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Acta Biochim Biophys Sin 2006, 38: 803-811

doi:10.1111/j.1745-7270.2006.00231.x

Design and Expression of a Synthetic phyC Gene Encoding the Neutral Phytase in Pichia pastoris

 

Li-Kou ZOU1,3, Hong-Ning WANG2,1*, Xin PAN4, Tao XIE1, Qi WU5, Zi-Wen XIE1, and Wan-Rong ZHOU1

 

1 Laboratory of Veterinary and Biotechnology, Sichuan Agricultural University, Ya'an 625014, China;

2 Sichuan University Bioengineering Research Center for Animal Disease Prevention and Control, Chengdu 610065, China;

3 Faculty of Environment and Resource of Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan 611830, China;

4 College of Earth Science, Chengdu University of Technology, Chengdu 610059, China;

5 College of Life Science, Sichuan Agricultural University, Ya'an 625014, China

 

Received: July 4, 2006�������

Accepted: September 10, 2006

This work was supported by the grants from the National Key Technologies� ��R&D Program of China during the 10th Five-Year Plan Period (No. 2002BA514A-12) and Innovative Fund for Distinguished Young Scholars of Sichuan Agricultural University

*Corresponding author: Tel/Fax, 86-28-85471599, 86-835-2886083; E-mail, [email protected]

 

Abstract������� The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed and synthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering the protein sequence. The expression vector, pP9K-phyCs, was linearized and transformed in P. pastoris. The yield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium (BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale, respectively, improving over 90 folds compared with the wild-type isolate. Purified enzyme showed temperature optimum of 70 �C and pH optimum of 7.5. The enzyme activity retained 97% of the relative activity after incubation at 80 �C for 5 min. Because of the heavy glycosylation the expressed phytase had a molecular size of approximately 51 kDa. After deglycosylation by endoglycosylase H (EndoHf), the enzyme had an apparent molecular size of 42 kDa. Its property and thermostability was affected by the glycosylation.

 

Key words������� design; expression; synthetic phyC gene; neutral phytase; Pichia pastoris

 

Cereals, legumes, and oilseed crops are grown in over 90% of the world harvested area. These crops serve as a major source of nutrients for human and animals. An important constituent in these crops is phytic acid (myo-inositol hexaphosphate). The salt form, phytate, is the major storage form of phosphorus and accounts for more than 80% of the total phosphorus in cereals and legumes [1,2]. Simple-stomached animals such as swine and poultry, are incapable of using phytate phosphorus due to little phytase (myo-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8 and 3.1.3.26) activity in their gastrointestinal tracts [3,4], necessitating supplementation of the feed with inorganic phosphorus. Phytic acid also acts as an antinutritional agent in simple-stomached animals by chelating various metal ions needed by the animal, such as calcium, copper, and zinc [5,6].

Phytases catalyze the hydrolysis of phytate, thereby releasing inorganic phosphate [7,8]. Supplemental microbial� phytase in corn-soybean meal diets for swine and poultry effectively improves phytate phosphorus use by these animals, decreases the addition of phosphorus of feedstuffs and reduces their fecal phosphorus excretion pollution by up to 50% [9-11].

Many phytases, including fungal phytase from Aspergillus� spp. (phyA and phyB) and bacterial phytase from Escherichia coli (appA), have been cloned, characterized and expressed in the host of Pichiapastoris and Saccharomyces cerevisiae [12-17]. These phytases are histidine acid phosphatases, which exhibit an optimum pH from 2.5 to 5.5, and have a pH activity profile ideally suited for maximal activity in the digestive tract of either pigs or poultry. The phytases from Bacillus spp. (phyC) are beta-propeller phytases which exhibit an optimum pH from 6.0 to 9.0, suitable for neutral animal tracts such as trout and cyprinids, and is more thermostable [18-20]. The phytase has been expressed in E. coli and B. subtilis [19,20], but the intracellular expression and endotoxin in E. coli, the instability of plasmid and the protease from B. subtilis restrict� its production.

