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11111
2
3
A model-based feeding strategy
4
5
for fed-batch fermentation of
6
7
recombinant Pichia pastoris
8
9 M.C. d’Anjou and A.J. Daugulis*
10111 Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada K7L 3N6
1
2 A two stage, exponential feeding strategy with mixed glycerol/methanol substrate was used in a fed-batch recombi-
3 nant Pichia pastoris fermentation. The feeding strategy was developed using a simple model based on mass balances,
4 Monod-type growth kinetics, and constant specific heterologous protein production rate. The model accurately
5 predicted cell growth, and demonstrated the usefulness of a rational, model-based approach for improving the produc-
6 tivity of recombinant P. pastoris fermentation.
7
8
9 Introduction gies (Brierley et al., 1990; Loewen et al., 1997). To this
20111 The methylotrophic yeast Pichia pastoris has been devel- point, much of the work done with this system is
1 oped into an effective expression system of heterologous preliminary and highly empirical resulting in subop-
2 proteins for bench and pilot-scale fermentation. A timal and often disappointing expression levels of
3 number of features of Pichia give it decided advantages heterologous proteins.
4 as an expression host over some more widely used
5 systems including: frequent high to very high expres- Significant enhancements to the productivity of recom-
6 sion levels; a lack of endotoxins which make produced binant fermentations in better characterized expression
7 protein suitable for therapeutic uses; correct folding and systems such as Escherichia coli and S. cerevisiae have been
8 secretion of eukaryotic proteins into the growth achieved by developing models accurately describing
9 medium; fewer problems of hyperglycosylation in Pichia system behaviour and using the models to improve
30111 systems compared to Saccharomyces cerevisiae; well devel- fermentation protocols to optimize heterologous protein
1 oped high cell density fermentation protocols; and, a expression (Baheri et al., 1997; Patkar et al., 1993). The
2 strong, tightly regulated promoter for the alcohol success of these techniques in recombinant E. coli and
3 oxidase gene (AOX1) which has been well characterized S. cerevisiae suggests that a rational, model-based
4 and exploited by a number of P. pastoris expression approach to developing an improved fermentation
5 vectors. A more detailed description of the features of protocol for recombinant P. pastoris systems is
6 this system are provided in two recent reviews (Cregg warranted. A P. pastoris strain that has been genetically
7 et al. 1993; Romanos, 1995). modified to produce and secrete sea raven anti-freeze
8 protein (SR-AFP) is used as a model system to demon-
9 However, despite its promise as a superior expression strate the use of a model-based feeding strategy in fed-
40111 system, the levels of expression of different heterologous batch recombinant P.pastoris fermentation. Fed-batch
1 proteins in P. pastoris vary over a wide range. A number mass balance equations, based on Monod-type kinetics
2 of molecular biology methods are being developed to and a constant specific heterologous protein production
3 improve on poor expression levels and overall produc- rate, were used to develop a feeding strategy and to
4 tivity (Cregg et al. 1993; Romanos, 1995). Enhance- predict biomass growth and protein production. A fed-
5 ments to process productivity in recombinant P.pastoris batch fermentation was carried out on a 10 l scale
6 fermentation can also be made by optimizing the according to the predicted feeding strategy to verify the
7 fermentation medium (Siegel and Brierley, 1989), and accuracy of the model.
8 improving the fermentation methodology. Examples of
9 the latter include substrate feeding strategies (Rodriguez Materials and methods
50111 Jimenez et al., 1997), oxygen supplementation Organism and growth
1 combined with feed back control on aeration to allow The Pichia pastoris strain GS115 (his4) was transformed
2 higher cell densities while avoiding oxygen limitation with plasmid pPIC9 containing the gene encoding for
3111 (Chen et al., 1997), and mixed-substrate feeding strate- SR-AFP according to the methodology presented in

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M.C. d’Anjou and A.J. Daugulis
11111 Loewen et al. (1997). The pPIC9 plasmid contains the
2 HIS4 gene for selection of his+ clones, and integrates
3 by homologous recombination into the AOX1 gene site
4 when linearized with BglII. This transformation
5 produces an AOX1-deficient clone characterized by a
6 MutS (Methanol utilization slow) his+ phenotype which
7 secretes SR-AFP into the fermentation broth.
