Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase
© Gmeiner et al.; licensee BioMed Central. 2015
Received: 7 September 2014
Accepted: 18 December 2014
Published: 8 January 2015
Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 MutS strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.
In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.
The methylotrophic yeast Pichia pastoris is an attractive host for the recombinant production of proteins and biopharmaceuticals (e.g. [1-3]). It can grow on inexpensive media to high cell densities , numerous molecular manipulation tools are available  and high production titers are possible [5,6]. Due to the capacity of performing posttranslational modifications, like glycosylation, P. pastoris is attractive for the production of eukaryotic proteins (e.g. [3,7-10]). However, the glycosylation capacity of this yeast also is a curse: native glycosyltransferases recognize the aminoacid motif N-X-S/T and link N-glycans to the asparagine [11,12]. In contrast to mammalians, however, no trimming reactions of the attached glycans happen, but the glycans are further extended, a phenomenon known as hyperglycosylation . The first reaction of this cascade is catalyzed by an α-1,6-mannosyltransferase (OCH1) localized in the Golgi apparatus [14,15]. Hyperglycosylation describes a huge problem since not only the physico-chemical properties of the target protein get masked leading to difficulties in the downstream process , but also yeast derived glycans are not compatible with the human organism and can cause immunogenic reactions . Consequently, there have been numerous attempts to manipulate the native glycosylation machinery of P. pastoris (e.g. [18-22]). In a recent study, we deleted OCH1 in a recombinant P. pastoris strain (Δoch1) and physiologically characterized the strain in the controlled environment of a bioreactor . We purified the recombinant product horseradish peroxidase (EC 220.127.116.11; HRP; e.g. ) and analyzed catalytic constants, thermal stability as well as protein glycosylation. Although the Δoch1 strain produced the recombinant protein with shorter glycans of considerably increased homogeneity, the strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and uncontrollable foam formation [25,26].
In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration (dO2) on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity in a multivariate manner to identify fed-batch operating conditions for the recombinant Δoch1 strain which give both high productivity and product purity, and hamper methanol accumulation as well as cell lysis and consequent foam formation.
Material and methods
A P. pastoris CBS7435 MutS Δoch1 strain carrying the gene coding for the HRP isoenzyme A2A was provided by Prof. Anton Glieder (University of Technology, Graz, Austria). Strain generation and isoenzyme characteristics were described previously [23,27]. A recombinant P. pastoris CBS7435 MutS strain with intact OCH1 expressing HRP A2A, hereafter called wildtype OCH1 strain, was included as reference.
Design of experiments
Experimental plan for the multivariate analysis of the 3 factors temperature, pH and dO 2 and their effects on different response parameters
dO 2 [%]
We analyzed the effects of the 3 factors on 1) strain physiology (specific substrate uptake rate during methanol adaptation (qs MeOH adapt), methanol accumulation during fed-batch (MeOHaccum), biomass yield (YX/S) and CO2 yield (YCO2/S)), 2) strain morphology (cell size distribution), 3) cell lysis (extracellular DNA content), 4) productivity (space-time-yield STY [U∙L−1∙h−1], specific productivity qp [U∙g−1∙h−1]) and 5) product purity (defined as the amount of target protein in relation to the amount of total extracellular protein [U∙mg−1]).
The P. pastoris Δoch1 strain expressing HRP isoenzyme A2A was cultivated in the controlled environment of a bioreactor. Batch and fed-batch phase were performed on glycerol, followed by a methanol adaptation pulse. Afterwards, a methanol fed-batch with a controlled feed rate corresponding to a certain specific substrate uptake rate of methanol (qs MeOH) was done.
Precultures were done in yeast nitrogen base medium (YNBM; 0.1 M potassium phosphate buffer pH 6.0, 3.4 g · L−1 YNB w/o amino acids and ammonia sulfate, 10 g · L−1 (NH4)2SO4, 400 mg · L−1 biotin, 20 g · L−1 glucose). Zeocine was added at a concentration of 100 μg∙L−1.
