Protein Expression and Purification 121 (2016) 112e117

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Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris Mert Karaoglan a, Fidan Erden Karaoglan a, Mehmet Inan a, b, * a b

Department of Food Engineering, Akdeniz University, 07058 Antalya, Turkey Food Safety and Agricultural Research Center, Akdeniz University, 07058 Antalya, Turkey

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 November 2015 Received in revised form 26 January 2016 Accepted 27 January 2016 Available online 2 February 2016

Recombinant protein production under the control of the PADH3 was compared with Pichia pastoris PAOX1 and PGAP. The single-copy-clones expressing Aspergillus niger xylanase (XylB) gene with the three different promoters were tested in shake flask and 5 L fed-batch fermentation processes. Recombinant protein production with PADH3, PAOX1 and PGAP were initiated by addition of ethanol, methanol and glucose, respectively in the culture medium. The fermentation process was carried out for 72 h at 30  C, pH 5 and 30% dissolved oxygen. Extracellular protein production yield for PADH3 (3725 U/mL) was higher than for PAOX1 (2095 U/mL) and PGAP (580 U/mL) at fermentor scale under the conditions tested. These results show that the PADH3 promoter is a promising tool for large scale production of recombinant proteins and can be an alternative to the PAOX1 and PGAP. © 2016 Elsevier Inc. All rights reserved.

Keywords: Pichia pastoris ADH3 promoter AOX1 promoter GAP promoter Xylanase

1. Introduction Pichia pastoris, a methylotrophic yeast, is a highly successful host system for recombinant protein production [1]. During the 1970s, P. pastoris was evaluated for production of single-cell protein because of its ability to achieve high cell density in fermentor cultures. However, after the 1973 oil crisis, increases in the cost of methanol made single-cell protein production uneconomical [2]. In the following decade, P. pastoris was developed as a host organism for heterologous protein expression along with the combination of the fermentation methods developed for the single-cell protein production process under the regulation of the alcohol oxidase promoter (PAOX1) [3]. This host system offers many advantages including the ability to reach very high cell densities using minimal media and perform higher eukaryotic protein modifications, such as glycosylation and disulphide bond formation providing proper folding [4]. P. pastoris secretes very low levels of its own proteins, therefore it is easier to recover the recombinant proteins produced extracellularly by filtration or centrifugation. In recent years, there have been many studies on the optimization of the P. pastoris system to improve productivity of

* Corresponding author. Akdeniz University, Department of Food Engineering, Dumlupinar Bulvari Campus, 07058 Antalya, Turkey. E-mail address: [email protected] (M. Inan). 1046-5928/© 2016 Elsevier Inc. All rights reserved.

recombinant proteins [5e8]. Increasing transcription efficiency using strong promoters and optimization of fermentation conditions are among the strategies employed [9]. Although several promoters are available, PAOX1 and PGAP are the most frequently used promoters in recombinant protein production with P. pastoris, the first being an inducible and the latter a constitutive promoter. However, for the expression of toxic proteins, inducible promoters are preferred [2,4]. During recombinant protein production via inducible promoters, accumulation of biomass occurs prior to protein expression and the protein production phase does not affect the growth phase [2]. Another strong promoter of the methanol utilization pathway is FLD1, which controls expression of formaldehyde dehydrogenase enzyme and is independently induced either by methanol as a carbon source or methylamine as a nitrogen source [10]. Other inducible promoters are PPEX8, a peroxisome membrane protein which is induced by both methanol and oleic acid, and PICL1, an ethanol inducible isocitrate lyase promoter. However, PPEX8 and PICL1 are not widely used due to low expression levels [11]. ADH genes of P. pastoris have recently been characterized by our research group [12]. The ADH3 (XM_002491337) is involved only in utilization of ethanol in P. pastoris and not in its production. The Dadh3 mutant strain has lost ability to grow on minimal ethanol media. However, it is able to produce ethanol in minimal glucose media. The ADH (FN392323) gene does not play any role in ethanol

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metabolism. The gene responsible for ethanol production in P. pastoris remains to be elucidated. Although a patent has been approved on the use of the ADH1 (named as ADH3 in NCBI database and also in this study) promoter of P. pastoris [13], there have been no studies comparing the strength of this promoter with other widely used Pichia promoters. To avoid confusion, it should be noted that the ADH1 gene that is stated in the patent is referred to as ADH3 in the NCBI database and also herein. In this study, the ADH3 promoter of P. pastoris was utilized and evaluated for the production of Aspergillus niger xylanase (XylB) as a reporter gene. Recombinant protein production under the control of PADH3 was compared to P. pastoris PAOX1 and PGAP in both shakeflask and large scale fermentor conditions. Promoter strengths were measured by quantifying the xylanase activity.