8 relies on the action of the strong, tightly regulated
9 Fed-batch fermentation was carried out in a 10 l
10111 Chemap fermenter under the following conditions:
1 temperature, 30°C; impeller speed, 750 rpm; aeration,
2 15 l/min; pH, 5.5 measured by an Ingold pH probe and
3 maintained at that level by the addition of 5 M KOH.
4 Dissolved oxygen was measured using an Ingold steril-
5 izable polarographic electrode. A 20% (v/v) methanol
6 feed and a 50% (v/v) glycerol feed were supplied during
7 the fed-batch phase by means of calibrated peristaltic
8 pumps with speed controllers. Inocula and fermentation
9 media are as follows:
20111 BMGY: yeast extract, 10 g/l; meat peptone, 20 g/l;
1 100 mM potassium phosphate buffer pH 6.0; yeast
2 nitrogen base without amino acids, 13.4 g/l; biotin,
3 400 mg/l; glycerol, 10 ml/l.
4 MGY: yeast nitrogen base without amino acids,
5 13.4 g/l; biotin, 400 mg/l; glycerol 10 ml/l.
6 Fermentation medium: glycerol 50 g/l; (NH4)2SO4,
7 20 g/l; KH2PO4, 12 g/l; MgSO4·7H2O, 4.7 g/l;
8 CaCl2·2H2O, 0.36 g/l; plus trace elements:
9 CaSO4·5H2O, 0.2 mM; KI, 1.25 mM; MnSO4·4H2O,
30111 4.5 mM; Na2MoO4·2H2O, 2 mM; H3BO3, 0.75 mM;
1 ZnSO4·7H2O, 17.5 mM, FeCl3·6H2O, 44.5 mM; pH
2 adjusted to 5.5 with 5 M KOH.
3 quickly while conditions favorable to the expression of
4 Inoculum cultures were started from MGY streak plates
5 in 10 ml of BMGY grown for 24 h at 30°C in a 50 mL
6 centrifuge tube at 250 rpm. 1 ml of the BMGY culture
7 was transferred to two 300 ml MGY inocula cultures
8 grown at 30°C at 250 rpm in 1 l Erlenmeyer flasks for
9 48 h. These cultures were used to inoculate 6l of fermen-
40111 tation medium in the 10 l Chemap fermenter.
1 medium. An exponentially increasing glycerol feeding
2 Cell dry weight was estimated from the optical density
3 measured at 650 nm (1 OD ≈ 0.3 g dry wt./l). Methanol
4 and glycerol concentrations in the feed solutions and
5 the fermentation broth were measured by HPLC using
6 a Sugar Pak I (Waters Millipore) column and a 50 mg
7 EDTA/l mobile phase 0.5 ml/min. Protein levels were
8 estimated from Western blots according the following
9 procedure: electrophoresis on a 15% SDS-PAGE con-
50111 taining 0.1 mM sodium phosphate and 4 M urea, at pH
1 6.8; blotting onto a nylon membrane (PVDF, NEN);
2 incubation with rabbit anti-SR-AFP antiserum and
3111 subsequently with horseradish peroxidase-linked goat
866 Biotechnology Techniques · Vol 11 · No 12 · 1997
anti-rabbit IgG; detection was by enhanced chemilu-
minescence; and, quantifying of protein concentrations
by visual inspection of band intensities relative to
known standards.
Results and discussion
Expression of heterologous proteins using this system
promoter for the AOX1 gene. Regulation of this
promoter is due to combined glycerol/ glucose repres-
sion/derepression and methanol induction mechanisms.
Expression of gene sequences downstream of the
promoter occurs when low levels of methanol are present
in the absence of repressing levels of glycerol or glucose.
The recombinant Pichia pastoris clone used in this study
exhibits a MutS phenotype due to the AOX1 gene being
transplaced by the SR-AFP gene. However, the strain
continues to have a limited ability to metabolize
methanol due to the presence of the transcriptionally
weak AOX2 gene. The maximum specific growth rate
on methanol alone of MutS strains lies between
0.01–0.04 h–1 (Brierley et al., 1990). As a consequence,
growth using methanol as the sole carbon source will
result in efficient induction of the heterologous protein
but very slow cellular growth rates, and low overall
productivity. Growth in the presence of excess glycerol
or glucose will rapidly increase biomass but repress the
expression of the desired protein. However, a fed-batch
fermentation using a mixed glycerol/methanol feed
where the glycerol is fed at limiting rates and the
methanol is fed to excess should lead to conditions
where the cell mass is allowed to increase relatively
the heterologous protein are maintained (Loewen et al.,
1997).
