Download Determination of Optimal Glucose Concentrations

Process Biochemistry, Vol. 31, No. 5, pp. 499-506, 1996
Couvrieht 0 1996 Elsevier Science Ltd
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0032-9592(95)00093-j
:LSEVIER
Determination of Optimal Glucose Concentrations
for Glucoamylase Production from PlasmidHarboring and Chromosome-Integrated
Recombinant Yeasts using a WC2 Promoter
Hyung Joon Cha & Young Je Yoo*
Department of Chemical
Seoul 151-742, Korea
(Received
4 September
Engineering
and The Institute
1995; accepted 21 December
for Molecular
Biology and Genetics,
Seoul National
University,
1995)
During foreign protein production using a promoter such as SUC2, which is
controllable by the glucose concentration in the culture broth, it is important to
determine the optimal glucose concentration for maximum protein production.
The optimal glucose concentrations for maximal production of glucoamylase in
plasmid-harboring recombinant yeast MMY2SUCSTA culture and in
chromosome-integrated
recombinant yeast MMY2SUCSTA-I
culture were 0.13
and 0.09 g litre - ‘, respectively. Using glucose feeding to maintain these optimal
glucose concentrations, the extracellular glucoamylase productivities of fedbatch culture were increased (12-fold in MMy2SUCSTA
and 23-fold in
MMY2SUCSTA-I)
over simulation results from batch cultures.
The yeast Saccharomyces cerevisiae is widely
used as a recombinant
host that expresses
foreign genes and secretes proteins.’ Glucoamylase (EC 3.2.1.3) which is used to saccharify
starchy feed stocks in commercial processes for
glucose and ethanol productions,
is not produced by S. cerevisiae. With this in mind, the
STA gene (glucoamylase gene of Saccharomyces
diastaticus) was chosen as a glucoamylase gene
source for this research.2 The STA gene used
was cloned from a wild type S. diastaticus
DS101.3
When a heterologous
gene is cloned into
yeast, a promoter of the heterologous gene may
usefully be replaced by a yeast promoter. Regulated promoters which control gene expression
INTRODUCTION
One of the principal tools in new biotechnology
is the recombinant
DNA technique
which
allows us to manipulate
directly the genetic
material of individual cells. By inserting forgein
genetic information into fast-growing microorganisms, foreign gene products such as proteins
may be produced at higher rates and yields than
possible with other cellular systems. Recombinant microorganisms
may be divided into
plasmid-harboring
recombinant
cells
and
chromosome-integrated
recombinant cells.
*To whom correspondence
should be addressed.
499
500
Hyung Joon Cha, Young Je Yoo
by changes of medium composition, addition of
chemicals, or change of cultural conditions are
commonly used for foreign protein production.4’5 In this research, the SUC2 promoter
regulated by glucose in the culture broth was
used6,7 and has the following advantages.
Firstly, the problems of the host’s growth inhibition and plasmid
instability
due to the
expression of the plasmid gene can be reduced
since the SUC2 promoter is a regulatable promoter. Secondly, since the SUC2 promoter is
affected only by glucose, the addition of an
inducer is unnecessary in comparison with other
inducible promoters.
Also, the problem
of
medium substitution does not exist in comparison with other repressible promoters.
When using a promoter controllable by the
glucose concentration in the culture broth (such
as SUC2 promoter), the close control of glucose
concentration
is essential and it is very importthe
optimal
glucose
ant
to
determine
concentration for maximum protein production,
In this research, optimal glucose concentrations
for higher production of foreign glucoamylase
from plasmid-harboring
and chromosome-integrated recombinant
yeasts were determined
with special consideration
for expression and
secretion.
MATERIALS AND METHODS
Strains and culture medium
Saccharomyces cerevisiae MMY2 (a, ura3-52,
sta’, stal0) strain was used as the host, Recombinant
plasmids YEpSUCSTA
and YIpSUCSTA containing the glucoamylase coding
STA gene fused with the SUC2 promoter and
original STA signal sequence were transformed
into the host. The transformants
were named
MMY2SUCSTA
(plasmid-harboring
recombiand
MMY2SUCSTA-I
nant
yeast)’
(chromosome-integrated
recombinant
yeast).’
