Biotechnology Letters 25: 251255, 2003.

2003 Kluwer Academic Publishers. Printed in the Netherlands.

251
Modeling of mini-proinsulin production in Pichia pastoris using the AOX
promoter
Jos e M. Pas

, Laura Varas, Jorge Vald es, Cecilia Cabello, Lester Rodrguez & Manuel Mansur
Center for Genetic Engineering and Biotechnology (CIGB), Biotechnology Development Unit, Fermentation De-
velopment Department, P.O. Box 6162, Havana 10600, Havana City, Cuba

Author for correspondence (Fax: (53-7) 271-8070; E-mail: jose.pais@cigb.edu.cu)

Received 10 September 2002; Revisions requested 9 October 2002; Revisions received 28 November 2002; Accepted 29 November 2002
Key words: fed-batch culture, fermentation, modeling, Pichia pastoris, recombinant human insulin
Abstract
An unstructured model based on mass balance equations for a recombinant methylotrophic yeast Pichia pastoris
Mut
S
(Methanol Utilization Slow) strain expressing the mini-proinsulin (MPI), was successfully established in
quasi-steady state fed-batch fermentations with varying total quantity of biomass in a 7 l fermenter. The model
describes the relationships between the total biomass and induction time, both in the batch and fed-batch phases.
In addition, good correlations were obtained when the total quantity of MPI was correlated with the total biomass,
demonstrating that the product of interest is associated with growth in the methanol phase. The parameters of the
fermentation model obtained are similar to those reported by other researchers.
Introduction
The recombinant human insulin has been produced
at large-scale using Escherichia coli (Goeddel et al.
1979) and Saccharomyces cerevisiae (Thim et al.
1986a,b, Markussen et al. 1986).
In 1986, Thim et al. (1986a,b) patented construc-
tions with a short basic spacer between the main
chains (A and B
130
) of human insulin. These B
130
-
XY-A constructions were named mini-proinsulin
(MPI), where XY is any combination of the basic
amino acids, lysine and/or arginine, which allowed
the in vitro processing of MPI to mature insulin by
a set of enzymatic treatments, separately or together,
of the precursor with trypsin and carboxypeptidase B
(CPB). Later, in order to avoid the use of the very
expensive CPB, a new construction, B
129
-Ala-Ala-
Lys-A, was employed (Markussen et al. 1986). The
alternative MPI construction has a fragment B
129
-
Ala corresponding to the pork insulin B chain, and
allows the in vitro processing with trypsin alone, to ob-
tain the mature insulin molecule in two steps: removal
of Ala-Ala-Lys spacer and addition of threonine in B
30
via a transpeptidation reaction.
Some authors have reviewed in detail the advan-
tages of Pichia expression system (Brierley et al.
1990, Cregg et al. 2000). High-level expression, the
possibility to achieve high-cell density cultivation us-
ing a non-expensive chemically dened media, strong
and tightly regulated promoters, as well as limited
or absent hyperglycosilation and its Generally Recog-
nized As Safe (GRAS) status, have all been argued as
the major advantages of P. pastoris (Kjeldsen et al.
1999, Cregg et al. 2000). Furthermore, other re-
searchers have investigated the expression of insulin
in P. pastoris employing the more recently devel-
oped MPI construction B
129
-Ala-Ala-Lys-A (Kjeld-
sen et al. 1999, Wang et al. 2001).
On the other hand, we have successfully employed,
at the rst time, the initial variant of MPI with a basic
spacer (B
130
-Lys-Arg-A), for its expression in the
P. pastoris system. The multimerization of expression
cassettes has been employed as the main strategy to
increase the gene dose and consequently the secretion
level in this case.
The purpose of this research is to establish an un-
structured model for a P. pastoris GS115 Mut
S
strain
expressing the MPI in the culture supernatant and to
252
know whether the MPI secretion is associated or not
to population growth during the induction phase. Once
established, this model could serve to perform dif-
ferent process control strategies, varying the model
parameters, like the feed ow of fresh medium, which
will inuence the expression level of MPI along the
fermentation.
Materials and methods
Experimental setup
Fermentations were performed with a 7 l fermenter
interfaced with the computer-based software, FER-
MACS, for data acquisition and supervisory control
(CIGB, Havana, Cuba). A methanol-feed pump P-1
(Pharmacia, Uppsala, Sweden) for stepwise methanol
supply in the induction phase was employed.
Strain and inoculums preparation
To generate recombinant P. pastoris clones secreting
the mini-proinsulin, the host strain GS115 was trans-
formed by electroporation with the BglII-linearized
pAO815 vector containing four consecutive copies
of expression cassette under pre-pro -factor secre-
tion signal. The MPI has been expressed in the cul-
ture supernatant and its molecular mass was well
characterized by low-energy electro-spray ionization
mass spectra (data not shown). Each fermentation
was inoculated with 400 ml growth medium (per
liter: 40 g glycerol, 10 g casein hydrolysate, 9 g
(NH
4
)
2
SO
4
, 4.3 g KH
2
PO
4
, 3.2 g MgSO
4
7H
2
O,
0.22 g CaCl
2
2H
2
O, and 5 ml of PTM
1
solution).
The inocula were agitated at 150 rpm, through 48 h
at 28