Batch and fed-batch cultivations were performed in 2-fold concentrated basal salt medium (BSM; 21.6 mL · L−1 85% phosphoric acid, 0.36 g∙L−1 CaSO4 · 2H2O, 27.24 g∙L−1 K2SO4, 4.48 g∙L−1 MgSO4 · 7H2O, 8.26 g∙L−1 KOH, 0.3 mL∙L−1 Antifoam Struktol J650, 4.35 mL∙L−1 PTM1, NH4OH as N-source). Trace element solution (PTM1) was made of 6.0 g · L−1 CuSO4 · 5H2O, 0.08 g · L−1 NaI, 3.0 g · L−1 MnSO4 · H2O, 0.2 g · L−1 Na2MoO4 · 2H2O, 0.02 g · L−1 H3BO3, 0.5 g · L−1 CoCl2, 20.0 g · L−1 ZnCl2, 65.0 g · L−1 FeSO4 · 7H2O, 0.2 g · L−1 biotin, 5 mL · L−1 H2SO4. Induction was carried out in presence of the heme-precursor Δ-aminolevulinic acid at a final concentration of 1 mM. The concentration of the base NH4OH was determined by titration with 0.25 M potassium hydrogen phthalate.
Frozen stocks (−80°C) were cultivated in 100 mL YNBM-Zeocine in 1,000 mL shake flasks at 30°C and 230 rpm for 48 h. Then, the preculture was transferred aseptically to the culture vessel. The inoculum volume was 10% of the final starting volume.
Batch and non-induced fed-batch
F0 = initial feed rate [g∙h−1]; X = calculated biomass concentration [g∙L−1]; V = volume in the bioreactor [L]; μ = specific growth rate [h−1]; δFeed = density glycerol feed [g∙L−1]; YX/S = biomass yield; cFeed = concentration feed [g∙L−1]; F = calculated feed rate [g∙h−1]; e, Euler constant; t = time [h]
X = calculated biomass [g∙L−1]; OD600 = optical density at 600 nm; α = correlation factor between DCW and OD600 value.
F = calculated feed rate [g∙h−1]; X = biomass concentration calculated from OD600 [g∙L−1]; V = volume in the bioreactor [L]; qs MeOH = specific methanol uptake rate [mmol∙g−1∙h−1]; CFeed = density of methanol feed [g∙L−1]; cFeed = concentration of methanol feed [g∙L−1].
Dry cell weight was determined by centrifugation of 5 mL culture broth (5,000 rpm, 4°C, 10 min) in a laboratory centrifuge (Sigma 4K15, rotor 11156), washing the pellet with 5 mL deionized water and subsequent drying at 105°C to a constant weight in an oven. The enzymatic activity of HRP was measured using an ABTS assay in a CuBiAn XC enzymatic robot (Innovatis, Germany). Ten μl of sample were mixed with 140 μl 1 mM ABTS solution (50 mM KH2PO4, pH 6.5). The reaction mixture was incubated at 37°C for 5 min before the reaction was started by the addition of 20 μl 0.078% H2O2 (v/v). Changes in absorbance at 415 nm were measured for 80 seconds and rates were calculated. The standard curve was prepared using a commercially available HRP preparation (Type VI-A, Sigma-Aldrich, USA) in the range from 0.02 to 2.0 U · ml−1. Protein concentrations were determined at 595 nm using the Bradford Protein Assay Kit (Bio-Rad Laboratories GmbH, Austria) with bovine serum albumin as standard. Extracellular DNA content was measured by the Nanodrop 1000 device (ThermoScientific, Austria). Concentrations of methanol and potential metabolites were determined in cell-free samples by HPLC (Agilent Technologies, USA) equipped with an ion-exchange column (Supelcogel C-610H Sigma-Aldrich, USA) and a refractive index detector (Agilent Technologies, USA). The mobile phase was 0.1% H3PO4 with a constant flow rate of 0.5 mL · min−1 and the system was run isocratically at 30°C. All measurements were done in duplicates.
