Enzyme and Microbial Technology 48 (2011) 209–216

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Enzyme and Microbial Technology

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Dynamic flux balance analysis for pharmaceutical protein production by

Pichia pastoris: Human growth hormone
Pınar Çalık ∗ , Merve Şahin, Hatice Taşpınar 1 , Elif Ş. Soyaslan 1 , Bahar İnankur 1
Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531 Ankara, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The influence of methanol feeding rate on intracellular reaction network of recombinant human growth
Received 30 June 2010 hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates,
Received in revised form namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04 h−1 (MS-0.04) where Period-I (33 ≤ t < 42 h) includes
21 September 2010
the early exponential growth phase; Period-II (42 ≤ t < 48 h) is the exponential growth phase where the
Accepted 29 September 2010
specific cell growth rate decreases; Period-III (48 ≤ t ≤ 51 h) is the exponential growth phase where rhGH
concentration was the highest; and Period-IV (t > 51 h) is the diminution phase for rhGH and cell synthe-
Keywords:
sis. In Period-I, almost all of the formaldehyde entered the assimilatory pathway, at MS-0.02 and MS-0.03,
Pichia pastoris
Flux
whereas, at MS-0.04 high methanol feeding rate resulted in an adaptation problem. In Period-III, only at
Sorbitol MS-0.02 co-carbon source sorbitol uptake-flux was active showing that sorbitol uptake does not affected
Methanol from the predetermined feeding rate of methanol at 0 > 0.02 h−1 . The biomass synthesis flux value was
Metabolism the highest in Period-I, -II and -III, respectively at MS-0.03 & MS-0.04, MS-0.04 and MS-0.02; whereas,
Fed-batch rhGH flux was the highest in Period-I, -II, and -III, respectively at MS-0.03, MS-0.02 and MS-0.03. Based
Human growth hormone on the fluxes, Period-I should start with MS-0.03 methanol feeding rate and starting from the middle of
Period-II methanol feeding rate should be shifted to MS-0.02.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction In the literature there is no work for intracellular reaction rate

The carbon source(s) used, bioreactor operation parameters and
the feeding strategy applied strongly affect product formation by
influencing the metabolic pathways and changing metabolic fluxes.
Therefore, to fine-tune bioreactor performance in relation with the
physiology of the microorganism, the effect of feeding strategy on
intracellular reaction network needs to be clarified. For Pichia pas-
toris, the methylotrophic yeast, fermentation, methanol (MeOH) is
the carbon and energy source as well as the inducer for the expres-
sion of recombinant proteins; however, at high concentrations, it
inhibits growth [1]. Thus, either a three- or a four-stage fed-batch
operation strategy is applied where MeOH is fed to the system in
the recombinant protein production stage [2]. On the other hand,
as sorbitol is a non-repressing carbon source for AOX1 promoter
[3–5], sorbitol and MeOH were utilized simultaneously, and MeOH
feeding rate did not affect the sorbitol consumption rate, batch-
wise sorbitol addition as a co-substrate at the induction phase of
MeOH fed-batch fermentation by P. pastoris (Mut+ ) was proposed
as a beneficial recombinant protein production stage strategy [6].
analysis for the production of non-glycoslated recombinant pro-
tein production by P. pastoris. However, recently Çelik et al. [7]
reported the effect of MeOH feeding in the presence and absence
of sorbitol on metabolic flux distributions of P. pastoris producing
the glycoprotein, recombinant human erythropoietin (rHuEPO), in
a fed-batch fermentation process, using a mass flux balance-based
analysis. Related with metabolic flux analysis (MFA) on P. pastoris
without an aim of analyzing the response of metabolism to recom-
binant protein production, Sola et al. [8] compared the amino acid
biosynthesis and central carbon metabolism fluxes of P. pastoris
with Saccharomyces cerevisiae by stable isotope labeling experi-
ments in glycerol-containing medium, and in their latter study, Sola
et al. [9] included the MeOH utilization pathway to their analysis.
In the present work, the effect of MeOH feeding rate on the
intracellular reaction rates of recombinant human growth hormone
(rhGH), non-glycosylated protein, producing P. pastoris were inves-
tigated by analyzing P. pastoris bioreaction network to determine
potential strategies for improving rhGH production by P. pastoris.
2. Materials and methods

2.1. Strain and growth conditions

∗ Corresponding author. Tel.: +90 312 210 43 85; fax: +90 312 210 26 00. P. pastoris hGH-Mut+ strain carrying hGH cDNA under the control of AOX1
E-mail address: pcalik@metu.edu.tr (P. Çalık). promoter was maintained and cultivated as described previously [6]. Fed-batch r-
1
Equal contribution. protein production was carried out in 3.0 L pilot-scale bioreactor (BBraun CT2-2) by

0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2010.09.016
210 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216

PRPP Nucleotides
(R92-110)
Peroxisome (55) (56)
(127) (16)
MeOH CARB G6P R5P
(1)
(7) (6) (17) (18) (19)
(5) (20)
FormAL SRB F6P
(2) Xyl5P Rib5P His
(4) (8) (9) (24) (21)
CO2 For
(3) G3P Pentose
One-C units (25) E4P Phosphate Pathway
(R111-115) S7P
Gly (10) (11)
(80) (49)
(48)
Ser 3PG Phospholipids (R134-138 )
(50)
(12) (13)