It is therefore necessary to develop a fermentation
protocol where the cells are grown on glycerol to a high
cell density relatively quickly while ensuring that no
glycerol is allowed to accumulate in the growth
rate matched to the growth of the cell biomass will
allow this to occur. Methanol need only be present to
induce translation of the cloned gene and should play
no role in cell growth of the MutS strain and can be
neglected in modeling cell growth. The feeding rate
profile during the production period was determined
using simple mass balances based on a Monod-type
kinetic model. The fed-batch mass balances are based
on a constant cell yield on glycerol and neglecting the
small contribution of consumed methanol to cell
growth. The model equations and the feeding rate which
allows exponential growth with a constant specific
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A model-based feeding strategy for fed-batch fermentation of recombinant Pichia pastoris

11111 growth rate are presented below: where X is cell biomass rate, it was expected that the system would become
2 (g dry wt./l); V is the fermentation volume (l); F is the oxygen-limited as the cell density became large. As
3 glycerol feed rate (l/h); m is the specific growth rate oxygen limitation is detrimental to heterologous protein
4 (h–1); S is the glycerol concentration in the growth expression in P. pastoris (Romanos, 1995), a second stage
5 medium (g/l); So is the glycerol concentration in the of slower growth (m = 0.01 h–1) was used to decrease
6 feed medium (g/l); YX/S is the cell yield on glycerol the culture’s oxygen requirement, and to allow a usable
7 (gX/gS); t and to are the run time and the run time at concentration of the product protein to accumulate, thus
8 the start of the fed-batch production period respectively facilitating downstream separation and purification. The
9 (h); mmax is the maximum specific growth rate on glyc- second stage of feeding was to be initiated when the
10111 erol (h–1); P is the heterologous protein concentration dissolved oxygen level reached 30% of saturation.
1 in the growth medium (gP/l); and qP is the specific
2 protein production rate (gP/gX·h). In our work, a batch growth phase was followed by a
3 24 h induction period of glycerol starvation and
4 d(XV) = m(XV) – FX methanol addition at a rate of 1.6 g/h. Though not
5 dt necessarily required, this induction phase was used to
6 d(SV) = FS – Y m(UV) ensure that the entire system was exposed to conditions
0 x/s
7 dt which induce expression of the heterologous protein.
8 d(PV) = q (XV) The methanol feed rate was kept constant throughout
p
9 dt the run such that the methanol concentration in the
20111 dV = F(t) growth medium was at inducing but non-inhibitory
1 dt levels (0.1–0.2%). Following the induction period, a
2 m = mmaxS glycerol feed following an exponential profile was started
3 S + Ks to initiate the higher growth rate production phase
4 F(t) = mV(t0)X(t0) exp[m(t – t0)] (stage 1). The glycerol feed rate was adjusted manually
5 S0Yx/s and discretely at regular intervals to reflect the predicted
6 exponential feed rate. The dissolved oxygen level was
7 Provided that the selected specific growth rate is signif- monitored and once the target of 30 % of saturation
8 icantly lower than the maximum and that no inhibitory was reached, the feed rate was lowered to initiate the
9 or otherwise unexpected effects occur due to the pres- second production phase (stage 2). Figure 1 shows the
30111 ence of methanol, this simple model should predict dissolved oxygen trace as well as the predicted and
1 cellular growth with acceptable accuracy. The necessary actual glycerol feed rates.
2 kinetic parameters were determined from batch and
3 continuous culture experiments (data not shown). As Figure 2 shows predicted and actual cell density and
4 well, the specific rate of heterologous protein produc- product protein concentration. Glycerol and methanol
5 tion, qP, was determined from these experiments and
6 assumed to be constant for the given conditions. These
7 kinetic parameters and the conditions at the start of the
8 fed-batch production period are presented as Table 1.
9
40111 Initially, a specific growth rate of 0.07 h–1 was chosen
1 as being sufficiently far removed from the maximum
2 specific growth rate (even if methanol does have some
3 inhibitory effect). However, even at this low growth
4
5
6 Table 1 Fed-batch model parameters
7
Maximum specific growth rate, mmax (h–1) 0.25
8 Cell yield on glycerol, YX/S (g dry wt/gS) 0.42
9 Monod saturation constant, KS (g/l) 0.005
50111 Feed glycerol concentration, So (g/l) 950
1 Initial volume, V(to) (l) 6.84
Initial cell density, X(to) (g dry wt/l) 16.2 Figure 1 Predicted and actual feed rate profile and
2 dissolved oxygen trace for fed-batch fermentation showing
Specific SR-AFP production rate, qP (ngP/gX· h) 5.1
3111 transition to reduced feed rate at 30% of saturation.