Yeast was grown at 30°C in semi-synthetic minimal medium containing 0.2% MgS04 - 7H20,
0.2% (NH4)$04,
0.1% KH2P04,
0.025%
CaC&. 2H20, 0.2% yeast extract, 0.3% bactopeptone, 1% glucose, 1% starch, and 50 mM
succinate. Starch was used as a carbon source
for glucoamylase biosynthesis.8
Measurements of cell mass and glucoamylase
activity
Estimations of cell mass as absorbance were
carried out with a spectrophotometer
(Kontron;
UVICON930)
at 600 nm. To measure glucoamylase activity, O-7 ml culture supernatant was
incubated in 0.1 ml 1 M sodium acetate buffer
(pH 5.0) and 0.2 ml 8% soluble starch (Junsei)
at 50°C for 30 min and boiled at 100°C for
5 min to inactivate glucoamylase. Glucose produced by the action of glucoamylase on soluble
starch was assayed using a glucose-diagnostic kit
(Sigma, No. 510). Units of glucoamylase activity
are defined as 1 ymol of glucose released per
1 min.
Cell fractionation
Cells were harvested,
washed with 10 mM
sodium azide, suspended in lysis buffer (0.1 M
sodium acetate (pH 5.0) 10 mM sodium azide,
1 mM EDTA and 0.1% (v/v) Triton X-100) and
mechanically lysed by vortexing with glass beads
(Sigma, 425-600 microns). After intermitent
vortexing and cooling on ice, the suspension
was harvested by centrifugation
at 12 000 rpm
at 4°C for 15 min, and the supernatant used for
enzyme assay.
Simulation of foreign glucoamylse production
in recombinant yeast culture
The parameters of model equations were estimated from experimental data of batch culture
using the least-squares
regression
method.
Determination
of optimal glucose concentration
and simulation of recombinant
yeast culture
were performed using MATLAB software (version 4.0, The MathWorks,
Inc.). Ordinary
differential equations of model equations were
solved using the 2nd and 3rd order RungeKutta method.
RESULTS AND DISCUSSION
Foreign glucoamylase production from plasmidharboring and chromosome-integrated
recombinant yeasts
In order to compare the characteristics of cultures of chromosome-integrated
recombinant
yeast (MMY2SUCSTA-I)
and plasmid-harboring recombinant
yeast (MMY2SUCSTA),
a
Glucoamylase production in recombinant yeasts
number of cultivations were carried out. Since
r,hanges of pH were very small and expression
of glucoamylase occurred normally in this pH
range of the culture, control of pH was not
liecessary in the fermentation
experiments (see
1.6
- 6.6
h
‘:
1.2 22 - 6.4
=
-7
ob
d- 6.2
i
X
S - 6.0 a
.4
6
0
- 5.8
??
0.;
.03
J 5.6
r
c
1
VI
.z
J
.02
.e
.g
Y
s
.a
75 0
70 z
e
65 ;
.O'
0
E
60 gj
a
55
50 +
Extra activi
d
E”
8 0.00
a
0
;;
2
”
4
6
8
10
Culture time (h)
(A)
10
1 1.6
, 6.6
7
1.2 t
2
8’1
s
gq
.a z3
4-
6.4
6.2
X
6.0 &
0
.4 s
2??
^
0+
.020
1
4
,015
i
0
I
5.6
0.0
100
3
95 k
E
12 .OlO
3
g
s
90 g
.$
jj ,005
a5
E"
g 0.000
0
s
B
5.8
2
4
6
8
d
80 +
10
Culture time (h)
(6)
Fig. 1. Batch profiles of plasmid-harboring
recombinant
yeast MMY2SUCSTA
culture (A) and chromosome-integratred recombinant yeast MMY2SUCSTA-I
culture (B).
501
Fig. 1). The patterns of cell growth and glucose
consumption
were similar in both types of
recombinant yeast culture. The profiles of pH
were similar to the pattern of glucose consumption in both recombinant yeasts. While the pH
in the culture broth decreased depending on the
cell growth, the decrease of pH was small
because succinate added to the medium played
a role in protecting the decrease of pH. In the
case of MMY2SUCSTA,
plasmid
stability
depended on glucoamylase expression. As the
expression of glucoamylase increased then plasmid stability decreased. In contrast, the genetic
stability of the chromosome-integrated
recombinant yeast MMY2SUCSTA-I
remained good.
