of alpha-galactosidase and of galactose uptake could be
detected in cells (Fig. I A) but it was not possible to detect any
galactose pathway enzyme activity. Once the feed line was
changed from the glucose medium to a medium containing
glucose and galactose, a transient accumulation of galactose
was observed during the first hour after the shift, correspond-
117
ll~
,
,
100
~.,A / /
2
bA--ZX--~
9 i.I
/
A
J-'/
~ --u--hi
/
|
o
I00
t'
61
~,,:: \~\
\
m/
i ~
~u
>o "o " D M
I
10
l-o--o--o
>.
i-
[]
p.-
<
o
I-
o/
\
o
<
LU
iii
)N
Z
UJ
>
NI(~
Z
l,iJ
:teady
state
i;~
0
i
i
i
I
1
2
3
4
TIME
Iii
5
steady
stab
(h)
Fig. 2. Induction kinetics of the Leloir pathway enzymes. UDP-glucose
4-epimerase (A), galactose-l-phosphate uridyltransferase (I) and galactokinase ([:3). The transition conditions were as in Fig. 1. All three
activities were undetectable in an uninduced steady state culture
ing to the theoretical curve of material accumulation in the
chemostat ~ = Sr (1 - e -~t) indicating that galactose was not
significantly consumed by cells within this time (Fig. 1 B).
Within this period the galactose pathway enzymes appeared
in cells, showing a simultaneous exponential increase with
maximum accumulation rate constants (K~) ranging from
/~ = 3.2 h - 1, for galactokinase to K a = 4.4 h 1, for epimerase
(Fig. 1A; Fig. 2).
These rates decreased when the galactose began to be
actively consumed by the yeast cells. When the culture had
reached a new steady state, Ka for all the enzyme activities
studied declined to zero.
During the entire period of transition of the culture there
was no change in glucose concentration, which remained at
non repressive levels (below 0.1mM); thus, the galactose
induction of the aforementioned enzymes could proceed and
both sugars were simultaneously consumed once a certain
level of these enzymes was reached.
This induced yeast culture growing in steady state under
galactose limitation, was subject to a step change in medium
composition by switching the feed line from the galactoseglucose medium to a glucose medium, the level of galactose in
the culture decay drastically (Fig. 3B). During the entire
period of transition of the culture there was no change in the
glucose concentration, which remained at non repressive
levels. All activities represented in Fig. 3A, decreased exponentially but showed distinct decay rate constant (Kd).
Alpha-galactosidase and the three Leloir pathway enzymes
decayed with a Ka = - 0 . 2 1 h 1 = - D ,
showing that the
synthesis of these enzymes had ceased immediately and there
was a simple dilution of the enzyme present in the culture as
consequence of removing the inductor from the culture
medium. In contrast, the galactose uptake decreased with a
higher absolute value of Kd = - 0.42 h - 1.
These results could suggest a high turnover rate for the
galactose uptake system and a low turnover rate for the
alpha-galactosidase and the Leloir pathway enzymes, al-
E
uJ
(..9
<
o
5
lli
i
I
I
I
I
2
//i
t
t
t
I
I
2
E
w
-!
uJ
I
\
,_I
<
O
x
0
O
_I
o
1
2
3
TIME
4
5
7
(h)
Fig. 3 A, B. Decay kinetics of the Leloir pathway enzymes, galactose
uptake system and alpha-galactosidase (A). Transient variations in
glucose and galactose levels during the inductor removing (B). Yeast cells
growing in steady state at D = 0.21 h- 1 under galactose limitation were
subjected to a step change in medium composition by switching the feed
line from a medium with glucose and galactose to a glucose medium (non
repressive levels). Galactose uptake (9 is expressed as gmol-g
cell -l-rain -1. The enzymes galactokinase (D), transferase (11), epimerase (A) and alpha-galactosidase (A) are expresed as units, g cell- 1
though these values could also mean a different glucose
sensibility for the galactose uptake system and the other four
enzymes.
Repression experiments
When an induced yeast culture growing in steady state under
galactose limitation, was subject to a gradual increase in the
glucose level by switching the feed line from a galactose
medium to a galactose plus glucose medium, the level of
galactose in the culture rose, indicating that the consumption
rate of this sugar had drastically diminished as a response to
the glucose present. All activities represented in Fig. 4A,
decreased exponentially, but showed distinct decay rate
constant (K~). Alpha-galactosidase and galactokinase decayed with a K d = --0.21 h - l = - D showing that the synthesis of these enzymes had ceased immediately and there was
a simple dilution o f the enzyme present in the culture
(catabolite repression but no catabolite inactivation). In
contrast transferase, epimerase and the galactose uptake
decreased with a higher absolute value of Kd (--0.33; --0.33
and --0.54 h - 1 respectively).
When an induced yeast culture growing in steady state
under nitrogen limitation (galactose, S t = 111raM), was
118
10C
; ,' , ~ . . . ?
9
.
,
.
,
,
tt
,
100
Il
l
I
u
, tl
'o \~,\,\,
A~OA
\ ",,,,
o~
p-
\o "~ ~
>-
0
\
iEl
).
N
Z
u
a,]i\m
A
>I-
,
l,~
ra
I-
\,
"-v
L)
10
El
i/ n
II
'
,
a
I
,
,
~
I
~
,
I
a
,
,
I
o\
El
~
0~11
N
z
It-'----
.