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M.C. d’Anjou and A.J. Daugulis

11111 the cell density is maximized prior to induction and

2 maintained at low growth conditions in the production
3 phase. As confirmation, we have also observed (data not
4 shown) that once the maximum cell density had been
5 reached no additional SR-AFP was produced despite
6 methanol being present in inducing concentrations.
7
8 Conclusion
9 A simple model was developed to predict growth and
10111 heterologous protein expression in recombinant Pichia
1 pastoris. The model was also used to develop a rational
2 two-stage exponential feeding strategy for a mixed-
3 substrate, fed-batch fermentation of a MutS strain. A
4 two-stage approach was necessary to avoid oxygen limi-
5 Figure 2 Predicted and experimental profiles for cell tation at higher cell densities and to allow the product
6 growth and protein production in fed-batch fermentation protein to accumulate to levels facilitating separation
7 using two stage feeding strategy. and purification. The model predicted cell growth with
8 reasonable accuracy, but the assumption of constant
9 specific heterologous protein production rate may have
20111 levels were evaluated off-line by HPLC as the run accounted for discrepancies between the model and
1 proceeded to ensure that no repressing levels of glyc- experimental data. It has been demonstrated that
2 erol had built-up, and that methanol was present at feeding strategies can be developed using a very simple
3 levels which induced heterologous protein expression model in order to improve process productivity in
4 but were non-inhibitory to growth. Glycerol was never fed-batch recombinant P. pastoris fermentations without
5 detected in the fermentation broth. Methanol was the need for complex model development and predic-
6 always present, but in levels (0.02–0.08%) much lower tive/feed-back compensated adaptive controller mecha-
7 than would be expected assuming that it was not being nisms.
8 consumed by the yeast. This is not entirely unexpected
9 as simultaneous utilization of methanol and some other References
30111 carbon source by methylotrophic yeast is well docu- Baheri, HR, Roesler, WJ, Hill, GA. (1997), Biotech. Techniques
1 mented (Egli et al., 1986). 11: 47–50.
Brierley, RA, Bussineau, C, Kosson, R, Melton, A, and Siegel,
2 RS. (1990), Ann. N.Y. Acad. Sci. 589: 350–362.
3 Cell growth was predicted by the model with reason- Chen, Y, Cino, J, Hart, G, Freedman, D, White, C, and Komives,
4 able accuracy. The contribution of methanol to cell EA. (1997), Process Biochem. 32: 107–111.
5 growth becomes more significant at lower growth rates Cregg, JM, Vedvick, TS, and Raschke, WC. (1993),
6 in methylotrophic yeast (Eggeling and Sahm, 1981). Bio/Technology. 11: 905–910.
Thus, the actual cell density is slightly higher than Eggeling, L, and Sahm, H. (1981). Arch. Microbiol. 130: 362–365.
7 Egli, T, Bosshard, C, and Hamer, G. (1986). Biotech. Bioeng. 28:
8 predicted by the model. Protein production was less 1735–1741.
9 well described, suggesting that the assumption of a Loewen, MC, Liu, X, Davies, PL, and Daugulis, AJ. (1997), Appl.
40111 constant specific expression rate may not be entirely Microbiol. Biotech. In press.
1 valid. The data suggest that the specific expression level Patkar, A, Seo, JH, and Lim, HC. (1993), Biotech. Bioeng. 41:
is higher at the higher growth rate and reduced during 1066–1074.
2 Rodriguez Jimenez, E, Sanchez, K, Roca, H, and Delgado, JM.
3 the slow growth period. This would preclude the use (1997), Biotech. Techniques 11: 461–466.
4 of more traditional approaches to fermentation opti- Romanos, M. (1995), Current Opinion in Biotech. 6: 527–533.
5 mization such as high cell density fermentation in which Siegel, RS, and Brierley, RA. (1989), Biotech. Bioeng. 34: 403–404
6
7
8 Received 22 August 1997;
9 Revisions requested 15 September 1997;
50111 Final Revisions received 30 September 1997;
1 Accepted 1 October 1997
2
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868 Biotechnology Techniques · Vol 11 · No 12 · 1997