(63) Aromatic
Cys PEP Chor Family Amino
Fatty
(36) (14) acids (R64-66)
Acids (R128-133)
Ac (28)
Acet (27)
Pyr (54) Ala Family Amino Acids (R51-54)
(30) (29)
(15) (32-33)
Lys AcCoA EtOH Lac
(31)
AcCoAm
(34) (35)
(37) TCA Cycle
Aspartic
OA Cit
Family Amino (38)
Acids (R57-62) (46-47)
ICit
(44) (39)
(45)
αKG Glutamic Family
(67)
(43) Amino acids (R67-72)
(40)
(41) (42)
Suc SucCoA
Mitochondria

Cytosol

Oxidative Phosphorylation
Biomass FADH2 (117) Maintenance
Synthesis ATP (141)
(R139) (116) ADP

(140)
hGH
Fig. 1. The metabolic pathway map of P. pastoris producing rhGH.
 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216 211

following the protocol given in detail elsewhere [6]. Before being fed to the biore-
actor, cells were grown in the BMGY precultivation medium (10 g L−1 yeast extract,
20 g L−1 peptone, 13.4 g L−1 YNB, 4 × 10−4 g L−1 biotin, 10 g L−1 glycerol, 0.1 M potas-
sium phosphate buffer pH = 6.0) until CX reached ca. 2.5 g L−1 , and then the cells
that were harvested from the medium were resuspended in 10 mL sterile water
[10]. In the bioreactor, the microorganism was first grown in the defined pro-
duction medium containing 40 g L−1 glycerol, 26.7 mL L−1 H3 PO4 (85%), 14.9 g L−1
MgSO4 ·7H2 O, 0.93 g L−1 CaSO4 , 18.2 g L−1 K2 SO4 , 4.13 g L−1 KOH, 4.35 mL L−1 PTM1
salt solution [11], and 0.1 mL L−1 antifoam (Y-30 emulsion, Sigma) in batch mode
until glycerol was totally consumed (glycerol batch (GB): Phase I). Thereafter, glyc-
erol feed (50% (v/v) glycerol containing 12 mL L−1 PTM1) was fed at limiting concen-
trations (glycerol fed-batch (GFB): Phase II) by a predetermined exponential feeding
profile, F(t), calculated for a constant specific growth rate, according to Eq. (1):
0 V0 CX0
F(t) = exp(0 t) (1)
CSO YX/S

where 0 is the desired specific growth rate, V0 is the initial volume, CX0 is the
initial cell concentration, CSO is feed substrate concentration and YX/S is the cell
yield on substrate. In the glycerol fed-batch phase, the pre-fixed parameters were
taken as, 0 = 0.18 h−1 ; YX/S = 0.5 and CSO = 790 g L−1 [11]. When a pulse if methanol
feed, CM0 = 1.5 g L−1 was given from 100% methanol containing 12 mL L−1 PTM1, a
short transition phase of methanol pre-induction (methanol transition (MT): Phase
III) was performed. After 6 h, in the beginning of r-protein production, t = 33 h
(rhGH production (PP): Phase IV), sorbitol was added to the medium in batch mode
(such that CSO = 50 g L−1 ), prior to feeding, and MeOH was fed to the system at
a predetermined rate for 0 = 0.02 h−1 , 0 = 0.03 h−1 and 0 = 0.04 h−1 , and these
conditions were denoted as MS-0.02, MS-0.03 and MS-0.04, respectively [11].

2.2. Analyses

Cell concentrations were measured with a UV–Vis spectrophotometer (Thermo

Spectronic, He␭ios-␣, Waltham, MA) at 600 nm [12]. The methods of Çelik et al. [6]
were used to measure the concentration of glycerol, MeOH, and sorbitol. RhGH con-
centration was determined by high-performance capillary electrophoresis (HPCE)
(Waters, Quanta 4000E, Milford, MA) using the method developed for the deter-
mination of the protein concentrations [13]. Excreted amino acid concentrations
were measured with an amino acid analysis system (Waters HPLC, Milford, MA),
using the modified Pico Tag method; excreted organic acid concentrations were
measured with an HPLC (Waters-2695); yeast viability was assessed by methylene
blue staining [6,13]. All analyses were performed at least in duplicate.
The intracellular-reaction-network of P. pastoris constructed by Çelik et al. [7]
was used by the addition of the rhGH synthesis reaction R140 instead of EPO
synthesis reaction. In writing the rhGH synthesis reaction (R140), the amino acid
components and the ATP requirements for peptide bond formation between these
amino acids were calculated by considering the intracellular composition of the pro-
tein, not the mature form. Knowing that 4 ATP-equivalents are required per peptide
bond formation [14], a total of 292 amino acids would require 1168 ATP (R140).
The simplified metabolic-pathway map of P. pastoris is shown in Fig. 1. As rhGH
is an extracellular recombinant gene product that is secreted to the fermentation
broth when the polypeptide chain synthesis is completed, the model was solved by
minimizing the rhGH accumulation-rate in the cell using the optimization program
GAMS 2.25 (General Algebraic Modeling System, GAMS Development Corp., Wash-
ington, DC), as described elsewhere [15]. The model variables (reaction-fluxes) were
expressed in mmol g DW−1 h−1 ; and the flux towards biomass was represented by Fig. 2. Variations in biomass, sorbitol, rhGH concentrations and specific AOX activ-
the specific-growth-rate () in h−1 . For the comparison of the results, actual flux val- ities with the cultivation time for (A) MS-0.02 condition and (B) MS-0.03 condition
ues were divided by MeOH-uptake-rate and multiplied by 100 to obtain normalized and (C) MS-0.04 condition, and the four periods of the bioprocess. RhGH concen-
flux values. tration, CrhGH (o); cell concentration, CX (); sorbitol concentration, CS (䊉); specific
AOX activity, UAOX ().