We have cloned the phyC gene from B. subtilis and expressed it in E. coli and P. pastoris [21,22]. Based on this, we synthesized the neutral phytase gene without altering� the protein sequence according to the P. pastoris codon usage bias. The recombinant phytase has been characterized� and compared with phytase expressed in E. coli and P. pastoris. Our aim was to study the property of the enzyme and to develop high production of phytase.

 

 

Materials and Methods

 

Strains, plasmids and medium

 

E. coli JM109 was used as a host for sub-cloning. P. pastoris GS115 was used as a host for expression, and GS115 albumin and b-gal as controls. Plasmid pMD18-T was used as a vector for cloning and sequencing, and plasmid pPIC9K was used for expression. E. coli was cultured in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar plate. When needed, ampicillin/kanamycin was added at a concentration of 100/80 mg/ml. The expression strains were screened in minimal dextrose (MD) medium [1.34% yeast nitrogen base (YNB), 0.00004% biotin, 2% dextrose] and minimal methanol (MM) medium (1.34% YNB, 0.00004% biotin, 0.5% methanol). Yeast was cultured in yeast extract peptone� dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose, 2% agar), buffered glycerol-complex� (BMGY) medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 0.00004% biotin, 1% glycerol) and induced in buffered methanol-complex (BMMY) medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 0.00004% biotin, 1% methanol).

Wheat Bran extract grown (WBEG) and wheat Bran extract induction (WBEI) media were our substitute media� for growing and induction. WBEG medium was prepared by dissolving 100 g of malt powder and 15 g of wheat bran in 600 ml of distilled water, incubated at 65 �C with constant stirring every 15 min until the starch hydrolyzed completely. The extract was filtered through a piece of cheesecloth, and the volume was adjusted to 600 ml with distilled water and was followed by autoclaving at 121 �C for 20 min.

WBEI medium was prepared by dissolving 100 g of malt powder and 15 g of wheat bran in 600 ml of distilled water, boiled for about 5 min, incubated at 90 �C with constant stirring every 15 min for about 3 h. The extract was filtered through a piece of cheesecloth, and the volume� was adjusted to 600 ml with distilled water and was followed� by autoclaving at 121 �C for 20 min. After cooling� to room temperature, methanol was added to a final concentration� of 1.5% (V/V).

 

Design and synthesis of the phyCs gene

 

The nucleic acid sequence of the synthetic gene was designed from the amino acid sequence of neutral phytase based on the P. pastoris preferred codons [23,24]. The DNAStar program was used to analyze the GC content, AT-rich stretches and the restriction enzyme sites of the resulting DNA sequence. In addition, the RNAstructure 4.2 program, Rensselaer bioinformatics system (http://www.bioinfo.rpi.edu), Apache (http://rna.tbi.univie.ac.at), GeneBee service (http://www.genebee.mus.su) and CodonW (http://bioweb.pasteur.fr/seqanal/interfaces/codonw.html) were used to analyze the mRNA structure, mRNA 5'- and 3'-untranslated region (UTR) and DG of the folding mRNA. The synthetic gene was designed with SnaBI and NotI restriction enzyme sites at the 5' and 3' terminal, respectively. The resulting sequence with the full length of 1074 bp (Fig. 1) was successfully synthesized by Shanghai Sangon Bioengineering Company (Shanghai, China) and cloned into pMD18-T named pMD-phyCs. The final synthetic gene, phyCs, was sequenced.

 

Construction of expression vector pP9K-phyCs

 

The DNA manipulations were carried out by using standard� procedures. All endonucleases were from TaKaRa (Dalian, China) unless stated otherwise.

The 1074-bp synthetic gene sequence fragment, phyCs, was prepared by digestion of pMD-phyCs with restriction endonucleases SnaBI and NotI, followed by agarose gel electrophoresis resolution and purification using the TaKaRa agarose gel DNA purification kit version 2.0. The expression vector was prepared by analogous procedures. The purified phyCs fragment was ligated to the purified SnaBI-NotI double-digested secretory expression vector pPIC9K with the correct orientation using TaKaRa DNA ligation kit version 2.1. E. coli strain JM109 was transformed with the ligation products. Bacterial transformants were selected for their ability to grow in LB medium in the presence of both 100 mg/ml ampicillin and 80 mg/ml kanamycin. The bacterial transformants were screened by colony-PCR using the primers phyCsA (5'-TACGTAAAGCAC�AAGTT�GTC�TGACC-3') and phyCsB (5'-GCTTACTTACC�AGAT�C�T��GTCAGTCAAC-3'). The recombinant expression vector, pP9K-phyCs, was prepared using a plasmid miniprep kit (W), identified by SnaBI-NotI double-digested restriction analysis and sequenced by TaKaRa.