resulting plasmid was named as pADH3ZaA-XylB. Diagram of the plasmid constructs are shown in Fig. 1. 2.4. Transformation of P. pastoris cells Expression plasmids including PADH3, PAOX1 and PGAP were linearized with ApaI, MssI and AvrII restriction enzymes, respectively and transformed into electrocompetent P. pastoris X33 cells using Eporator electroporator (Eppendorf, Germany) [14]. Transformed cells were spread on YPD plates (supplemented 100 mg/L zeocin) and incubated at 30  C. After approximately two days colonies were selected and streaked on YPD plates for single colony isolation. Single colonies for each different clone were then used to inoculate 3 mL of YPD broth for genomic DNA isolation followed by Southern blot analysis.

2. Materials and methods 2.5. Southern blot analysis 2.1. Chemicals, enzymes and reagents Cultivation media ingredients for Escherichia coli XL1-Blueand P. pastoris strains were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). The primers used in this study were synthesized by Metabion International AG (Martinsried, Germany). Restriction enzymes were purchased from Fermentas (Fermentas, MD, USA) and Hot-start KOD DNA polymerase for PCR experiments was obtained from Novagen (Darmstadt, Germany). All chemicals were purchased from SigmaeAldrich (MO, USA). The DIG-High Prime DNA Labeling and Detection Starter Kit II for Southern blot analysis and DNA ligation and Dephosphorylation Kit were purchased from Roche Applied Sciences (Indianapolis, IN, USA). 2.2. Strains and cultivation medium E. coli XL1-Blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F0 proAB lacIq ZDM15 Tn10 (Tetr)] were used for subcloning and plasmid reproduction. P. pastoris X33 (WT) strain was used as host organism for recombinant protein production. E. coli cells which containing plasmids were grown in LB Lennox medium (1% tryptone, 0.5% yeast extract and 0.5% sodium chloride) supplemented with 25 mg/mL zeocin. P. pastoris cells were grown in YPD broth medium containing 1% yeast extract, 2% peptone and 2% dextrose, whereas YPD agar plates were supplemented with 1.5% agar. Zeocin (100 mg/L) was added to YPD agar plates to select transformants.

Expression clone genomic DNA was extracted from the freshly grown 3 mL YPD cultures using the MasterPure™ Yeast DNA Purification Kit (Epicenter, Madison, WI, USA). Genomic DNA concentrations were quantified using the Qubit® dsDNA BR Assay Kit, (Invitrogen). One microgram genomic DNA of each clone was digested with ClaI (for ADH3 and GAP clones) and EcoRV (for AOX1 clone) restriction enzymes. Digested genomic DNA was separated in 0.8% TAE agarose gel by electrophoresis. The DNA was transferred to a positively charged nylon membrane (Roche, Germany). DIG labeled specific DNA fragments for each promoter region were used as probes to identify the transformants containing a single copy expression plasmid. DIG-High Prime DNA Labeling and Detection Starter kit II was employed for scanning the chemiluminescence signal on X-ray films. The clones containing single copy expression plasmids were selected. 2.6. Shake-flask experiments The single copy plasmid containing clones were inoculated into 3 mL of YPD broth and grown overnight at 30  C and 225 rpm. The

2.3. Reporter gene and plasmid constructions A. niger xylanase gene (XylB) was used as a reporter gene for comparison of recombinant protein production with the different P. pastoris promoters. The XylB gene, excluding its signal peptide, was synthesized based on P. pastoris codon usage by Genescript (NJ, USA). The codon-optimized XylB gene coding for endo-b-D-1,4xylanase was cloned in pUC57. Subsequently, the gene was cloned into pPICZaA (including PpAOX1 promoter) and pGAPZaA (including PpGAP promoter) expression plasmids at the XhoI and XbaI restriction sites. The newly constructed plasmids were named as pPICZaA-XylB and pGAPZaA-XylB. Expression plasmid containing PpADH3 promoter region was created from pPICZaA-XylB. ThePADH3 was amplified by PCR from the P. pastoris X33 genomic DNA using the ADH3pBamHI-F 50 -AAAGGATCCTGACGGTACTAGAGGACTCT-30 and ADH3pAsuII-R 50 -TTTCGAAAGTAAATAAGATAAAAGCTAGTAG30 primers. The 1200 bp PCR product was digested with BamHI and AsuII restriction enzymes. The PAOX1 was excised from pPICZaAXylB plasmid using BglII and AsuII restriction sites and the residue plasmid was ligated to the digested PCR product (PADH3). The

Fig. 1. Diagram of plasmid constructs of XylB gene with different promoter (PADH3, PAOX1 and PGAP). a-MF; Alpha-mating secretion sequences, XlyB; Aspergillus niger Xylanase B gene, AOX1 TT; AOX1 transcription termination, Zeocin; Sh ble selection marker.