C in 2 l bafed ask. The PTM
1
solution is the
same described in Pichia Fermentation Guidelines of
Invitrogen Co. (San Diego, CA).
Fermentation
The batch phase was performed with 4 l de-
ned medium (per liter: 50 g glycerol, 20 g
(NH
4
)
2
SO
4
, 12 g KH
2
PO
4
, 4.7 g MgSO
4
7H
2
O,
0.36 g CaCl
2
2H
2
O, and 5 ml of PTM
1
) similar to
medium employed by DAnjou & Daugulis (2000).
Prior the inoculation, the pH was adjusted to 5.1 with
30% (v/v) ammonium hydroxide. Batch phase condi-
tions were temperature 30 0.2

C, pH 5.1 0.1,
agitation was increased stepwise from 500 to 900 rpm
in order to keep constant the pO
2
value over 20% with
a constant aeration rate of 6 l air min
1
. The end of
batch phase was indicated by a sharp increase in the
pH and pO
2
caused by the exhaustion of glycerol as
a carbon source. Previous experiments performed to
test the stability of human insulin in the supernatant
of non-transformed strains showed that the molecule
was more stable at pH values below 5, maybe due to
the lower proteolytic degradation by the host extra-
cellular proteases (data not shown). For this reason,
when the culture restored the original pH value after
induction, a new pH value of 4.6 was xed.
Mini-proinsulin (MPI) determination
MPI concentration was determined by reverse-phase
HPLC, employing a 5.4 150 mm C8 208TP5415
column from Vydac (Hesperia, CA) kept constant at
45

C. A gradient from 30 to 45% (v/v) of buffer
B in 30 min at a ow of 0.62 ml min
1
was em-
ployed. Buffers A and B are 0.1% (v/v) triuoroacetic
acid (TFA), and 90% (v/v) acetonitrile plus 0.05%
(v/v) TFA, respectively. Previously, a linear calibra-
tion curve with recombinant human insulin (I-2767,
Sigma) as internal standard was constructed.
Modeling of the fed-batch growth
The fed-batch system can be characterized by a set
of mass balance equations concerning the global,
cell, substrate and recombinant product balances. The
equations are:
d(
C
V)
dt
=
C

dV
dt
+V
d
C
dt
=
=
F
F +
WA
Q
WA

EA
Q
EA
, (1)
d(X V )
dt
=
FB
X V, (2)
d(M V)
dt
= M
F
F

FB
X V
Y
XM
, (3)
d(P V)
dt
=
dC
MPI
dt
=

FB
X V
Y
xp
, (4)
where t : time (h);
F
,
C
: fresh media and culture
densities (g ml
1
);
EA
,
WA
: densities of exhausted
and wet air (g ml
1
); F: ow of feeding fresh medium
(l h
1
); Q
EA
, Q
WA
: volumetric ow of exhausted and
wet air (ml h
1
); X: cell concentration (g dry wt l
1
);
V: culture volume (l);
FB
specic growth rate at fed-
batch phase (h
1
); M, M
F
: methanol concentration in
253
Table 1. Correlations and coefcients of determination (R-squared values) obtained from the fermentation
data.
Correlation CD
a
Correlation CD
a
V = 4.0 0.01279 t R
2
= 0.7998 X V = 148.29 e
0.0926t
i
R
2
= 0.9874
1
Y
XM
= 0.9801 +
0.0261