El
~
IO
O ~O......----O--......4
9
20
o/
o/
B
/o--o,
20
/
/x~x
e
10
120
O
0
~E Io0
-I
lo~
~/
x,......~x~
//I
E
El
,,
N
I
I
I
I
I
I~1
I
I //
I
-
,,, 80
co
~ eo
Ii ",
0
I
1
9
x/
.......0- - o - o
3
TIME
4
5
Acknowledgements. We wish to thank Prof. S, Gasc6n for his critical
reading of the manuscript. This ,work was supported in part by a grant
from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica.
60
0
O
_1
/-o-o-4 40
-- 20
o
oa
0
1
2
3
TIME
subject to a gradual increase in the glucose level by switching the feed line from a galactose medium to a galactose
(S, = 111 mM) glucose (S, = 83 mM) medium, the level of
galactose rose, indicating that the consumption rate of this
sugar had drastically disminished as a response to the glucose
present (Fig. 5 B). The results shown in Fig. 5 A, were identical
to the obtained in the repression experiments carried out
under galactose limitation.
This could mean that transferase and epimerase were
subject to catabolite inactivation. The inactivation was not a
response of a hight turnover rates for these enzymes, how it
was shown in Fig. 3 A. The galactose uptake system was also
subject to catabolite inactivation although this value could
also suggest a hight turnover rate for this uptake system
(Fig. 3 A).
The fact that yeast cells growing on galactose suddenly
ceased to consume it when glucose entered into the chemostat
would be difficult to explain only as a function of the decay of
the galactose pathway enzymes; however, if we add to this the
inhibitory effect of glucose on the galactose uptake by
yeast cells (Fig. 6), both combined effects clearly explain this
fact.
~
9
O/
(h)
Fig. 4 A, B. Decay kinetics of the Leloir pathway enzymes, galactose
uptake system and alpha-galactosidase (A). Transient variations in
glucose and galactose levels during repression (B). Induced cells growing
in steady state at D = 0.218 h - 1 under galactose limitation were subjected
to a gradual increase in the glucose level. Galactose uptake (9 is
expressed as gmol.g cell-1, m i n - 1 The enzymes galactokinase (n),
transferase (11), epimerase (zX)and alpha-galactosidase (A) are expressed
as units 9 g cell- ~
80
--I
o/
o/
,i
.
.
2
E
x~
~ 40
t;
I00
X ****~.X.*-X
x~
4
5
I
I /,~
6
7
(h)
Fig. 5 A, B. Decay kinetics of the Leloir pathway enzymes, galactose
uptake system and alpha-galactosidase (A). Transient variations in
glucose and galactose levels during repression (B). Induced ceils growing
in steady state at D=0.21 h -1 under nitrogen limitation (galactose,
S, = 111 mM), were subjected to a gradual increase in the glucose level by
switching the feed line to a galactose (St = 111 raM) glucose (St = 83 raM)
medium. Galactose uptake (9 is expressed as v m o l ' g c e l l - 1 min-J.
The enzymes galactokinase (rn), transferase (i), epimerase (A) and alphagalactosidase (A) are expressed as units, g cell- 1
100
t
[
!
I
I
I
I
!
I
I
i
I
111
lo
I-n
\
\
50
0
El
0
I-
0
U
...I
\
o\
~
I
0
I
I
I
0
GLUCOSE
I
50
25
(ram)
Fig. 6. Galactose uptake inhibition by glucose. Cells growing in steady
state at D = 0.218 h-1 on galactose were collected, centrifuged and the
galactose uptake determined in the presence of L-glucose
119
References
Ashby RE, Harrison DEF (1980) Studies on the induction and turnover
of the citrate-oxidizing capacity in Klebsiella aerogenesusing chemostat culture. J Gen Microbiol 120:465-473
Blume KG, Bender E (1975) Galactokinase from human erytrocytes. In:
Wood WA (ed) Methods in enzymology, vol 42. Academic Press,
New York, pp 4 7 - 53
Dean ACR (1972) Influence of enviroment on the control of enzyme
synthesis. J Appl Chem Biotechnol 22:245-259
Douglas HC, Hawthorne DC (1964) Enzymatic expression and genetic
linkage of genes controlling galactose utilization in Saccharomyees.
Genetics 49: 837- 844
Douglas HC, Hawthorne DC (1966) Regulation of genes controlling
synthesis of the galactose pathway. Genetics 54: 911 - 916
Gilliland RB (1969) The raffinose fermentation of Saccharomyces
pastorianus and Saecharomyees bayanus. Antonio van Leeuwenhoek
J Microbiol Gerol 35:13 - 23
Kew OM, Douglas HC (1976) Genetic co-regulation of galactose and
melibiose utilization in Saccharomyces. J Bacteriol 125:33-41
Maxwell ES, Kurahashi K, Kalckar HN (1962) Enzymes of the Leloir
pathway. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 5. Academic Press, New York, pp 174-189
Moreno F, Herrero P, Parra F, Gasc6n S (1979) tnvertase and alphagalaetosidase synthesis by yeast. Cell Mol Biol 2 5 : 1 - 6
Moreno F, Rodicio R, Herrero P (]981) A new colorimetric assay for
UDP-glucose 4-epimerase activity. Cell Mol Biol 27:589-592
Ryu DY, Matetes RI (1968) Transient response of continuous cultures to
changes in temperature. Biotechnol Bioeng 10 : 385- 397
Tempest DW, Neijssel OM (1976) Microbial adaptation to low nutrient
enviroments. In : Dean ACR, Ellwood DC, Evans CGT, Melling J
(eds) Continuous culture 6: Applications and new fields, chap 22.
Ellis Horwood Ltd, Chichester
Received January 25, 1983/Accepted May 3, 1983