 

Transformation and selection of P. pastoris expression strains

 

pP9K-phyCs used for transformation, was linearized by SacI. P. pastoris GS115 strains were made competent and transformed with SacI-linearized pP9K-phyCs by electroporation following the manufacturer's recommendations (Manual of multi-copy Pichia expression kit, Invitrogen, Carlsbad, USA). About 10 mg of linearized DNA pP9K-phyCs and 80 ml of competent GS115 cells were used for each transformation. Transformants were plated onto MD plate and incubated at 28 �C for 3-4 days. All plates were checked daily. Using a sterile toothpick, a single colony was selected and the His+ transformant was patched in a regular pattern on both MM plates and MD plates, ensuring to patch the MM plate first. For testing the effectiveness of expression conditions, GS115 albumin� and GS115 b-gal were grown as a control.

 

Expression of recombinant neutral phytase

 

Single colonies and recombinant P. pastoris expression strains were grown at 28 �C in 10 ml BMGY/WBEG medium� in 100-ml flasks for 16-20 h with vigorous shaking. Next, 3% (V/V) cells were inoculated into 100 ml BMGY/WBEG medium. Cells were grown at 28 �C for 17-20 h and shaken at 225-250 rpm until an A600 value of approximately 20 had been reached, then harvested by centrifugation� at 3000 g and 4 �C for 5 min. Supernatant was decanted and the cell pellet was washed with potassium� phosphate buffer (100 mM, pH 6.0). The pellet was resuspended� in 25 ml induction medium BMMY/WBEI in separate 250 ml flasks. Methanol (1.5%, V/V) was added to the flask every 12 h in order to induce phytase production� during the induction period. Supernatants were taken after induction for 24 h, 48 h, 72 h, and 96 h for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

 

Purification of recombinant neutral phytase

 

All purification steps were carried out at 4 �C unless otherwise stated.

Recombinant P. pastoris strains induced in WBEI were collected by centrifugation at 5000 g for 15 min. CaCl2 was added to a final concentration of 2 mM in the collected� supernatant. Initial purification was achieved by mixing the culture supernatant with three volumes of cold (-20 �C) enthanol. Constant stirring was carried out for 30 min, and the precipitation was continued overnight at -20 �C. The precipitate was collected by centrifugation at 1800 g for 20 min. The collected precipitate was washed once with cold ethanol and once with cold acetone. The drying was completed at room temperature. Dried precipitate� was dissolved in 50 mM Tris-HCl (2 mM CaCl2, 10% glycerol, pH 7.5). Then ammonium sulfate was added slowly with constant stirring to give 45% saturation. The solution was incubated overnight and centrifuged at 10,000 g for 15 min, and the supernatant was collected. The supernatant was dialyzed against 25 mM Tris-HCl (pH 8.1, 2 mM CaCl2) overnight and concentrated by PEG20000. Aliquots of enzyme preparation were stored at -20 �C.

Final purification of the enzyme preparation was performed by Chromatography Systems BioLogic LP and BioFracTM Fraction Collector (Bio-Rad, California, USA). Three milliliters of the thawed sample was passed through an Econo-PacR High Q Cartridge column equilibrated with buffer A (25 mM Tris-HCl, 2 mM CaCl2, pH 8.1). The bound protein was eluted with buffer B (25 mM Tris-HCl, 2 mM CaCl2, 0.5 M NaCl, pH 8.1) using the following gradient: 0-10 min, 100% A; 10-22 min linearly increased from 0% to 50% B; 22-27 min, 50%-100% B; and all steps were at a flow rate of 1.5 ml/min. The collected� protein fraction was stored at -20 �C for the next step analysis.