Changes in the morphology of the recombinant P. pastoris Δoch1 strain during the bioprocess were monitored by a Malvern Mastersizer 2000, measuring the cell size distribution in all samples. Frozen cell pellets (stored at −20°C) with a biomass concentration of 10–20 g∙L−1 were thawed and resuspended in 10 mL deionized water. After dispersing the samples with module Hydro2000S, 6 to 20 drops of cell suspension were dripped into the water tank of the Malvern Mastersizer 2000 until the laser diffraction reached a value between 10–12%. Samples were analyzed between 0 – 10,000 μm. Furthermore, all samples were analyzed microscopically using a Zeiss Epifluorescence Axio Observer Z1 deconvolution microscope (Carl Zeiss, Germany) equipped with a LD Plan-Neofluar 63x objective (+10x ocular) and the LED illumination system Colibri.
Electrophoresis was done with aliquots of supernatants obtained at the end of the cultivation. SDS-PAGE was performed using a 6% stacking gel and a 12% separating gel in 1x Tris-glycine buffer. Gels were run in the vertical electrophoresis Mini-PROTEAN Tetra Cell apparatus (Biorad; Austria) at 150 V for about 2 h. Gels were stained with Coomassie blue. The protein mass standard used was the PageRuler Prestained Ladder (Fermentas; Austria).
Protein identification and glycopeptide analysis by LC-ESI-MS
Relevant protein bands were cut out and digested in gel. S-alkylation with iodoacetamide and digestion with sequencing grade modified trypsin (Promega) were performed. The peptide mixture was analysed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion, DDA mode (= switching to MSMS mode for eluting peaks). MS-scans were recorded (range: 150–2200 Da) and the 6 highest peaks were selected for fragmentation. Instrument calibration was performed using ESIcalibration mixture (Agilent). For separation of the peptides a Thermo BioBasic C18 separation column (5 μm particle size, 150°0.360 mm) was used. A gradient from 95% solvent A and 5% solvent B (Solvent A: 65 mM ammonium formiate buffer, B: 100% ACCN) to 32% B in 45 min was applied, followed by a 15 min gradient from 32% B to 75% B, at a flow rate of 6 μL∙min−1. The analysis files were converted using Data Analysis 4.0 (Bruker) to XML files, which are suitable to perform MS/MS ion searches with MASCOT (embedded in ProteinScape 3.0, Bruker) for protein identification. Only proteins identified with at least 2 peptides with a protein score higher than 80 were accepted. For searches the SwissProt database was used.
Results and discussion
In the present study we developed a fed-batch bioprocess for a recombinant P. pastoris Δoch1 strain. We analyzed the effects of 3 process parameters (temperature, pH and dO2) on strain physiology and morphology , as well as on productivity and product purity in a multivariate manner.
Dynamic batch cultivation to determine qs max MeOH
We performed a dynamic batch cultivation with methanol pulses to determine the maximum specific methanol uptake rate (qs max MeOH) of the Δoch1 strain at 20°C, 25°C and 30°C [25,26]. The average qs max MeOH determined for at least 3 consecutive 1% (v/v) methanol pulses were 0.43 mmol∙g−1∙h−1 at 30°C, 0.74 mmol∙g−1∙h−1 at 25°C and 0.85 mmol∙g−1∙h−1 at 20°C. Interestingly, qs max MeOH was strongly temperature dependent. At 20°C, cells specifically consumed the double amount of methanol per time compared to 30°C. In previous studies with different microorganisms it was shown that substrate uptake usually declines with lower temperature [35-37]. Thus, we also analyzed a reference strain with intact OCH1, hereafter referred to as wildtype OCH1 strain, and indeed observed decreasing qs MeOH with decreasing temperature, namely 1.30 mmol∙g−1∙h−1 at 28°C, 1.20 mmol∙g−1∙h−1 at 24°C and 0.95 mmol∙g−1∙h−1 at 20°C. So far, we do not have a physiological explanation for the opposite behaviour of the recombinant Δoch1 strain. However, since the subsequent DoE covered a range from 20-30°C we designed the fed-batch strategy for the recombinant Δoch1 strain in a way to constantly feed at a rate corresponding to a rather low qs MeOH = 0.2 mmol∙g−1∙h−1. This was less than half of the lowest qs max MeOH determined in the dynamic batch experiment, and thus guaranteed a certain safety margin to avoid methanol accumulation.