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cells were used to inoculate 50 mL of BMGY (contained 2% glycerol, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids, 4  105% biotin and 100 mM phosphate buffer, pH 6) with starting OD600nm of 0.1 in baffled 250 mL flasks. After 18 h, the cells reached approximately 18e20 OD600nm. The cultures were centrifuged (2000  g) at room temperature and resuspended in 50 mL of BMEY, BMMY and BMDY (BMGY containing either 1% ethanol, 1% methanol or 1% glucose, respectively, instead of glycerol) for PADH3, PAOX1 and PGAP, respectively. The cells were induced for 72 h. Ethanol, methanol and glucose were added for PADH3, PAOX1 and PGAP as inducers to a final concentration of 1% at every 12 h, respectively. One milliliter of cell culture was separated by centrifugation at 6000  g and supernatants were kept at 4  C for total protein and xylanase activity assays. The shake-flask experiments were performed in triplicate. Optical densities of cell culture throughout fermentation were determined spectrophotometrically at 600 nm wavelength. 2.7. Fed-batch cultivation in 5 L bioreactors Fermentation was carried out as a three-step fed-batch process. The cells were inoculated into 3 mL of YPD broth and grown overnight at 30  C and 225 rpm. The cells were used to inoculate 100 mL of BMGY. One hundred mL of the BMGY over-night culture (at approximately 10 OD) was used as inoculum. The batch phase was cultivated in 2 L Basal Salt Medium (4% glycerol, 26.7 mL 85% H3PO4, 0.93 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4$7H2O, 4.13 g KOH and 2 mL 5% antifoam) containing 4.35 mL/L PTM1 (2 g/L CuSO4.5H2O, 7 g/L ZnCl2, 0.08 g/L NaI, 22.0 g/L FeSO4.7H2O, 3.0 g/L MnSO4$H2O, 0.2 g/L biotin, 0.2 g/L Na2MoO4.2H2O, 0.02 g/L boric acid, 0.5 g/L CoCl2, 2 mL H2SO4) and continued for about 16 h. Glycerol fed-batch was initiated with 50% glycerol (w/v) at 16 mL/L/ h. After 2 h 2 mL/L ethanol and methanol was added for ADH3 and AOX1 promoters, respectively, and feed rate was linearly reduced to 0 mL/L in 3 h. The induction phase was started with ethanol for the PADH3, methanol for the PAOX1 and glucose for the PGAP, and continued for 72 h. Carbon sources used in the induction phase contained 12 mL/L of PTM1. The feed rate was gradually increased from 1.5 mL/L/h to 9.0 mL/L/h for PADH3 and PAOX1 fermentations during the first 48 h of fermentation and continued to 72 h. The feed rate applied for glucose was kept at twice the rates applied for methanol and ethanol due to the fact that methanol and ethanol feeds were pure whereas glucose was supplied as a 50% w/v aqueous solution. The temperature and pH during fermentation were maintained at 30  C and 5.0, respectively. The agitation speed was set to 1000 rpm with airflow being 1.5 vvm. Dissolved oxygen (DO) level was controlled at 30% saturation by supplying pure oxygen as necessary. The cell density was quantified as grams of wet cell weight (WCW) per liter. The cells in 1 mL samples were separated by centrifugation. Supernatants were collected at the same time intervals and analyzed by SDS-PAGE. The supernatants were analyzed for total protein and xylanase enzyme activity. The fed-batch fermentation experiments were run in triplicate for each promoter clone. 2.8. Xylanase activity assay The xylanase activity of supernatants was determined by measuring liberated reducing groups by the 3,5-dinitrosalicylic acid (DNS) method [15]. The substrate solution contained 1.0% beechwood xylan in 50 mM sodium citrate buffer at pH 5. The supernatants were diluted appropriately in the citrate buffer if required. An aliquot of 100 mL of enzyme solution was added to 900 mL 1% substrate solution. After incubation at 50  C for 5 min, the