R
2
= 0.8299 X V = 149.22 e
0.0114t
i
R
2
= 0.9471
V
S
V
= 1.0121 0.001 X
W
R
2
= 0.8126 C
MPI
= 0.9132 X V 138.77 R
2
= 0.8650
X = 0.3388 X
W
R
2
= 0.9205 F = 0.048 X V
a
Coefcient of determinations.
the culture and feeding medium (g l
1
); P: concen-
tration of MPI (mg MPI l
1
); C
MPI
: total quantity
of MPI (mg); Y
XM
biomass/methanol yield (g dry
wt g
1
); Y
XP
: biomass/MPI yield (g dry wt mg
1
).
The inuence of the last two elements of the right
side of Equation (1) can be neglected in well-designed
bioreactors with an adequate heat transfer area in the
top condenser that greatly reduces the water loss from
fermentation media. Nevertheless, in the case under
study, this difference is approximately equal to the
water loss in the culture. Finally, assuming almost con-
stant the culture density throughout the fermentation,
the expression (1) can be rewritten as:

C

dV
dt
=
F
F
H
2
O
Q
Lost
, (5)
where:
H
2
0
: density of water lost during fermentation
(g ml
1
); Q
Lost
: average volumetric owof water loss
during fermentation (l h
1
).
From Equation (3) must be demonstrated that
methanol-feeding rate is equal to:
F =

FB
X V
M
F
Y
XM
. (6)
Equation (6) can be rearranged and equalized ac-
cording to Pirts equation (Pirt 1975) or the relation
between 1/Y
XM
and 1/. Thus, the new equation is:
1
Y
XM
=

FB
X V
M
F
F
=
1
Y
T
XM
+
m
M

, (7)
where: m
M
: maintenance coefcient (g g dry wt
1
h
1
); Y
T
XM
: true Biomass/methanol yield (g dry
wt g
1
).
Differential Equations (2), (4) and (5) must be
rewritten in an integral form, and the following equa-
tions are obtained:
C
MPI
=

1
Y
XP

X V

1
Y
XP

(X V)
0
, (8)
X V = (X V)
o
e

FB
(t t
0
)
= (X V)
0
e

FB
t
i
,
(9)
V = V
0

H
2
O

0
Q
Lost
dt

0
F dt

, (10)
where (X V)
0
: total biomass obtained in batch phase
(g dry wt); t
0
: duration of batch phase (h); t
i
: induction
time (h).
Finally, the relation between the culture and super-
natant volumes is:
V
S
V
=