3. Results and discussion

3.1. MeOH feeding strategy and evaluation of metabolic fluxes
Our research group recently reported the effects of MeOH feed-
ing strategy on rhGH production, where sorbitol was used as the
co-substrate at the induction phase of MeOH fed-batch fermenta-
tion [11]. The data of MS-0.02, MS-0.03 and MS-0.04 were used to
determine the influence of MeOH feeding rate on the intracellular
reaction rates of rhGH producing P. pastoris. The variations in sor-
bitol, cell, rhGH concentrations and alcohol oxidase activities with
the cultivation time and the MeOH feeding strategy are given for all
cases for the exponential methanol feeding phase in Fig. 2. Sorbitol
was consumed at a constant rate and totally depleted at t = 48 h for
MS-0.03 and MS-0.04; t = 51 h for MS-0.02. In relation with sorbitol
consumption, cell concentrations increased between t = 33–48 h
and the maximum values were obtained between t = 48–51 h,
then reached to the stationary phase. Moreover, while rhGH con-
centrations increased in a similar manner until t = 48 h, due to
sorbitol depletion, after t = 48 h the difference between rhGH con-
centrations for different MeOH feeding rates were significant, and
remained almost constant through the stationary phase (t > 51 h).
Considering the cell, rhGH, protease, sorbitol concentrations
and specific alcohol oxidase activity profiles, the bioprocess was
divided into four periods. Period I (33 ≤ t < 42 h) includes the expo-
nential phase, where rhGH synthesis just starts to increase; Period
II (42 ≤ t < 48 h) is the exponential growth phase where the specific
growth rate decreases; Period III (48 ≤ t ≤ 51 h) is the exponential
growth phase where the rhGH concentration was the highest; and
Period IV (t > 51 h) is the diminution phase for rhGH and cell syn-
thesis. Extracellular concentration data from representative points
of each period were used in the metabolic flux analysis in order to
analyze each period. These points were chosen as t1 = 40 h, t2 = 47 h,
t3 = 50 h and t4 = 53 h to obtain the intracellular metabolic flux dis-
tributions in Periods I, II, III, and IV, respectively.
212 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216