 

Recombinant phytase activity assay

 

The crude enzyme preparations and culture supernatant after induction were assayed for phytase activity. All chemicals were analytical reagents and purchased from Shanghai Sangon Bioengineering Company or TaKaRa. Phytase activity was assayed by measuring the rate of increase in inorganic orthophosphate (Pi) using the method as described by Mikio Shimizu [25], Kim et al. [26] and Bae et al. [27]. One unit of enzyme activity was defined as the amount of enzyme hydrolyzing 1 mmol of Pi per minute under assay conditions. The crude enzyme was diluted in suitable volume of 50 mM Tris-HCl buffer (2 mM CaCl2, 10% (v/v) glycerol, pH 7.5). All enzyme assays�� were run in duplicate. A reaction mixture containing 100 ml enzyme preparation and 400 ml 2 mM sodium phytate in 100 mM Tris-HCl buffer (2 mM CaCl2, pH 7.0) was incubated� at 37 �C for 30 min. The reaction was stopped by adding 500 ml of 15% (W/V) trichloroacetic acid (TCA). The released inorganic phosphate was measured by adding� 1 ml of a coloring reagent (freshly prepared by mixing four volumes of 1.5% (W/V) ammonium molybdate in a 5.5% (V/V) sulfuric-acid solution and one volume of a 2.7% (W/V) ferrous sulfate solution). The control was incubated� with TCA at 37 �C for 30 min. And the solution's absorbance at 700 nm was measured.

 

Property of recombinant neutral phytase

 

Enzyme activity assays were performed in defined buffers� for various pH and temperature tests. The optimal pH of the expressed phytase was determined (37 �C) using� buffers of 0.1 M glycin-HCl (pH 2.0 and 2.5), 0.1 M sodium� citrate (pH 3.5, 4.0, 5.0 and 5.5), 0.1 M Tris-maleate (pH 6.0 and 6.5), 0.1 M Tris-HCl (pH 7.0, 7.5, 8.0 and 9.0), and 0.1 M glycin-NaOH (pH 10.0, 11.0, 12.0 and 13.0). The purified enzyme was diluted in assay buffers with 2 mM CaCl2. The optimal temperature (pH 7.0) was determined at 16, 26, 37, 45, 50, 55, 60, 65, 70, 75, 80, 90 and 100 �C. For enzyme thermostability tests, the enzyme was incubated at seven different temperatures (37, 75, 80, 85, 90, 95 and 100 �C) in buffer of 50 mM Tris-HCl [2 mM CaCl2, 10% (V/V) glycerol, pH 7.5] for 5 min and 10 min and then cooled to room temperature before the enzyme assay.

 

Deglycosylation and SDS-PAGE analysis

 

Endoglycosylase H (EndoHf, New England Biolabs, Beijing, China) was used to deglycosylate the phytase. The reaction was carried out by incubating purified phytase with 100 U EndoHf in 0.5 M sodium citrate (pH 5.5) for 5 h at 37 �C and stopped by chilling at 4 �C. After the reaction, the mixture was subjected to SDS-PAGE. The molecular weight was determined by using 4.4%-12.5% gradient SDS-PAGE. The properties of the enzyme before and after� deglycosylation were compared.

 

 

Results

 

Design and synthesis of phyCs gene

 

The synthetic gene and expression vector inserted the synthetic gene encoding phytase are shown in Fig. 2. The synthesized 1074-bp phyCs gene showed 73.5% homo�logy� with the wild type phyC gene (Fig. 1). All codons in phyCs were designed to be preferential for methylotrophic yeast P. pastoris. The GC content was improved from 46.2% to 49.1% compared with the original phyC gene. AT-rich stretches were eliminated to avoid premauture termination.

The mRNA secondary structure around the AUG start codon was adjusted, so that AUG is relatively free of the secondary structure to ensure efficient translation of mRNA as predicated by the RNA fold software analysis (Fig. 3). The DG of the folding mRNA was increased to 235.7 kkcal/mol, which is higher than that of the original mRNA -244.0 kkcal/mol.