Glucoamylase was produced late in the culture
in both recombinant
yeast cultures since the
SUC2 promoter was regulated by glucose concentration in the culture broth as previously
reported.‘.‘. I0 In the case of MMY2SUCSTA,
glucoamylase synthesis began below 7 g litre-’
glucose concentration
and secretion occurred
below 5 g litre ~ ’ (Fig. l(A)). Expression of
glucoamylase then increased rapidly but secretion increased slowly as glucose concentration
in the culture broth decreased. In the case of
MMY2SUCSTA-I,
glucoamylase
synthesis
began below 4 g litre- ’ glucose concentration
and secretion below 2 g litree’ (Fig. l(B)).
Expression
of glucoamylase
then
rapidly
increased
and
secretion
of glucoamylase
increased slowly as with MMY2SUCSTA.
The
behaviour of the two yeasts with respect to
glucose concentration
clearly differed. These
might be due to a gene dosage (the number of
copies of a given gene present in a cell) effect.’ ’
Since MMY2SUCSTA had a high plasmid copy
number (20-40) transcription regulation of the
SUC2 promoter by glucose in the recombinant
plasmid was not tight. In contrast, MMY2SUCSTA-I had three copy numbers of the desired
gene, expression of glucoamylase was tightly
regulated by glucose and the optimal glucose
concentration
for maximum expression of glucoamylase
was lower
from
that
in the
plasmid-harboring
recombinant yeast culture.
Determination of optimal glucose concentration
for glucoamylase production
The determination
of optimal glucose concentration for protein production is important from
a biotechnological
point of view. In this
research, we considered glucoamylase secretion
502
Hyung Joon Cha, Young Je Yoo
as well as glucoamylase expression in the determination of optimal glucose concentration.
The
optimal glucose concentration
for maximum
production of foreign glucoamylase, the specific
expression rate and the specific secretion rate of
each recombinant yeast were calculated as functions of glucose concentration
in the culture
broth. The mass balance for total glucoamylase
protein can be written asi2,13
d(PTX)
x
=F
dt
”
d(f’d)
dt
Fern
G
(kp + G + G2/kpi) ’
(3)
where
K= specific
secretion
rate
(h-l),
PMX=extracellular
glucoamylase activity (units
ml-‘).
The specific secretion rate K was related to
cell growth condition
(data not shown).‘3,‘4
(1)
where Fp=specific expression rate (units mg ~ ’
cell h-l),
PTX=total
glucoamylase
activity
(units mll’),
t=operation
time (h), X=cell
mass (g litre-‘).
The SUC2 promoter used in this work, is
regulated by glucose concentration
in the culture broth. When the glucose concentration
is
high, the expression
of SUC2 promoter
is
repressed. However, when glucose is decreased
to a threshold concentration
then expression is
not repressed. Thus, to consider glucose repression on the expression of SK2
promoter, a
glucose repression term (G’/kpi) is introduced
into the specific expression rate Fp’2,13
Fp=
=K(P-&-PM@,
0.0
(2)
where FP,,,=maximum specific expression rate
(units mgg’ cell hh’), G=glucose
concentration
in the
culture
broth
(g litre-‘),
kp=constant
of specific expression
rate (g
litre - ’), kpi = repression
constant
of specific
expression rate (g litre-‘).
The specific glucoamylase expression rate FP
of MMY2SUCSTA is indicated in Fig. 2(A) and
its maximum value calculated to be O-054 units
mg-’ cell h- ’. Maximum expression of plasmid-harboring
yeast occurs
at a glucose
concentration
of 0.17 g litre-’ in the culture
broth.
Similarly,
the specific glucoamylase
expression
rate
of chromosome-integrated
recombinant
yeast MMY2SUCSTA-I
is indicated in Fig. 2(B) and its maximum value
calculated as O-026 units mg-’ cell hh’. In this
case, maximum expression occurs at a glucose
concentration
of 0.13 g litre-‘.
The mass
balance for secreted glucoamylase13 is
*-
.025
I
.5
1.0
1.5
2.0
2.5
3.0
, .4
Fig. 2. Optimal glucose concentrations
for maximum
specific expression rate and specific secretion rate in plasmid-harboring
recombinant
yeast MMY2SUCSTA
(A)
and chromosome-integrated
recombinant
yeast MMY2SUCSTA-I
(B). SER: specific expression
rate; SSR:
specific secretion rate; SOR: specific optimal rate.
503
Glucoamylase production in recombinant yeasts
Since specific growth rate was a function of
glucose concentration,
the specific secretion
rate may also be described as a function of
glucose concentration.‘”
described below and the optimization was performed in fed-batch culture as shown in Fig. 3.