3.2. Metabolic flux analysis of the dynamic process Table 1

Normalized metabolic flux distributions with respect to methanol uptake flux (%,
C-mol/C-mol methanol) around the glycolysis pathway.
3.2.1. Period I (33 ≤ t < 42 h): early exponential phase
In Period I, formaldehyde formation from MeOH (R1) flux val- Normalized fluxes
ues were 1.29, 2.33 and 2.88 mmol gDW−1 h−1 at MS-0.02, MS-0.03 Period I Period II Period III Period IV
and MS-0.04, respectively. With the increase in MeOH feeding
R# MS-0.02
rate R2 flux, formation of formic acid (For) from formaldehyde 1 100.00 100.00 100.00 100.00
(FormAl) increased whereas R4 flux, formation of glyceraldehyde- 2 0.16 63.68 0.28 0.27
3-phosphate (G3P) from FormAl, value decreased. At MS-0.02 and 3 0.00 63.48 0.00 0.00
MS-0.03 conditions, respectively 99 and 98% of FormAl entered 4 98.84 36.32 99.72 99.63
5 39.21 39.77 40.25 0.00
the assimilatory pathway was converted to the other metabolites. 6 15.14 39.19 47.38 6.76
Whereas at MS-0.04, 13.4% of FormAl entered the assimilatory 9 40.06 0.00 53.28 70.59
pathway and 86.6% was discarded by the dissimilatory pathway 10 87.03 71.98 69.01 26.12
indicating the shift in metabolism from the assimilatory pathway 12 81.91 71.98 54.03 22.83
14 80.43 70.83 51.31 37.08
to the dissimilatory pathway at the predetermined MeOH feeding
16 14.05 38.37 45.97 6.21
rates greater than those obtained at MS-0.03 (Table 1). The flux MS-0.03
values of R5, formation of F6P from sorbitol, were 0.505, 0.623 1 100.00 100.00 100.00 100.00
and 0.464 mmol gDW−1 h−1 , respectively at MS-0.02, MS-0.03 and 2 1.93 79.40 0.58 42.29
MS-0.04 conditions. The glycolysis, gluconeogenesis and PPP path- 3 0.00 12.46 0.00 42.02
4 98.07 20.60 99.42 57.71
ways worked uninterruptedly at all conditions. However, only at
5 26.75 15.57 0.00 0.00
MS-0.03 pyruvate (Pyr) synthesis was achieved via serine (Ser) syn- 6 58.57 23.04 17.62 10.77
thesis through the reactions R48 and R86 instead of the glycolysis 9 60.67 7.92 76.21 43.21
pathway reactions, R12 and R14. 10 60.15 24.91 16.94 12.66
12 1.07 24.51 13.75 12.44
From the anaplerotic reactions, the Pyr and OA replenishment
14 0.00 23.58 13.03 11.91
reactions (R34, R35) were active at MS-0.03 and MS-0.04; whereas 16 57.75 22.33 17.13 10.42
at MS-0.02 only reaction R34 formation of Pyr from malate (Mal) MS-0.04
was active. The TCA cycle was not complete at MS-0.02 condition, 1 100.00 100.00 100.00 100.00
as R38, R39 and R42 reactions were not active; however due to 2 86.60 14.99 27.12 43.10
3 85.07 0.00 0.00 0.00
fumarate (Fum) synthesis by R71 and R100, the reactions R44 and
4 13.40 85.01 72.88 56.90
R46 were active to complete the TCA cycle as (Table 2). At MS-0.03 5 16.11 16.51 0.00 0.00
condition, the reactions R37–42 were active and Fum was synthe- 6 13.37 18.43 9.17 0.37
sized by R71, R89 and R100, further the TCA cycle reactions R44 9 0.00 48.18 54.62 38.11
10 24.06 50.34 13.75 18.68
and R46 were also active. On the other hand, in MS-0.04 condition,
12 20.83 49.97 11.99 18.65
only R37–39 and R44 were active and Mal synthesized by R44 was 14 15.31 49.16 11.73 18.51
used in anaplerotic reaction R34 for pyruvate synthesis. 16 12.47 17.82 8.97 0.34
In Period I, the highest total intracellular amino acid flux was
obtained at MS-0.03 as 8.52 mmol gDW−1 h−1 . Among the fluxes
through amino acid groups, glutamate-group amino acids have the substrate-level phosphorylation reactions (R10, R14 and R41) and
highest flux values as glutamate and glutamine serve as the donor oxidative phosphorylation reactions (R116 and R117) throughout
of virtually all amino and amide groups in the cellular compo- the cultivation (Table 4). ATP generation increased with increase
nents, either by the direct participation of glutamine in biosynthetic in MeOH feeding rate, and the highest value was obtained as
reaction or through the action of glutamate as a substrate for 14.72 mmol gDW−1 h−1 at MS-0.04 being 1.35- and 3.11-fold higher
transamination reaction. Thus, excluding glutamate-group amino than that of MS-0.03 and MS-0.02 conditions, respectively. At
acids, alanine- and aspartic acid-group amino acids have the high- MS-0.02 condition while 53% of ATP was produced by oxidative
est flux values while aromatic acid group amino acids have the phosphorylation reactions (R116, R117), at MS-0.03 and MS-0.04
lowest values (Table 3). The nucleotide (R92–110), carbohydrate conditions 87 and 93% of ATP was produced by R116. The main-
(R127) and lipid synthesis (R128–138) pathway fluxes were the tenance energy requirement was the highest at MS-0.04 condition
lowest at MS-0.02; where the fluxes obtained were close to each (R141 = 10.946 mmol gDW−1 h−1 ) being ca. 12-fold higher than that
other at MS-0.03 and MS-0.04 conditions. ATP was supplied by the at MS-0.02.

Table 2
Actual and normalized metabolic flux distributions with respect to methanol uptake flux in the TCA cycle at t = 14 h in Period II.

R# Actual fluxes Normalized fluxes Actual fluxes Normalized fluxes Actual fluxes Normalized fluxes

MS-0.02 MS-0.03 MS-0.04

15 0.000 0.00 0.000 0.00 0.000 0.00
35 0.569 46.80 0.503 22.40 0.511 17.20
36 0.000 0.00 0.000 0.00 0.000 0.00
37 0.553 45.40 0.106 4.70 0.121 4.10
38 0.553 45.40 0.105 4.70 0.121 4.10
39 0.553 45.40 0.105 4.70 0.121 4.10
40 0.621 51.00 0.000 0.00 0.000 0.00
41 0.621 51.00 0.030 1.30 0.000 0.00
42 0.000 0.00 0.000 0.00 0.000 0.00
43 0.618 50.80 0.000 0.00 0.000 0.00
44 0.676 55.50 0.153 6.80 0.180 6.10
45 0.000 0.00 0.000 0.00 0.000 0.00
46 0.308 25.30 0.000 0.00 0.000 0.00
47 0.000 0.00 0.000 0.00 0.000 0.00
 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216 213