DoE fed-batch cultivations
Effects on strain physiology
Strain characteristic parameters of the recombinant P. pastoris Δ och1 strain induced under different conditions
q s Gly batch
q s MeOH adapt
q s MeOH fed-batch
[mmol∙g −1 ∙h −1 ]
[mmol∙g −1 ∙h −1 ]
[mmol∙g −1 ∙h −1 ]
[g∙L −1 ]
[C-mol∙C-mol −1 ]
[C-mol∙C-mol −1 ]
Specific substrate uptake rate during methanol adaptation (qs MeOH adapt)
Methanol accumulation during fed-batch (MeOHaccum)
During induction the feed rate was regulated to correspond to a constant qs MeOH = 0.2 mmol∙g−1∙h−1, which was 2-fold lower than the lowest qs max MeOH at 30°C determined in the dynamic batch experiment. However, methanol still accumulated in cultivations DoE3, DoE4 and DoE10. For DoE3 methanol accumulation set on after around 80 h and for DoE10 after around 70 h of induction, whereas for DoE4 methanol accumulated right from the start. In Table 2 methanol concentrations at the end of cultivation are shown. Methanol only accumulated in cultivations at 30°C which is in agreement with both the results from the dynamic batch cultivation at 30°C where qs max for methanol was the lowest, as well as with the low qs MeOH adapt in fed batch cultivations at 30°C (Figure 2). However, we could not identify a significant factor causing this phenomenon since in DoE5 (30°C, pH 7.0, 10% dO2) no methanol accumulated (Table 2). Apparently, induction at 30°C favours methanol accumulation over time, a phenomenon which was observed before . However, using the current DoE screening approach based on only 3 factors and the underlying linear regression model, we could not identify significant factors and their interactions causing methanol accumulation. Apparently, a temperature of 30°C favours changes in cell physiology and a reduction of methanol uptake over time which is why a lower cultivation temperature of 20°C for fed-batch production processes with this Δoch1 strain is highly recommended. In contrast, the wildtype OCH1 strain can be cultivated at 30°C without experiencing changes in methanol uptake.
We investigated the influence of the 3 process parameters on both biomass yield (YX/S) and CO2 yield (YCO2/S). However, yields were not affected by varying the 3 process parameters in the design space (Table 2).
Effects on strain morphology
Cell-size distribution at different time points of the fed-batch cultivation. Single and budding cells were categorized with a size of < 15 μm, whereas cell clusters were classified to be > 15 μm
Size distribution after batch [%]
Size distribution after non-induced fed-batch [%]
Size distribution after induced fed-batch [%]
< 15 μm
> 15 μm
< 15 μm
> 15 μm
48.8 ± 7.6
51.3 ± 7.6
59.8 ± 12.3
40.2 ± 12.3
78.6 ± 7.8
21.4 ± 7.8
wildtype OCH1 strain
Apparently, cell cluster formation of the recombinant Δoch1 strain was a function of the C-source and not of any of the 3 investigated process parameters. When grown on methanol, cell clusters disappeared over time. However, Δoch1 cells stayed much smaller than wildtype OCH1 cells (Figure 4) which might result from the altered cell wall composition with enhanced chitin deposition .