PI =mF
K=
GIG
a+pG
’
{Productivity} =mp
{‘$V,!)”
1,
(4)
where K,=maximum
specific secretion
rate
I h-r),
m=constant of specific secretion rate (g
litreel), /?=constant of specific scretion rate.
The dependence of specific secretion rates on
glucose concentration
in both recombinant
yeasts is indicated in Fig. 2. In both recombihant yeasts, the specific secretion rate increased
with increase in glucose and finally reached
s,aturation.
The optimal glucose concentration
might
exist between the specific expression rate and
rhe specific secretion rate. The optimal glucose
concentration
for maximum extracellular gluco<imylase activity was calculated through two
equations of specific expression rate (eqn 2)
tend specific secretion rate (eqn 4). In the case
,)f MMY2SUCSTA,
the optimal glucose con::entration for specific expression rate was 0.17 g
litre-’ (Fig. 2(A)) but, the optimal glucose conzentration for both specific expression rate and
specific secretion rate was 0.76 g litree’. In the
zase of MMY2SUCSTA-I,
the optimal glucose
concentration
for specific expression rate was
9.13 g litreel and the optimal glucose concentration for both specific expression rate and
specific secretion rate was O-25 g litre-’ (Fig.
2(B)). In this case, the difference between two
optimal glucose concentrations was smaller than
that of MMY2SUCSTA
since the slope of the
specific secretion rate of MMY2SUCSTA-I was
larger in the region of low glucose concentration. This calculation was performed using only
eqn (2) for specific expression rate and eqn (4)
for specific secretion rate. Values calculated in
this manner can be used in simulation or real
experiment for fed-batch culture. However, in
the real culture, many other factors that affect
final extracellular product concentration
exist
including, expression level, secretion capacity,
genetic stability, final operation
time, final
vessel volume, and culture type. Therefore, in
order to determine real optimal glucose concentration, glucoamylase
productivity
(unit h-‘)
was used using a performance
index (PI) as
(5)
where V,=final vessel volume (litre).
A fixed final vessel was used as a constraint
condition, the initial vessel volume being set as
0.5 litre and final vessel volume set as 2 litre. In
the case of MMY2SUCSTA,
the optimal
glucose concentration
for the desired extracellular glucoamylase
productivity
was 0.16 g
1itre-l and its value was close to the optimal
Glucose concentration
(g litre-‘)
(4
I
I
I
Glucose concentration
I
I
(g litre-‘)
W
of optimal glucose concentrations
Fig. 3. Determination
for maximum
glucoamylase
productivity
in fed-batch
culture of plasmid-harboring
recombinant
yeast MMY2SUCSTA (A) and chromosome-integrated
recombinant
yeast MMY2SUCSTA-I
(B). Initial and final conditions;
X+=0.1 g litre-‘,
X- =O*Og litre-‘,
X=0*1 g litre-‘,
G=S g litre-‘, Gr=20 g litre- , V,,--05 lure, and l/f=2 litre. X + : plasmid-harboring
cells, X - : plasmid-free cells,
X: chromosome-integrated
cells, P,W/t:
extracellular
glucoamylase productivity and P,XV/t: total glucoamylase
productivity.
504
Hyung Joon Cha, Young Je Yoo
value for specific expression rate (O-17 g litre-‘)
(Fig. 3(A)), while that for total glucoamylase
productivity was 0.13 g litre-‘. In the case of
MMY2SUCSTA-I,
the optimal glucose concentrations
for
the
desired
extracellular
glucoamylase productivity and for total glucoamylase productivity were equal and their value
was 0.09 g litre-’ (Fig. 3(B)). These optimal
glucose concentrations
(O-16 and 0.09 g litre-‘)
were closer to the glucose concentrations
for
maximum specific expression rate (O-17 and
0.13 g litre-‘) than those for maximum specific
expression rate and specific secretion rate (0.76
and 0.25 g litre-‘). The reason for this may be
that expression was rate-limiting
for overall
foreign glucoamylase production
compared to
secretion in both recombinant yeasts.