 

Activity of recombinant phytase produced from P. pastoris strains

 

From hundreds of transformants of P. pastoris, one colony that exhibited the highest phytase activity among the colonies examined was selected for shake-flask expression. As shown in Fig. 4, after 120 h of methanol induction, the yield of total extracellular phytase activity was 17.6 U/ml induced in BMMY and 18.5 U/ml in WBEI medium at the flask scale, respectively, increased over 90-fold compared to the wild-type isolate B. subtilis WHNB02 [28].

 

Purification and property of recombinant phytase

 

The expressed protein was obtained in gradual resolved� fractions, four of these demonstrated high phytase activity (18 to 21; Fig. 5). Therefore, the four fractions were taken together for enzymatic property analysis. Before and after deglycosylation, the enzymatic activity of the expressed phytase both showed temperature optima of 70 �C as shown in Fig. 6(A). They both showed high relative activity at pH between 6.0 and 9.0 and optima� pH of 7.5, as shown in Fig. 6(B). However, less phytase activity was detected at pH below 4.0 and above 11.0. The glycosylated and deglycosylated enzyme activity retains� 97% and 77% respectively, of the relative activity after incubation at 80 �C for 5 min. At 80 �C for 10 min the phytase lost 17% and 31% of its activity, as shown in Fig. 6(C).

 

SDS-PAGE analysis of culture supernatant and deglycosylated product

 

The molecular mass of the mature phyCs phytase was determined by SDS-PAGE. As shown in Fig. 7(A), phyCs phytase demonstrated an apparent molecular size of nearly 51 kDa in SDS-PAGE. Three major bands were found at 47.95 kDa, 50.23 kDa and 51.17 kDa after 24 h induction. The glycosylation of P. pastoris phytase appears to have increased with induction time. After deglycosylation by EndoHf, the enzyme had an apparent molecular size of 42 kDa [Fig. 7(B)]. Thus, the percentage of glycosylation was approximately 14.17%, 19.6% and 21.83%.

 

 

Discussion

 

Although phytase genes have been cloned from plants, animals, bacteria and fungi, and expressed in different hosts, many of these are acidic phytases belonging to the histidine acid phosphatases families and exhibit an optimum pH of below 7.0, which is only suitable for animal acid digestive tracts such as swine and poultry. The phytases from Bacillus spp. are beta-propeller phytases, which exhibit an optimum pH around 6.0 to 9.0 and is suitable for neutral animal tract such as trout and cyprinids and are more thermostable. However, low expression levels in wild type isolates restrict its production.

The synthesized 1074-bp phyCs gene showed 73.5% homology with the wild type phyC gene. In synthetic gene, 246 bases were changed compared with the original phyC gene. Almost 202 amino acid codons were substituted by the P. pastoris optimal codons including 32 amino acid codons whose relative synonymous codon usage (RSCU) was 0 in the original phyC gene [23]. To eliminate the AT-rich stretches, the GC content was improved from 46.2% to 49.1% in comparison to the original phyC gene. In addition� to codon usages, the free energy and secondary structure also affected the expression level. The more stable the mRNA second structure, the lower the mRNA free energy. Therefore the free energy of mRNA was increased compared with the original gene. Secondary structure in the mRNA has a negative effect on expression of the recombinant� protein, so online service was adopted to analyze� the 5'-UTR of the mRNA for secondary structure formation to ensure efficient translation of mRNA. An AUG sequence should be avoided in the loop of the 5'-UTR to ensure efficient translation of mRNA from the actual translation� initiation site. This was accomplished by re�designing the initial portion of the coding sequences with yeast preferential codons.