Effects on cell lysis
Extracellular DNA content measured at the end of cultivation
DNA content [ng∙μL −1 ]
Effects on productivity and product purity
Specific productivity (q p ), space-time-yield (STY) and specific enzyme activity at the end of cultivation
q p [U∙g −1 ∙h −1 ]
STY [U∙L −1 ∙h −1 ]
Spec. activity [U∙mg −1 ]
The only significant factor for all 3 responses was the temperature, whereas pH and dO2 had no effect (p-values for qp: temperature = 0.000072, pH = 0.77, dO2 = 0.33; p-values for STY: temperature = 0.000026, pH = 0.37, dO2 = 0.90; p-values for specific activity: temperature = 0.000024, pH = 0.49, dO2 = 0.45). The summary of fit plots indicated valid models (for qp: R2 = 0.94, Q2 = 0.88, Model Validity = 0.77, Reproducibility = 0.97; for STY: R2 = 0.95, Q2 = 0.81, Model Validity = 0.55, Reproducibility = 0.99; for specific activity: R2 = 0.95, Q2 = 0.93, Model Validity = 0.87, Reproducibility = 0.95). To visualize the effects, we plotted the responses versus temperature and dO2 at pH 5.0 (Additional file 1: Figure S1). Summarizing, both highest productivity and product purity were obtained at 20°C and a low dO2 at pH 5.0. Apparently, at lower temperature the recombinant Δoch1 strain secreted more active HRP and less contaminating proteins compared to higher temperatures. This is not only important to increase the total amount of active product over time, but also for the subsequent downstream process. We observed a similar trend for the wildtype OCH1 strain where qp and product purity were both higher at lower temperature: (20°C: qp = 15 U∙g−1∙h−1, specific activity = 95 U∙mg−1; 24°C: qp = 11 U∙g−1∙h−1, specific activity = 50 U∙mg−1; 28°C: qp = 6 U∙g−1∙h−1, specific activity = 35 U∙mg−1).
Electrophoresis and glycopeptides analysis by LC-ESI-MS
To identify the different HRP proteins, respective bands were excised (indicated in Figure 5) and glycopeptides were analyzed by HPLC-ESI-MS. In fact, we were able to identify the different HRP preparations (Additional file 2: Table S1). We observed apparent size differences between the 2 HRP A2A preparations from the Δoch1 strain and the wildtype OCH1 strain, which might result from the different degree of glycosylation (lanes 1 and 2), and between HRP A2A and HRP C1A from the wildtype OCH1 strains (lanes 2 and 5; Additional file 2: Table S1).
Verification runs at 15°C
The results of the DoE revealed a low cultivation temperature to be highly beneficial for productivity and product purity. To check if both responses could be further increased, 2 additional fed-batch cultivations at 15°C, pH 5.0 and dO2 of 10% were done. The average qs MeOH adapt was 0.18 mmol∙g−1∙h−1, which was comparable to the results at 20°C and 25°C (Table 2). Apparently, significant changes in qs MeOH adapt of the recombinant Δoch1 strain only happened between 25°C and 30°C. Surprisingly, at 15°C productivity and product purity were only half compared to 20°C. We determined a qp of 1.50 [U∙g−1∙h−1], a STY of 66 [U∙L−1∙h−1] and a specific activity of 23.3 [U∙mg−1] at 15°C, whereas in DoE2 (20°C, pH 5.0, dO2 10%), qp of 3.46 [U∙g−1∙h−1], a STY of 153 [U∙L−1∙h−1] and a specific activity of 56.5 [U∙mg−1] were measured. Thus, we concluded that the highest values for productivity and product purity, which are the main target variables in production processes, might be obtained between 15°C and 20°C, a pH of 5.0 and a dO2 of 10%.
In the present study we developed a fed-batch bioprocess for a recombinant P. pastoris Δoch1 strain. High productivity and product purity were reached when this strain was cultivated at pH 5.0, dO2 of 10% and a temperature of 20°C. At 30°C methanol accumulated over time due to apparent changes in cell metabolism. Cell cluster formation, which was accompanied by cell lysis, was dependent on the C-source. To avoid intensive foam formation during glycerol batch and fed-batch, aeration and stirrer speed had to be reduced.
Currently, we are investigating cell cluster formation on other C-sources, like glucose and sorbitol, and analyze the temperature range between 15-20°C in more detail to find the true optimum for the recombinant Δoch1 strain. However, the present study already describes a good basis for bioprocess engineers working with glyco-engineered Δoch1 yeast strains.
The authors thank the Austrian Science Fund FWF (project: P24861-B19) for financial support and Prof. Anton Glieder (University of Technology, Graz, Austria) for providing the recombinant P. pastoris Δoch1 strain. Furthermore we want to thank Simona Capone for great assistance in the lab.
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