Simulation of foreign glucoamylase production
using optimal glucose concentration
Simulations of foreign glucoamylase production
in fed-batch cultures of plasmid-harboring
and
chromosome-integrated
recombinant
yeasts
were performed using optimal glucose concentrations for 30 h operation times as shown in
Fig. 4. Glucose feeding rate was calculated
using growth conditions to maintain the optimal
glucose concentration
in the culture broth as
shown below.‘”
!g-G&
1
P
-++m
Y
f=
Cultu~
time(b)
(4
xv’
(6)
where Gf=glucose concentration
in the feed (g
litre-‘),
GOPT=optimal
glucose concentration
for glucoamylase production (g litre-‘), (O-16 g
litre-’ for MMY2SUCSTA
and 0.09 g litre-’
MMY2SUCSTA-I),
m =maintenance
coeffivolume
(litre),
YXG=yield
cient, V=vessel
coefficient, p=specific growth rate (h-l). This
equation includes state variables (cell mass and
vessel volume) and feed rate was calculated
using these state variables. As shown in Fig.
4(A), cell mass reached a maximum at 20 h in
the MMY2SUCSTA
culture. A larger fraction
of total cells was occupied by plasmid-free cells
in the final culture period and the internal
glucoamylase expression was therefore maximal
at 25 h and then decreased. The final extracellular glucoamylase
activity was O-215 units
ml-’ and the productivity was 13.0 units h-‘.
5
10
15
20
Cnlbtime(h)
2s
30
1
(61
Fig. 4. Simulation results for fed-batch culture of plasmid-harboring
recombinant
yeast MMY2SUCSTA
(A)
and chromosome-integrated
recombinant
yeast MMY2SUCSTA-I
(B). Initial conditions;
X+ =O.l g litre-‘,
X-=O.Og
litre-‘,
X=0*1 g litre-‘,
G=S g litre-‘,
Gf=20 g litreP’, V,=O5 lit re, and t,=30 h. X+: plasmidharboring cells, X - : plasmid-free cells, XT: total cells, X:
chromosome-integrated
cells, G: glucose concentration,
P,X: intracellular
glucoamylase
activity, PMX: extracellular glucoamylase activity and PTX: total glucoamylase
activity.
505
Glucoamylase production in recombinant yeasts
Table 1. Summary of simulation results for glucoamylase
chromosome-integrated
recombinant yeasts
production
in fed-batch
culture
Chromosome-integrated
MMy2SlJCSTA-I
Plasmid-harboring yeast
MMYZSUCSTA
Batch culture
Fed-batch culture
(30 h culture time)
Fed-batch culture
(2 litre final vessel volume)
using plasmid-harboring
and
yeast
Productivity
(units h ‘)
Relative
increase
Productivity
(units h _ ‘)
Relative
increase
1.2
13.0
1.0
10.8
0.8
1I.9
1.0
14.9
14.0
11.7
18.4
23.0
This pattern became more marked with an
increase of plasmid-free
cell fraction in the
initial culture. In contrast, cell mass steadily
increased in the case of MMY2SUCSTA-I
(Fig.
4(B)). The glucoamylase activity continued to
increase and the final extracellular glucoamylase
activity was 0.274 units ml-‘; while the extracellular
glucoamylase
productivity
reached
11.9 units hh ‘. When using final operation time
as a constraint
condition,
the extracellular
glucoamylase
productivities
of both recombinant yeasts were similar. This resulted from a
final small vessel volume of the chromosomeintegrated yeast culture. In the case of using
final vessel volume as a constraint condition,
the final extracellular glucoamylase productivity
of the chromosome-integrated
yeast culture was
31.4% larger (18.4 units hh’) than that of plasmid-harboring
yeast culture (14.0 units hh’).
This resulted from higher genetic stability and
higher secretion efficiency (68.1% compared to
48.7% of MMY2SUCSTA).
These simulation results of foreign glucoamylase production using fed-batch culture of the
plasmid-harboring
recombinant
yeast MMY2SUCSTA
and the chromosome-integrated
recombinant yeast MMY2SUCSTA-I
are summarized in Table 1. By using glucose feeding to
maintain optimal glucose concentrations
(0.16 g
litre - ’ in MMY2SUCSTA and 0.09 g litre - ’ in
MMY2SUCSTA-I),
the final glucoamylase productivities of fed-batch cultures were increased
(11*7-fold in MMY2SUCSTA
and 23-O-fold in
MMY2SUCSTA-I)
compared to those of the
batch culture when using final vessel volume as
a constraint condition.
ACKNOWLEDGEMENT
This research was supported by the Genetic
Engineering Research Fund from the Ministry
of Education, Korea.
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