C
X
W

S
, (11)
where V s: supernatant volume (l);
s
: supernatant
density (g ml
1
); X
W
: cell density (g wet wt. l
1
).
Results and discussion
The cell mass at the end of the batch phase was ap-
prox. 38 g dry wt l
1
and it was reached after 25 h.
After the initial glycerol was consumed, a methanol
concentration of 1% (v/v) was suddenly established in
the culture medium. After adaptation to the new C-
source, the pH controller was positioned in a new set
point value (4.6 0.1). At this moment, a continuous
methanol feeding (M
F
= 775.8 g l
1
, supplemented
with 12 ml PTM
1
l
1
), with periodic increment in the
ow was initiated. Every 12 h, a new feed ow rate
was xed after calculating an average value, substitut-
ing in Equation (6) the values of 0.012-h
1
and 0.4-g
dry wt g
1
for the desired specic growth rate and
biomass/methanol yield, respectively.
Applying the above explained mass balance equa-
tions and the experimental fermentation data, a set of
254
Fig. 1. Experimental data of four 7 l fermentations (B.E. Marubishi, Tokyo, Japan) performed under the same conditions as described in
Materials and methods (the symbols , , , correspond to fermentation from 1 to 4) and moldel prediction (solid lines) are obtained from
the application of the unstructured model. The dotted lines represent the 95%-condence intervals in each model. (A) Variation of culture
volume in four fermentations, (B) relation between de l/Y
XM
and 1/
FB
in the induction phase, for fermentations 1 and 2, (C) experimental
relations between dry and wet well wt. concentrations () and between the supernatant and culture volumes ratio (V s/V) and wet cell weight
concentration () for the four fermentations performed, (D) kinetics for total biomass at different induction times for fermentations 1 and 2,
(E) kinetics for total MPI in the supernatant at different induction times (for fermentation 1 only), (F) a typical pH prole and constant ow
steps xed for all fermentations during the fed-batch phase of each fermentation process (bold solid lines).
correlations was obtained, which correspond to the
searched unstructured model (Table 1). In Figure 1
can be observed the experimental data and tted the
achieved correlations, observing a good correspon-
dence between them. Other authors have employed
this kind of models for simulation of the fermenta-
tion process at large-scales and to optimize the process
(Zhang et al. 2000a, DAnjou & Daugulis 2001).
From the determined unstructured model, some
fermentation parameters could be obtained, which are
presented in Table 2.
The specic growth rate and biomass/methanol
yield used to calculate the feed ow in Equation (6)
are in good correspondence with the same parameters
shown in Table 2 obtained from the dry cell concen-
tration time chart (Figure 1D) and the Pirts relation
(Figure 1B). The specic growth rate is typical for
the Mut
S
phenotypes as reported by Brierley et al.
(1990). A value of biomass/glycerol yield higher than
the value reported by Roels (1983) for other methy-
lotrophic species was obtained, maybe due to the
incomplete consumption of glycerol in the inocula. On
the other hand, the biomass/methanol yield and main-
255
Table 2. Fermentation parameters obtained from the achieved
unstructured model.
Fermentation parameter Value
a
Specic growth rate
Batch phase
B
= 0.093 0.013 h
1
Fed-batch phase
FB
= 0.011 0.002 h
1
Biomass substrate yields
On glycerol Y
XG
= 0.619 0.08 g dry wt g
1
On methanol Y
XM
= 0.306 0.06 g dry wt g
1
Y
T
XM
= 1.02 0.56 g dry wt g
1
Biomass product yield Y
XP
= 1.09 0.24 g dry wt mg
1
Maintenance coefcient m
M
= 0.026 0.012 g dry wt
1
h
1
Densities
Culture
C
= 1.01 0.06 g ml
1
Supernatant
S
= 1.00 0.01 g ml
1
a
Average values standard deviation.
tenance coefcient were similar to those obtained by
Zhang et al. (2000b) for a Mut
+
(Methanol Utilization
Plus) GS115 strain expressing intracellularly a heavy-
chain fragment C of botulinum neurotoxin, serotype
A. The expression levels obtained were signicantly
lower than the values obtained by Wang et al. (2001),
reaching up-to 1.5 g MPI l
1
in the culture super-
natant, but higher than results reported by Kjeldsen
et al. (1999).
Conclusions
An unstructured model was developed for a Mut
S
P. pastoris strain expressing the MPI. The model de-
scribes the relationships between the total biomass
grown under a specic growth rate, both in batch and
in fed-batch phases. The average values of specic
growth rates of 0.093 and 0.011 h
1
for the batch
and fed-batch phases respectively, was obtained. The
calculated biomass/substrate yields showed that there
is a little overestimate of this variable in the batch
phase, but in the fed-batch phase, the results are in
clear correspondence with values reported by other
authors. In the analyzed case the water loss during
the fermentation is signicant and for this reason the
variation of the volume along the fermentation intro-
duces changes in the traditional presentation of mass
balance equations. However, robustness of unstruc-
tured model based on mass balance equations permits
the accurate representation of the experimental data
values. Studies are in progress to test this model for
other secreted MPI constructions in P. pastoris.
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