reaction was stopped by adding 900 mL of DNS solution to 100 mL of reaction mixture. The mixture was boiled for 5 min and cooled on ice. The absorbance was measured at 540 nm and a standard curve was created with xylose (1e10 mmole). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1.0 mmole of reducing sugars per minute measured as xylose equivalents from xylan at pH 5.0 at 50  C. 2.9. SDS-PAGE analysis The collected supernatant samples were treated with SDS-PAGE loading buffer and incubated at 70  C for 10 min. After incubation, 15 mL of samples were separated by electrophoresis in 10% (w/v) SDSePAGE gels using 1  TGS (Tris-Glycine-SDS) buffer. Protein bands were visualized by Coomassie Blue (G-250) staining. The gels were scanned with Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA). The protein molecular weight markers used was PageRuler unstained protein ladder (Thermo Fisher Scientific, MA, USA). 3. Results and discussion 3.1. Selection of single-copy clones pADH3ZaA-XylB, pPICZaA-XylB and pGAPZaA-XylB expression plasmids were linearized by ApaI, MssI and AvrII digestion in AOX1, GAP and ADH3 promoter regions, respectively and transformed into P. pastoris X33 competent cells by electroporation using Eppendorf Eporator System (Eppendorf AG, Germany). Transformants were selected on YPD supplemented with100 mg/mL zeocin and inoculated into YPD broth. The cultures were incubated overnight and genomic DNA of the transformants was isolated. Genomic DNA samples were digested with ClaI for the PADH3 and PGAP clones and EcoRV for thePAOX1 clones. The digested samples were subjected to Southern blot analysis. Theoretical band sizes were 3889 bp, 3527 bp and 1552 bp in control clones; 8429 bp, 1850 and 5774 bp, and 5192 bp in single copy clones for the PADH3, PAOX1 and PGAP, respectively (Fig. 2). All the selected clones contained a single copy of the expression cassettes, which was necessary to compare the promoter strength [16]. Recombinant protein production under the regulation of the PADH3, PAOX1 and PGAP promoters were compared in the P. pastoris X33 strain. Ethanol, methanol and glucose were used as carbon sources for the PADH3, PAOX1 and PGAP regulated protein expression, respectively. 3.2. Protein production in shake-flask culture The selected single-copy plasmid containing clones were tested in shake flask culture conditions. The single colonies from YPD plates were inoculated into YPD broth and incubated over-night at 30  C. For growth phase, the over-night cultures were inoculated into 50 mL of BMGY medium to start with OD600nm of 0.1 and grown for 18 h (~18e20 OD600nm). The cells were harvested at 3000  g and resuspended in 50 mL of BMEY, BMMY and BMDY for PADH3, PAOX1 and PGAP regulated protein expression, respectively. Ethanol, methanol and glucose were added to final concentration of 1% every 12 h and continued for 72 h. The samples were taken to follow cell growth and enzyme production levels. Xylanase activity measured in 72-h samples was ~228 U/mL, 416 U/mL and ~219 U/mL for the PADH3, PAOX1 and PGAP induced clones, respectively (Fig. 3b). Results showed that the cells grown in ethanol media (~117 OD600) and glucose media (~107 OD600) reached higher cell densities than that of methanol media (~70 OD600) (Fig. 3a). However, enzyme production under the regulation

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Fig. 2. Southern blot analysis for gene copy number. a. ADH3 promoter; b. AOX1 promoter; c. GAP promoter. Genomic DNA of ADH3 and GAP promoter clones were digested with ClaI. Genomic DNA of AOX1 promoter clones were digested with EcoRV restriction enzyme. M, DIG marker; C, control strain; 1e5, selected clones.

Fig. 3. Results of time-course samples of shake-flask experiments with ADH3, AOX1 and GAP promoter. a. Optical density (600 nm), b. Xylanase activity (U/mL). Ethanol, methanol and glucose were used as carbon source in complex media for ADH3, AOX1 and GAP promoter, respectively. Close circles: ADH3 promoter; open circles: AOX1 promoter and close triangles; GAP promoter. The error bars represent standard deviation of three runs.