Table 3 (R4); whereas, 63.5% discarded by the dissimilatory pathway (R2,

Variations in amino acid fluxes with the cultivation time for all feeding conditions.
R3) (Table 1). At MS-0.03 conditions, while 20% of FormAl entered
Fluxes (mmol g−1 DW h−1 ) the assimilatory pathway (R4) whereas 79.4% converted into For
(R2) but only 12.46% of MeOH entered was further oxidized to CO2
Period I Period II Period III Period IV
(R3). At MS-0.04 condition, 85% of FormAl entered the assimila-
Amino Acids MS-0.02
tory pathway (R4) whereas 15% was converted into For (R2) but
Serine family 0.1253 0.0003 0.3114 0.0673
Alanine family 0.0350 0.3710 0.2610 0.0180 did not go through complete oxidation by reaction, R3 (Table 1).
Aspartic acid family 0.2210 0.5940 0.4956 0.0760 The formation of F6P from sorbitol reaction flux (R5) values were
Aromatic acid family 0.0089 0.0077 0.0150 0.0054 0.484, 0.350 and 0.490 mmol gDW−1 h−1 , respectively at MS-0.02,
Glutamic acid family 0.6459 0.4857 1.6256 0.2064 MS-0.03 and MS-0.04 conditions. In Period II, the glycolysis, gluco-
Total 1.0361 1.4587 2.7086 0.3731
MS-0.03
neogenesis and PPP pathways worked uninterruptedly at MS-0.03
Serine family 1.4624 0.0093 0.1254 0.0053 and MS-0.04; whereas at MS-0.02 condition formation of F6P from
Alanine family 0.0700 0.1000 0.0270 0.0250 G3P reaction (R9) was inactive as F6P supplied through the reaction
Aspartic acid family 1.5300 0.5950 0.4340 0.3240 R5 was sufficient. At all the conditions, R6 flux G6P formation from
Aromatic acid family 0.0120 0.0100 0.0077 0.0055
F6P was active with fluxes of 0.48, 0.52 and 0.55 mmol g DW−1 h−1 ,
Glutamic acid family 5.4470 0.8810 0.6827 0.3175
Total 8.5214 1.5953 1.2767 0.6773 respectively. At MS-0.03 and MS-0.04 in addition to R6, R9 was
MS-0.04 active with a value of 0.18 and 1.43 mmol gDW−1 h−1 , respectively.
Serine family 0.1773 0.0113 0.1061 0.0006 From the anaplerotic reactions, the Pyr and OA replen-
Alanine family 0.1760 0.1070 0.1690 0.0022 ishment reactions (R34, R35) were active at all the condi-
Aspartic acid family 0.2750 0.6010 0.2980 0.0166
tions. At MS-0.02 condition, R34 = 0.37 mmol gDW−1 h−1 and
Aromatic acid family 0.0790 0.0120 0.0034 0.0024
Glutamic acid family 0.9200 0.9860 1.0334 0.0168 R35 = 0.57 mmol gDW−1 h−1 flux values were 30 and 47% of the
Total 1.6273 1.7173 1.6099 0.0385 MeOH uptake flux (R1); whereas at MS-0.03 and MS-0.04 condi-
tion R34 flux values were 9 and 6% and R35 flux values were 22
The biomass synthesis flux (R139) was 0.034, 0.045 and and 17% of the MeOH uptake flux, respectively. The TCA cycle was
0.044 h−1 at MS-0.02, MS-0.03 and MS-0.04, respectively. The complete only at MS-0.02 condition. However, at MS-0.03 and MS-
highest rhGH synthesis flux (R140 = 0.014 ␮mol gDW−1 h−1 ) was 0.04 conditions R37, R38, R39 and R44 were active; additionally at
obtained at MS-0.03 condition being 1.22- and 1.85-fold higher MS-0.03 reactions R41 and 47 and at MS-0.04 R43 were also active.
than that of MS-0.02 and MS-0.04, respectively. The TCA cycle flux values at MS-0.02 were ca. 8-fold higher than
the other two conditions (Table 2).
3.2.2. Period II (42 ≤ t < 48 h): exponential growth phase In Period II, the total intracellular amino acid fluxes increased
In Period II, formaldehyde formation from MeOH (R1) flux values with the increase in MeOH feeding rate, being the lowest and
were 1.22, 2.25 and 2.97 mmol gDW−1 h−1 at MS-0.02, MS-0.03 and the highest at MS-0.02 and MS-0.04 conditions as 1.46 and
MS-0.04, respectively. In Period II, R1 flux values were close to those 1.71 mmol gDW−1 h−1 , respectively (Table 3). The nucleotide, car-
obtained in Period I at all conditions, being the lowest at MS-0.02 bohydrate and lipid synthesis pathway fluxes were the lowest
and the highest at MS-0.04 condition. At MS-0.02 condition, 36% at MS-0.02 and the highest at MS-0.04. At Period II, ATP gen-
of FormAl formed from MeOH entered the assimilatory pathway eration increased with the decrease in MeOH feeding rate, the

Table 4
Variations in ATP generated throughout the bioprocess for all cases.

