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Mixed-feed exponential feeding for fed-batch culture of recombinant

methylotrophic yeast

Article  in  Biotechnology Letters · March 2000

DOI: 10.1023/A:1005612415737

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 Biotechnology Letters 22: 341–346, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
341

Mixed-feed exponential feeding for fed-batch culture of recombinant

methylotrophic yeast

Marc C. d’Anjou & Andrew J. Daugulis∗

Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada K7L 3N6
∗ Author for correspondence (Fax: +6135456637; E-mail: daugulis@chee.queensu.ca)

Received 24 December 1999; Accepted 10 January 2000

Key words: fed-batch culture, glycerol, methanol, mixed substrates, Pichia pastoris

Abstract
A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate
fed-batch fermentation of MutS recombinant Pichia pastoris was developed and was used to design an exponential
feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate
feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth
rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.

Introduction hol oxidase promoter. This promoter is regulated by

Fed-batch fermentation, where substrates, nutrients,
and/or inducers are fed into the fermenter either in-
termittently or continuously, is the prevalent mode
of operation in recombinant bioprocesses as it mini-
mizes such deleterious effects as substrate inhibition
and catabolite repression while allowing the engineer
to control the specific growth rate of the cells and
achieve high cell densities and product concentrations.
During fed-batch culture, it is essential to design a
feeding strategy to prevent overfeeding or underfeed-
ing of substrate (or inducer) to the culture. In microbial
fermentation, feeding a single limiting substrate at
an appropriate exponentially increasing rate results
in a constant substrate concentration and a constant
specific growth rate. This strategy has been used to
successfully boost the productivity of both conven-
tional and recombinant E. coli systems (Yee & Blanch
1992, Paalme et al. 1990).
Methylotrophic yeast, such as Pichia pastoris and
Hansenula polymorpha, are finding increasing use
in recombinant gene expression due to their many
favourable features as hosts (see reviews by Vedvick
1991, Cregg & Higgins 1995). The expression of
heterologous proteins in the Pichia system is driven
by the action of the strong, tightly regulated alco-
a combination of repression/derepression by glycerol
and induction by methanol. This combined regulation
mechanism allows Pichia pastoris to simultaneously
metabolize both methanol and glycerol. Mixed glyc-
erol/methanol feeding has been shown to improve the
productivity of recombinant Pichia fermentation sys-
tems, especially those exhibiting a MutS (Methanol
Utilization Slow) phenotype (Brierley et al. 1989,
Loewen et al. 1997). However, fed-batch culture of
recombinant Pichia cultures is complicated due to
methanol’s role as both an inhibitory substrate and an
inducer for recombinant gene expression. Methanol
must be present in quantities sufficient to fully in-
duce heterologous gene expression without inhibiting
growth or product formation. The design of an opti-
mal mixed feed fed-batch fermentation protocol for
culturing recombinant Pichia requires some means
of maintaining a constant residual methanol concen-
tration while controlling the specific growth rate to
optimize product formation. This can be accomplished
with an effective mixed substrate exponential feeding
strategy. In this study, a MutS P. pastoris strain that
has been genetically modified to produce and secrete
sea raven anti-freeze protein (SR-AFP) is used as a
model system to demonstrate the implementation of
this strategy.
342