of the PADH3 was approximately two-fold lower compared to that of PAOX1. PADH3 and PGAP regulated enzyme production levels were almost the same under shake flask conditions. 3.3. Protein production in 5 L bioreactor scale Expression of xylanase under the regulation of the PADH3, PAOX1 and PGAP was also compared at fermentor scale using a three-step fed-batch fermentation scheme. The first step included a glycerol batch phase to generate cell mass, followed by the second step of a glycerol fed-bath phase, where glycerol was added at growth limiting rate. These first two steps were identical for all promoters. The third step was an induction phase which differed depending on

the promoter used for protein expression. The induction phase was maintained for 72 h for all promoters. The enzyme activities measured in the final supernatant samples were ~3725 U/mL, ~2095 U/mL and ~580 U/mL for PADH3, PAOX1 and PGAP cultures, respectively (Fig. 4c). Interestingly, contrary to shake flask results, the enzyme production at fermentor scale with the PADH3 was approximately 1.8-fold higher than that of the PAOX1. Biomass accumulation of the PADH3 cultures (408 g/L) was also higher than that of PAOX1 (287 g/L) cultures. However, the wet cell weight of PADH3 and PGAP (392 g/L) was similar while enzyme production levels highly differed (Fig. 4a). Total protein contents of all three cultures were proportional to their wet cell weights (Fig. 4b). Based on the enzyme activity, specific activities (U/mgprotein) for


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Fig. 4. Results of time-course samples of fermentor-scale experiments with ADH3, AOX1 and GAP promoter. a. Wet cell weight (g/L), b. Amount of total protein (mg/L) c. Xylanase activity (U/mL). Ethanol, methanol and glucose were used as carbon source in BSM (Basal salt media) for ADH3, AOX1 and GAP promoter, respectively. Close circles: ADH3 promoter; open circles: AOX1 promoter and close triangles; GAP promoter. The error bars represent standard deviation of three runs.

PADH3, PAOX1 and PGAP cultures were 8.85 U/mgprotein, 9.07 U/ mgprotein and 1.55 U/mgprotein, respectively. Specific productivities for PADH3, PAOX1 and PGAP cultures were calculated as 0.126 U/gWCW/ h, 0.101 U/gWCW/h and 0.021 U/gWCW/h, respectively. Supernatant samples were analyzed by SDS-PAGE for each promoter culture and Fig. 5 clearly shows that xylanase band intensities increased by time for each promoter.

Results achieved herein demonstrate that relative xylanase activities differed between the flask and fermentor cultures. Moreover, the highest enzyme production was obtained with PAOX1 cultures at shake-flask level while PADH3 cultures were more productive at fermentor scale. Although few recombinant proteins have been expressed well in P. pastoris under shake-flask conditions, the expression levels in

Fig. 5. SDS-PAGE analysis of xylanase expression under ADH3, AOX1 and GAP promoter in fermentor-scale experiments. a. ADH3 promoter; b. AOX1 promoter; c. GAP promoter. M, protein marker; 0e72, sampling times.

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shake flask have been generally shown to be lower than that of fermentor cultures [17]. Our results also showed that the protein production under the regulation of the PADH3 at fermentor conditions was ~17-fold higher than that of shake flask conditions, while PAOX1 showed a ~5-fold increase and PGAP showed a ~2.5-fold increase. This is the first study which utilizes P. pastoris PADH3 to express recombinant protein with comparison to the commonly used promoters, PAOX1 and PGAP. Although there is a patent on the use of PADH1 of P. pastoris, the performance of this promoter was not known since absolute expression values were not previously reported [13]. 4. Conclusions Recombinant protein production under the regulation of the PADH3 promoter in P. pastoris was compared with the commonly used promoters, PAOX1 and PGAP. Promoter strengths were measured indirectly by xylanase activity. Applying standard fermentation conditions resulted in higher expression rates for the PADH3 than for the PAOX1 and PGAP clones, however future studies on optimization of fermentation parameters such as feed profile, agitation speed and oxygen levels could be effective in further increasing productivity and yield.

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Acknowledgments [13]

This project was supported by the grant 111T905 from the Scientific and Research Council of Turkey (TUBITAK). The authors wish to thank Lynne A. Becker and Barcin Karakas for critical reading of this manuscript. References [1] D.R. Higgins, J.M. Cregg, Introduction to Pichia pastoris, in: D.R. Higgins, J.M. Cregg (Eds.), Pichia Protocols, Methods in Molecular Biology vol. 103, 1998, pp. 1e15. [2] M. Ahmad, M. Hirz, H. Pichler, H. Schwab, Protein expression in P. pastoris:






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Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris.

Recombinant protein production under the control of the PADH3 was compared with Pichia pastoris PAOX1 and PGAP. The single-copy-clones expressing Aspe...
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