Amount of ATP produced (mmol g−1 DW h−1 ) Percent of ATP produced (%)

Period I Period II Period III Period IV Period I Period II Period III Period IV

R# MS-0.02
10 1.121 0.876 0.737 0.286 23.725 6.122 13.100 14.137
14 1.036 0.862 0.548 0.406 21.926 6.025 9.740 20.069
41 0.066 0.621 0.003 0.211 1.397 4.340 0.053 10.430
106 0.002 0.001 0.002 0.001 0.042 0.007 0.036 0.039
108 0.001 0.001 0.001 0.000 0.021 0.005 0.018 0.022
116 2.438 10.984 4.108 0.912 51.598 76.768 73.018 45.082
117 0.061 0.963 0.227 0.207 1.291 6.731 4.035 10.232
Total 4.725 14.308 5.626 2.023 100 100 100 100
MS-0.03
10 1.401 0.560 0.351 0.288 12.865 6.808 8.795 4.970
14 0.000 0.530 0.270 0.271 0.000 6.443 6.765 4.676
41 0.004 0.030 0.218 0.082 0.037 0.365 5.462 1.415
106 0.003 0.002 0.001 0.001 0.028 0.024 0.025 0.017
108 0.001 0.001 0.001 0.001 0.009 0.012 0.018 0.010
116 9.456 7.010 2.978 5.120 86.832 85.218 74.618 88.352
117 0.025 0.093 0.172 0.032 0.230 1.131 4.310 0.552
Total 10.890 8.226 3.991 5.795 100 100 100 100
MS-0.04
10 0.693 1.494 0.429 0.663 4.789 20.678 9.009 14.892
14 0.441 1.459 0.366 0.657 3.047 20.194 7.686 14.757
41 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000
106 0.002 0.002 0.001 0.000 0.014 0.028 0.017 0.003
108 0.001 0.001 0.000 0.000 0.007 0.014 0.010 0.001
116 13.452 4.186 3.806 3.124 92.958 57.938 79.924 70.171
117 0.132 0.083 0.160 0.008 0.912 1.149 3.360 0.180
Total 14.721 7.225 4.762 4.452 100 100 100 100
214 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216
values were 14.3, 8.2 and 7.2 mmol gDW−1 h−1 at MS-0.02, MS-
0.03 and MS-0.04, respectively. While at MS-0.02 and MS-0.03
conditions 82 and 86% of ATP was produced by oxidative phospho-
rylation reactions (R116, R117), respectively, at MS-0.04 condition
59% of ATP was produced by R116 and R117 (Table 4). In Period
II, the highest (R141 = 10.95 mmol gDW−1 h−1 ) and the lowest
(R141 = 0.91 mmol gDW−1 h−1 ) maintenance energy requirements
were observed at MS-0.02 and MS-0.04, respectively. The results
indicate the higher impact of substrate-level phosphorylation at
higher MeOH feeding rates and less ATP waste for maintenance
energy requirements.
The biomass synthesis fluxes (R139) were 0.024, 0.037 and
0.044 h−1 at MS-0.02, MS-0.03 and MS-0.04, respectively. Hence,
at MS-0.02 and MS-0.03 conditions lower fluxes than the ones at
Period I were obtained; at MS-0.04 the flux values at Periods I and
II were equal to each other. The rhGH synthesis flux (R140) val-
ues were obtained as 0.032, 0.017 and 0.0094 ␮mol gDW−1 h−1 ,
respectively at MS-0.02, MS-0.03 and MS-0.04 conditions.
3.2.3. Period III (48 ≤ t ≤ 51 h): exponential growth and highest
rhGH synthesis phase
In Period III, FormAl formation from MeOH (R1) flux values were
1.07, 2.07 and 3.12 mmol gDW−1 h−1 at MS-0.02, MS-0.03 and MS-
0.04, respectively. In MeOH metabolism for Period III at MS-0.02
and MS-0.03 condition, almost all FormAl formed from MeOH;
entered the assimilatory pathway (R4); whereas, at MS-0.04 condi-
tion, while 73% of FormAl entered the assimilatory pathway (R4);
27.12% of MeOH entered was converted to For (R2) but not go
through complete oxidation by reaction, R3 (Table 1). In Period III,
sorbitol uptake flux (R5 = 0.432 mmol gDW−1 h−1 ) was active only
at MS-0.02. Thus, at Period III due to the inactive R5 reaction at MS-
0.03 and MS-0.04 conditions, the predetermined feed rate of MeOH
does not need to be higher than 0 = 0.03 h−1 . On the other hand,
both glycolysis and gluconeogenesis worked efficiently in Period III
at all the conditions (Table 1), but R16 flux, formation of R5P from
G6P, decreased with the increase in MeOH feeding rate.
From the anaplerotic reactions, the Pyr and OA replenishment
reactions (R34, R35) were active for all conditions, and the flux
values (R34 = 0.40 mmol gDW−1 h−1 , R35 = 0.43 mmol gDW−1 h−1 )
were the highest at MS-0.02 condition; whereas TCA cycle was
inactive, only R37 and R41 were active. On the other hand, the
TCA cycle was complete at MS-0.03 condition; whereas just R40–42
were inactive at MS-0.04 condition (Table 2).
In Period III, the amino acids were most effectively synthe-
sized (Table 3). The amino acid of central importance, glutamic
acid family amino acid fluxes had the highest synthesis flux val-
ues at MS-0.02, MS-0.03, and MS-0.04 conditions as 1.62, 6.38
and 1.03 mmol gDW−1 h−1 , respectively. In Period III, the synthesis
fluxes for one-carbon units (R111–115), carbohydrates (R127), and
lipids (R128–138) were the lower than those obtained in Period
II; furthermore, in Period III, the lowest and highest values were