Kerovuo and Tynkkyen [29] reported that the phyC gene was expressed in Lactobacillus plantarum 755 using L. amylovorus a-amylase secretion signals, but the recombinant� phytase was secreted at a lower rate in comparison� to the native proteins of L. plantarum 755. Xiong et al. [30] synthesized an acidic phytase gene and the expressed phytase activity increased 14.5 folds compared� with the wild type phytase of the P. pastoris strain. But the recombinant phytase was a histidine acid phosphatase that had two pH optima (pH 2.5 and pH 5.5) and was less thermostable. In 2004 and 2005 [21,22], Wu et al. reported the expression of B. subtilis WHNB02 phyC gene in E. coli and P. pastoris. The recombinant phytase expressed in E. coli showed optimum temperature and pH of 50 �C and 7.0 and the residual activity with treatment at 80 �C for 5 min was about 50%. The phytase activity expressed in P. pastoris reached 2.4 U/ml whose activity was also low and not suitable for production. The purified enzyme revealed temperature and pH optima of 65 �C and 7.0 and the residual activity with treatment at 80 �C for 5 min was about 95%. In this study, we successfully expressed� the synthetic neutral phytase gene (phyCs) in P. pastoris, producing phytase at a level of 18.5 U/ml at shake-flask scale, 90-fold over the wild-type isolate B. subtilis WHNB02 [28]. The recombinant neutral phytase secreted showed maximal activity at 70 �C and pH 7.5, and the enzyme activity retained 97% of the relative activity� after incubation at 80 �C for 5 min. Compared with the phyC gene expressed in E. coli, the optimum temperature of purified phytase (phyCs) improved from 50 �C to 70 �C, and the phytase was more thermostable. Compared with the phyC gene expressed in P. pastoris, the optimum temperature of purified phytase (phyCs) improved from 65 �C to 70 �C. Although the enzyme revealed optimum pH of 7.5, it also showed high activity around pH 6.0 to 9.0, just as phyC gene expressed in E. coli and P. pastoris. It seemed that the increased production affected the property� of the recombinant phytase. This is the first report of a synthetic neutral phytase gene in methylotrophic yeast P. pastoris which has been shown to be suitable for high-level expression of various heterologous proteins either intracellulary or secretory. Although P. pastoris secretes low levels of endogenous proteins, it has the capacity to secrete grams per liter of foreign proteins [31]. In addition, protein expression levels in P. pastoris can be scaled up by 100-fold when the cells are grown in a fermenter [32,33]. Expression of recombinant proteins in P. pastoris is based on the use of the alcohol oxidase gene. So there is a huge potential for the fermentation of synthetic neutral phytase. Compared with the expensive BMMY medium, WEBG is an excellent medium for the growth of P. pastoris and WEBI is also suitable for protein expression.

The expressed protein was obtained by chromato�graphy systems without a sharp peak (Fig. 4) due to the glycosylation and the low flow eluted rate. But the four fractions (18 to 21) all showed high phytase activity. Therefore, the four were taken together for phytase activity� analysis. Three N-glycosylated potential motifs that occurred� within the Asn-Xaa-Ser/Thr sequon were found by the online analysis software NetNglyC 1.0, and a prediction� score larger than 0.5 was recommended (http://www.cbs.dtu.dk/services/NetNGlyc). Three major bands were found at 47.95 kDa, 50.23 kDa and 51.17 kDa after 24 h induction. After deglycosylation by EndoHf, the enzyme� had an apparent molecular weight of 42 kDa, the same as the phytase from the wild-type isolate [21,28]. The glycosylated and deglycosylated phytase both revealed a temperature optimum of 70 �C and a pH optimum of 7.5 [Fig. 5(A,B)]. As shown in Fig. 5(C), the glycosylated phytase resulted in the enhancement of thermostability. Before deglycosylation, the enzyme activity retained 97% of the relative activity after incubation at 80 �C for 5 min; enough for the pelleting of feedstuff. However, after deglycosylation the residual activity of phytase was 77%. The purified phytase showed streaking [Fig. 6(B)] in SDS-PAGE, but after deglycosylation the streaking was eliminated.

In conclusion, the phyCs gene was highly expressed as an active, thermostable and extracellular phytase in P. pastoris, and its enzyme property and thermostability was affected by glycosylation.

 

 

Acknowledgements

 

We are grateful to Prof. Wen-Jun LIU, Mr. Wei-Jie DENG, Ms. Wen SHAO and Dr. Yan LUO for critically reading and correcting the manuscript. Many thanks to Chang-Tai LU, Zhi-Xiang QIN, Cui ZHAO, Bei LI, Zhong-Ying LIAO and Feng LIU for their technical assistance.

 

 

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