Materials and methods HPLC using a Sugar Pak I (Waters Millipore) column
and a 50 mg EDTA l−1 mobile phase at 0.5 ml min−1 .
The Pichia pastoris strain GS115 (his4) was trans-
formed with plasmid pPIC9 containing the gene en-
coding for SR-AFP according to the methodology Results and discussion
presented in Loewen et al. (1997). The pPIC9 plasmid
contains the HIS4 gene for selection of his+ clones, Earlier work done examining this model system
and integrates by homologous recombination into the (d’Anjou & Daugulis 1997), demonstrated that
AOX1 gene site when linearized with BglII. This methanol contributes significantly to cell growth, even
transformation produces an AOX1-deficient clone in MutS strains of P. pastoris, and must be considered
characterized by a MutS (Methanol utilization slow) as a second substrate in the fed-batch cell and mass
his+ phenotype which secretes SR-AFP into the fer- balances for this system. Therefore, the following
mentation broth. mass balances need to be considered for this fed-batch
Fed-batch fermentation was carried out in a 10 l system:
Chemap fermenter under the following conditions: d(XV )
= µXV − F X, (1)
temperature, 30 ◦ C; impeller speed, 750 rpm; aera- dt
tion, 15 l min−1 ; pH 5.5 measured by an Ingold pH d(GV )
probe and maintained by the addition of 5 M KOH. = F (G0 − G) − qG XV , (2)
dt
Dissolved oxygen was measured using an Ingold ster-
ilizable polarographic electrode and was maintained at d(MV )
= F (M0 − M) − qM XV , (3)
greater than 30% of saturation by supplementing with dt
oxygen gas if necessary. The methanol/glycerol mixed d(V )
feed was added via a Gemini PC-1 linear peristaltic = F, (4)
dt
pump (Alaris Medical, San Diego, CA).
where X is the cell density in gdry wt l−1 ; V is the
The media used in this study were:
volume in l; F is the substrate feed rate in l h−1 ;
BMGY: yeast extract, 10 g l−1 ; meat peptone, 20 g l−1 ;
G is the glycerol concentration in g l−1 ; G0 is the
100 mM potassium phosphate buffer pH 6.0; yeast ni-
feed glycerol concentration in g l−1 ; qG is the spe-
trogen base without amino acids, 13.4 g l−1 ; biotin,
cific glycerol consumption rate in gG /gX · h; M is
400 µg l−1 ; glycerol, 10 ml l−1 .
the methanol concentration in g l−1 ; M0 is the feed
MGY: yeast nitrogen base without amino acids,
methanol concentration in g l−1 ; qM is the specific
13.4 g l−1 ; biotin, 400 µg l−1 ; glycerol 10 ml l−1 .
methanol consumption rate in gM /gX · h.
Fermentation medium: glycerol 50 g l−1 ; (NH4 )2 SO4 ,
It is possible to represent the cell density as a func-
20 g l−1 ; KH2 PO4 , 12 g l−1 ; MgSO4 · 7H2 O, tion of the total mass of substrate added and the cell
4.7 g l−1 ; CaCl2 · 2H2 O, 0.36 g l−1 ; plus trace yields. During mixed substrate growth, the formation
elements: CaSO4 · 5H2 O, 0.2 µM; KI, 1.25 µM; of biomass from glycerol and methanol is additive
MnSO4 · 4H2 O, 4.5 µM; Na2 MoO4 · 2H2 O, 2 µM; (Egli et al. 1986). Therefore:
H3 BO3 , 0.75 µM; ZnSO4 · 7H2 O, 17.5 µM, FeCl3 · !
Rt
6H2 O, 44.5 µM; pH adjusted to 5.5 with 5 M KOH. X(T0 )V (t0 ) + YX/G G0 F (t) dt − G(t)V (t)
Inoculum cultures were started from MGY streak t0
X(t)=
plates in 10 ml of BMGY grown for 24 h at 30 ◦ C in a V (t)
!
50 ml centrifuge tube at 250 rpm. 1 ml of the BMGY Rt
YX/M M0 F (t) dt − M(t)V (t)
culture was transferred to two 300 ml MGY inocula t0
cultures grown at 30 ◦ C at 250 rpm in 1 l Erlenmeyer +
V (t)
, (5)
flasks for 48 h. These cultures were used to inocu-
late 6 l of fermentation medium in the 10 l Chemap where, YX/G is the cell yield on glycerol; YX/M is the
fermenter. cell yield on methanol; t0 is the time at the start of the
Cell dry weight was estimated from the optical fed-batch period.
density measured at 650 nm (1 OD ≈ 0.3 g dry wt l−1 ). In order to solve this series of differential equa-
Methanol and glycerol concentrations in the feed so- tions, it is necessary to make some assumptions and
lutions and the fermentation broth were measured by simplifications. The methodology of solving this se-
ries of equations is presented below:
 343