obtained at MS-0.04 and MS-0.02 conditions, respectively.
In Period III, due to the active R5 flux, the highest total ATP
synthesis flux was achieved at MS-0.02 as 5.62 mmol gDW−1 h−1
(Table 4). At MS-0.02, MS-0.03 and MS-0.04 conditions 77, 79, and
83% of ATP was produced by oxidative phosphorylation reactions
(R116, R117), respectively.
The biomass synthesis flux (R139) was 0.035, 0.023 and
0.015 h−1 at MS-0.02, MS-0.03 and MS-0.04, respectively. At
MS-0.02 condition due to lower MeOH feeding rate, sorbitol
utilization flux decreased in Periods I and II, resulting in contin-
uation of the sorbitol uptake; thus growth continued in Period
III. The rhGH synthesis flux values (R140) were 0.044, 0.048 and
0.001 ␮mol gDW−1 h−1 , respectively at MS-0.02, MS-0.03 and
MS-0.04.
3.2.4. Period IV (t > 51 h): the diminution phase rhGH synthesis
phase
In Period IV, FormAl formation from MeOH (R1) flux values were
1.10, 2.28 and 3.55 mmol gDW−1 h−1 at MS-0.02, MS-0.03 and MS-
0.04, respectively. In Period IV, in MeOH metabolism at MS-0.02
condition, 99% of FormAl, formed from MeOH; entered the assimi-
latory pathway (R4); whereas at MS-0.03 and MS-0.04 conditions,
58 and 57% of FormAl entered the assimilatory pathway (R4). At
MS-0.03 and MS-0.04 conditions while 42 and 43% of FormAl con-
verted into For (R2); at MS-0.03 condition 42% of MeOH entered
was further oxided to CO2 (R3) (Table 1), showing that the pre-
determined feed rate of MeOH does not need to be higher than
0 = 0.02 h−1 in Period IV, similar to the findings of Çelik et al. [7].
In Period IV, sorbitol uptake flux was zero at all the conditions.
Both glycolysis and gluconeogenesis worked efficiently in Period IV
(Table 1). R16 flux, formation of R5P from G6P, was 0.068, 0.237 and
0.012 mmol gDW−1 h−1 at MS-0.02, MS-0.03 and MS-0.04, respec-
tively.
From the anaplerotic reactions, the Pyr and PEP replenishment
reactions (R34, R36) were active at MS-0.02 condition with the flux
values of 0.017 and 0.166 mmol gDW−1 h−1 ; whereas at MS-0.03
and MS-0.04 conditions Pyr and OA replenishment reactions (R34,
R35) were active and the highest flux values were obtained at MS-
0.03 condition as 0.05 and 0.25 mmol gDW−1 h−1 , respectively. The
TCA cycle was complete at MS-0.02 and incomplete at MS-0.04 con-
dition; on the other hand, at MS-0.03 condition R42, R43, R45–47
reactions were inactive (Table 2).
The amino acid fluxes were the lowest in Period IV for all the con-
ditions compared to Periods I–III. The highest flux was Glu synthesis
flux and the total amino acid synthesis flux was obtained at MS-
0.03 condition (Table 3). The synthesis fluxes for one-carbon units
(R111–115), carbohydrates (R127), and lipids (R128–138) were the
lower than those obtained at Periods I–III.
The lowest total ATP synthesis flux was achieved at MS-0.02
as 2.02 mmol gDW−1 h−1 ; whereas the highest total ATP synthesis
flux was achieved at MS-0.03 as 5.80 mmol gDW−1 h−1 (Table 4).
At MS-0.02, MS-0.03 and MS-0.04 conditions 55, 89, and 70% of
ATP was produced by oxidative phosphorylation reactions (R116,
R117), respectively. High maintenance energy requirements (R141)
were observed at MS-0.03 and MS0.04, respectively as 3.18 and
2.35 mmol gDW−1 h−1 .
The biomass synthesis flux (R139) was 0.014, 0.018 and
0.002 h−1 at MS-0.02, MS-0.03 and MS-0.04, respectively. The
rhGH synthesis flux values (R140) were 0.054, 0.031 and
0.0025 ␮mol gDW−1 h−1 , respectively at MS-0.02, MS-0.03 and MS-
0.04 condition.
3.2.5. Metabolic flux distribution at principal nodes
Normalized metabolic flux distributions for Period II at the pyru-
vate and glyceraldehyde-3-phosphate nodes are given in Fig. 3 for
all the conditions, as normalized by assigning 100% to the fluxes
for the reactions R14 and R4, respectively. Period II was selected
for the analysis, as high rhGH synthesis starts in this period and
as R5 reaction is active at all the conditions. The metabolic branch
points that highly affect product yield due to significant changes
in flux partitioning, were selected as the principal nodes and Pyr
is a generally accepted candidate [16]; whereas G3P node, which
directly affects r-protein yield, was selected for its importance in
MeOH metabolism [7].
The incoming flux to the pyruvate node was replenished by the
anaplerotic pathway from malate, with the highest rate being in
the MS-0.02 condition during Period II (Fig. 3A). The byproduct
synthesis was the highest at MS-0.04 condition. Overall, MS-0.03
condition seems to be the most favorable, considering effective
operation of the bioreaction network.
 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216 215