1. The system is initially considered as a batch sys- strategy. For a single substrate (S) fed-batch system
tem growing on glycerol alone. During this batch where a quasi-steady state exists for the residual sub-
growth phase, where the initial glycerol charge strate concentration, the specific growth rate of the
is consumed, the system volume balance may be culture at time t is given:
neglected as the volume is not changing. The YX/S S0 F (t)
methanol balance is not required. As well, during µ(t) = . (6)
X(t)V (t)
the batch growth phase, the specific growth rate of
the cells follows Monod-type kinetics and the cell If the specific growth rate is controlled at a constant
yield on glycerol is assumed to be constant. The rate, µ, and the fed-batch cell balance is integrated, it
end-point solution (i.e. time at glycerol exhaus- is possible to solve for F(t):
tion) of the batch growth model is used to generate X(t)V (t) = X(t0 )V (t0 ) exp(µ(t − t0 )), (7)
initial conditions (X(t0 ), V(t0 )) for the fed-batch
period. µX(t)V (t)
2. The cell yield on glycerol is assumed to be F (t) =
S0 YX/S
constant and set at the value determined during
µX(t0 )V (t0 ) exp(µ(t − t0 ))
the batch growth phase throughout the fed-batch = . (8)
phase. While this is not necessarily a true rep- S0 YX/S
resentation of the cellular behaviour reported in For two substrates, provided that the yield coefficients
the literature (Egli et al. 1986), the model should are constant at the fixed growth rate. It is necessary
accurately predict cell growth on a mixed-feed pro- to develop a compound cell yield (based on an av-
vided that this assumption is consistently applied. erage weighted by the relative concentrations of the
3. To ensure that the methanol metabolic pathway is methanol and glycerol in the feed) and substrate feed
active and that the AOX1 promoter driving heterol- concentration terms which transforms F(t) to:
ogous protein production is induced, a 12 h induc- µV (t0 )X(t0 )
tion phase where the cells are fed only methanol F (t) = exp[(µ(t − t0 )], (9)
S0 YX/S
is initiated immediately after the end of the batch
growth phase.
S0 = (G0 + M0 ), (10)
4. Methanol concentrations are maintained at levels
sufficient to fully induce heterologous protein pro-  
G0 M0
duction, yet not so high as to be inhibitory to YX/S = YX/G + YX/M . (11)
G0 + M0 G0 + M0
cell growth or heterologous protein expression.
Therefore, it is desirable to keep methanol levels Returning to the fed-batch mass balances, it is now
between 1–2 g l−1 . possible to solve for V, X, and M as functions of time
5. Due to glycerol’s repression of the foreign pro- from the start of the fed-batch production phase:
tein production, the average specific growth rate
of the fed-batch culture must be kept sufficiently Zt
V (t0 )X(t0 )
far from µmax to ensure that no residual glycerol F (t) dt = exp[(µ(t − t0 )], (12)
S0 YX/S
is allowed to accumulate in the broth. Therefore, t0
µ < 12 µmax .
6. Because the residual glycerol concentration is es- Zt
sentially kept at zero, it is possible to assume a V (t) = V (t0 ) + F (t) dt, (13)
quasi-steady state on the glycerol balance, where t0
G ≈ 0 g l−1 .
7. It is necessary to determine the specific methanol !
Rt
consumption rate and the cell yield on methanol X(t0 )V (t0 ) + YX/G G0 F (t) dt
(based on the constant cell yield on glycerol as- t0
X(t)=
sumption) as functions of the specific growth rate. V (t)
This was accomplished by a set of steady-state !
Rt
CSTR experiments (data not shown). YX/M M0 F (t) dt − M(t)V (t)
It is possible to maintain µ at a constant value in t0
+ , (14)
a fed-batch culture by using an exponential feeding V (t)
344
Table 1. Parameters for mixed substrate fed-batch fermentation model.

Parameter Run No. 1 Run No. 2 Source

Maximum specific growth rate, µmax (h−1 ) 0.26 0.27 Batch data
Cell yield on glycerol, YX/G (gCDW g−1 ) 0.40 0.44 Batch data
Monod saturation constant, KS (g l−1 ) 0.005 0.005 Batch data
Lag period, tlag (h) 1.5 1.5 Batch data
Feed glycerol concentration,G0 (g l−1 ) 630 630 Measured
Feed methanol concentration, M0 (g l−1 ) 255 65 Measured
Cell yield on methanol, YX/M (gCDW g−1 ) 0.61 1.73 CSTR data
Volume at start of fed-batch phase, V0 (l) 6.65 6.73 Calculated
Cell density at start of fed-batch 20.1 21.3 Batch data
production phase, X0 (gCDW l−1 )
Specific growth rate during fed-batch 0.03 0.07 Chosen
production phase, µ (h−1 )
Compound substrate feed concentration during fed-batch production phase, S0 (g l−1 ) 885 695 Calculated
Compound cell yield on substrate during
fed-batch production phase, YX/S (gCDW g−1 ) 0.46 0.51 Calculated
Specific methanol consumption rate during
fed-batch production phase, qM (gM /gX · h) 0.020 0.015 CSTR data

model and compared to fed-batch experimental data

! to evaluate the accuracy of the model. Using the
Rt Rt
M0 F (t) dt − qM X(t)V (t) dt methanol metabolism parameters derived from CSTR
t0 t0 experimental work, the mixed-feed exponential feed-
M(t) = ,
V (t) ing strategy was employed to design two fed-batch
= G(t) ∼= 0. (15) runs at different specific growth rates. These two runs
As M(t) and X(t) both appear in both the cell and were then carried out experimentally to validate the
methanol balances, it is necessary to solve this system accuracy of the model and the validity of the mixed
of equations numerically according to: substrate exponential feeding strategy.
! The parameters used in the fed-batch fermentation
Rti
M0 F (t) dt + Mi−1 Vi−1 − qM Xi−1 Vi 1t runs are presented in Table 1. The fermentations were
ti −1
Mi = , (16) carried out according to the production-phase sub-
Vi
strate feed rate trajectory required by the exponential
! feeding strategy. The predicted and actual cell density,
Rt residual methanol concentration, and substrate flow
X(t0 )V (t0 ) + YX/G G0 F (t) dt rates are plotted as functions of time in Figures 1A
t0
X(t) = and 1B.
V (t)
! In run No. 1, the experimental cell density was very
Rt close to that predicted by the model. Therefore, the
YX/M M0 F (t) dt − Mi V (t)
t0 mixed substrate exponential feeding strategy assump-
+ . (17) tion was feasible and it correctly supported cell growth
V (t)
with an average specific growth rate of 0.03 h−1 during
The use of the cell density from the previous time the fed-batch production phase. As well, the predicted
step should not introduce a significant error provided cell yield on methanol correctly estimated the result-
that the time step is sufficiently small compared to ing cell growth from the mixed feed. The methanol
the change in the cell density (i.e., 1 t  1/µ). consumption rate was slightly underestimated by the
This is a simple model, where all of the variables model, which resulted in the experimental residual
can be solved for explicitly. Time profiles for each
of the relevant outputs can be determined with the
 345

Fig. 1. (A) Experimental (xpt) and Model prediction of cell density (X) and residual methanol concentration (M) and substrate flow rate (F) as
a function of time. Fed-batch run No. 1: µ = 0.03 h−1 ; G0 = 630 g l−1 ; M0 = 255 g l−1 ; other parameters listed in Table 1. (B) Experimental
(xpt) and Model prediction of cell density (X) and residual methanol concentration (M) and substrate flow rate (F) as a function of time.
Fed-batch run No. 1: µ = 0.07 h−1 ;G0 = 630 g l−1 ; M0 = 65 g l−1 ; other parameters listed in Table 1.
 346
methanol concentration being lower than the predicted
value.
Comparing the experimental data to the model pre-
dictions for Run No. 2, the actual cell density appeared
slightly higher at the end of the fed-batch production
phase than predicted. During the methanol induction
phase, there was a significant drop in the cell density
as the culture adapted to using methanol as a sub-
strate. The low cell density at the start of the fed-batch
production phase resulted in the initial cell density
used to define the exponential feeding rate being too
high such that the resulting specific growth rate was
slightly higher than 0.07 h−1 . The residual methanol
concentration prediction more closely matched the ex-
perimental data compared to the lower growth rate run.
The estimate of the specific methanol consumption
rate, derived from the CSTR data at a dilution rate of
0.07 h−1 , was likely more accurate. There is a jump
in the residual methanol level at the very end of the
fermentation as the cell growth reached its maximum
and growth began to slow due to a limitation of some
other component, possibly nitrogen.
Conclusion
This paper describes a simple accurate model capa-
ble of predicting cell growth and methanol utilization
during the mixed substrate fed-batch fermentation of
MutS recombinant Pichia pastoris. This model was
used to design an exponential feeding strategy for
mixed substrate fed-batch fermentation at a constant
specific growth rate. Maintaining a constant growth
rate during the production phase is an important fea-
ture in optimization of fed-batch fermentation. This
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simple predictive approach is a useful tool in achieving
this end. While this approach was developed specif-
ically for a MutS Pichia pastoris host, it should be
equally applicable to Mut+ Pichia strains, and to
mixed substrate Hansenula polymorpha systems.
Acknowledgement
The authors are grateful to the Lloyd Carr-Harris
Foundation for supporting this work.
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