A D
PEP F6P
100.0 FormAl 0.0 0.0
0.0 76.8 100.0 0.8
Acet Pyr Aa G3P PPP
0.0 0.1 0.0
66.0 99.1
42.7
Lac Trp Lipids
Mal OA 3PG
B E
PEP F6P
100.0 FormAl 0.0 39.4
41.3 100.0 1.1
0.0
Acet Pyr Aa G3P PPP
0.0 0.1 0.1
94.9 60.5
36.4
Lac Trp Lipids
Mal OA 3PG
C F
PEP F6P
100.0 FormAl 0.0 56.7

0.0 13.8 100.0 13.7

Acet Pyr Aa G3P PPP
63.5 0.0 0.0
35.0 29.6
12.3
Mal Lac Trp Lipids
OA 3PG
Fig. 3. Normalized metabolic flux distributions at Pyr (at t = 14 h) and G3P (at t = 14 h) nodes for the bioprocesses with different feeding conditions. (A and D) MS-0.02
condition; (B and E) MS-0.03 condition; (C and F): MS-0.04 condition.

In addition to MeOH, sorbitol also supports formaldehyde enter-
ing the metabolism from glyceraldehyde-3-phosphate node and at
MS-0.02 condition F6P supplied through sorbitol (R5) and F6P for-
mation from G3P reaction (R9) was inactive. On the other hand, R9
was active at MS-0.03 and MS-0.04 conditions. As a result; when
compared to other conditions at MS-0.02 the highest flux from the
G3P node towards glycolysis and the lowest flux towards the PPP
were obtained (Fig. 3D).
The investigated principle nodes (Pyr and G3P nodes) were flex-
ible (Fig. 3), thus could adapt to changes in MeOH feeding rate
supporting the results of Çelik et al. [7].
4. Conclusion
Since methanol feeding rate affects product and by-product for-
mations in the bioprocess for the rhGH production, well-defined
perturbations were achieved by three different predetermined
MeOH feeding rates which are 0.02, 0.03, and 0.04 h−1 , and an in-
depth insight was provided by applying metabolic flux analysis.
On the basis of the proposed bioreaction network that is valid for
rhGH production in P. pastoris with the carbon source MeOH and
the co-carbon source sorbitol, the mass flux balance based stoi-
chiometric model that contains 102 metabolites and 141 reaction
fluxes has been set up. Thereafter, metabolic flux distributions were
obtained from the model, based on the minimum in vivo rhGH accu-
mulation rate assumption that is in combination with the elaborate
experimental data.
In the MeOH utilization pathway, almost all of the formalde-
hyde (>98%) converted from MeOH in R1 entered the assimilatory
pathway (via R4), in Periods I and III at MS-0.02 and MS-0.03 con-
ditions and also in Period IV at MS-0.02. On the other hand, at
MS-0.04 since high MeOH feeding rate resulted in an adaptation
problem, a shift in the metabolism was observed; and in Period II
formaldehyde (>85%) entered the assimilatory pathway (via R4).
In Period I, the highest sorbitol uptake flux was obtained at MS-
0.03 condition; whereas in Period II the highest value was obtained
at MS-0.04 condition. In Period III, only at MS-0.02 condition sor-
bitol uptake-flux was active showing that sorbitol uptake was not
affected from the predetermined feed rate of MeOH higher than
0 = 0.02 h−1 .
The nucleotide (R92–110), one-carbon units (R111–115), car-
bohydrates (R127), and lipids (R128–138), and biomass synthesis
flux (R139) were the highest at in Periods I, II and III, respectively
at MS-0.03, MS-0.04 and MS-0.02; while at Period IV all the fluxes
216 P. Çalık et al. / Enzyme and Microbial Technology 48 (2011) 209–216
were low due to the diminished growth. Furthermore rhGH synthe-
sis flux (R140) was the highest in Periods I, II, and III, respectively
at MS-0.03, MS-0.02 and MS-0.03.
Thus considering central metabolism, biomass and rhGH syn-
thesis fluxes, induction phase (Period I) should start with methanol
feeding rates at MS-0.03 condition and from the middle of Period II
until the end of the bioprocess, MeOH feeding rate should be shifted
to that at MS-0.02.
Acknowledgements
This work was supported by the Scientific and Technological
Research Council of Turkey (TÜBİTAK) through the project 109R015
and METU Research Fund. B.İ, E.Ş.S, MŞ and HT were awarded
scholarships by Scientific and Technical Research Council of Turkey
(TÜBİTAK).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.enzmictec.2010.09.016.
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