Cloning and expression of recombinant purine nucleoside phosphorylase in the methylotrophic yeast Pichia pastoris

Nucleoside phosphorylases (NPs) are widely applied for the synthesis of modified nucleosides, which have used as potential anticancer, antiviral, and antibacterial drugs [1-4]. Based on their substrate spectrum, NPs are classified into purine nucleoside phosphorylases (PNP, EC 2.4.2.1) and pyrimidine nucleoside phosphorylases (PyNP, EC 2.4.2.2). Both catalyze the reversible phosphorolysis (transglycosylation) of nucleosides to nucleobases and α-D-pentofuranose-1-phosphates [5-6]. Enzymatic modification of nucleosides promises increased regioselectivity and exquisite control of the transglycosylation and is a better alternative to the commonly used chemical synthesis [6-7]. 
In recent years, E. coli purine nucleoside phosphorylases have attracted increasing interest as a biocatalyst for the synthesis of purine-type nucleosides [8-10]. PNP catalyzes the reversable hydrolysis of the glycosidic bond of pentofuranoses in the presence of inorganic phosphate [10-11].
In transglycosylation reactions, bacterial nucleoside phosphorylases are used either as part of cell lysates (raw) or as recombinant enzymes obtained by genetic engineering. In this regard, obtaining new strains of microorganisms producing recombinant enzymes with a high yield is relevant. Pichia pastoris is one of the advanced expression systems used in biotechnology, characterized by a high yield of recombinant proteins compared to other expression systems and the absence of endotoxins/pyrogens. Therefore, the aim of this study was to clone and express recombinant purine nucleoside phosphorylase in Pichia pastoris as a producer on an industrial scale [12-14].

Chemicals
Restriction enzymes (FastDigest EcoRI, FastDigest XbaI and FastDigest SacI) and T4 DNA ligase used in DNA clonning were purchased from Intivrogen (Thermo Scientific, USA). Inosine (Ino), hypoxanthine (6-hydroxypurine), D-ribose, and purine nucleoside phosphorylase (10 U/mg) were purchased from Sigma (Germany). All other chemicals were purchased from Sigma in the highest purity.

Escherichia coli genomic DNA extraction
Bacterial genomic DNA was isolated from pure cultures of Escherichia coli strain RKMUz – 221 (the strain was obtained from the collection of industrial microorganisms of the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan) by using the previously published protocol [15]. Bacterial cells were grown in liquid nutrient medium (LB Broth Miller, Invitrogen) at 37°C for 12 h. Isolation was carried out in a 1.5 ml tube containing 1 ml of the overnight E. coli culture. Centrifuged at max speed for 1min to pellet the cells. Remove as much of the supernatant as we can without disturbing the cell pellet. The cell pellet resuspended in 600 µl lysis buffer (9.34 ml TE buffer pH 8, 600 µl of 10% SDS, 60 µl of proteinase K (20 mg/ml)) and then vortexed. The mixture was incubated 1 h at 37°C. After that, lysate treated an equal volume 2 times using phenol/chloroform without vertex and 1 time chloroform. After centrifuged at max speed for 5 min the upper aqueous phase was carefully transferred to a new tube. For precipitating the DNA, it was added 3 volumes of cold 96% ethanol and mixed gently. Finally, 1μl of samples were used directly for PCR in 20 µl reactions.

Preparation of transfer vector and cDNA (gene) for ligation
1 μg of the transfer vector pPICZαA was sequentially treated using FastDigest EcoRI and FastDigest XbaI restrictases. Linear DNA 3536 bp was isolated after 0.7% agarose gel electrophoresis using PureLink^TM Quick Gel Extraction Kit [16].
Purine nucleoside phosphorylase cDNA (deoD gene, insert), size of 720 bp was amplified by PCR using as a template genomic DNA isolated from cells of Escherichia coli strain RKMUz–221 using the following primers: 
Forward – 5’-ccaagaattcatggctaccccacacattaatgc-3’, 
Reverse – 5’-cttgtctagatattactctttatcgcccagcagaac-3’
After electrophoresis in 1% agarose gel the amplificon (insert) was purified using PureLink^TM Quick Gel Extraction Kit [16] and treated with the same enzymes which used for the transfer vector.

Ligation of the vector with cDNA (purine nucleoside phosphorylase gene)
Ligation of the pPICZαA vector with the purine nucleoside phosphorylase gene (E. coli PNP) was carried out in a volume of 10 μl in a molar ratio of 1:3, accordingly, using recombinant the phage T4 DNA ligase (Thermo Scientific^TM) [17-18]. 

Transformation of E. coli TOP10 with a ligation mixture
After precipitation with the ligation mixture, the pellet (recombinant DNA) was dissolved in 10 µl of de-ionized water. Dissolved DNA was transformed into electrocompetent plasmid-free strain E. coli TOP10 cells (Invitrogen) according to the method [19].

Identification of recombinant clones containing pPICZαA- PNP
The molecular weight of plasmid DNA isolated from clones obtained after transformation with a ligation mixture was determined using 0.8% agarose gel electrophoresis. Clones containing DNA with a length of 4256 bp (deoD gene + pPICZαA transfer vector fragment) were investigated by PCR analysis. PCR amplicons were analyzed by electrophoresis in 1% agarose gel [20].

Sequencing of cloned cDNA
Recombinant cDNAs were sequenced as described [21] in DNA Sequencing with the SeqStudio EDDU protocols using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). Three identical cycle sequencing reactions (containing 50 ng of the template DNA); 5 pmol of the gene-specific primers; 4 µL of BigDye Terminator v3.1 Ready reaction Mix; and deionized water to a final vol of 10 µL were subjected to an initial denaturation step of 96°C for 2 min, followed by 30 cycles of 96°C for 20 s, 60°C for 10 s, 65°C for 4 min in a QuantStudio PCR System (Applied Biosystems, Thermo Fisher Scientific, USA). Sequencing purification was performed using ZR DNA Sequencing Clean-Up Kit as described previously [22]. Automated sequencing traces were obtained in SeqStudio Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, USA).

Accession numbers for target gene nucleotide sequences
The nucleotide sequences of the PNP (deoD gene) presented in this study have been entered to the National Centre for Biotechnology Information (NCBI) as GenBank accession number OQ133467.1.

Transformation of the pPICZαA-PNP vector into Pichia pastoris GS115
The recombinant pPICZαA-PNP vector was linearized with FastDigest SacI and the mixture was precipitated with absolute ethanol in the presence of 3M Sodium acetate pH 5.2 to purify and concentrate the DNA. The purified linear DNA vector was transformed into Pichia pastoris GS115 yeast using an Electroporator 2510 (Eppendorf, USA). Then, the yeast transformants was grown in YPD selection medium containing the antibiotic Zeocin™ (100 μg/mL).

Expression, purification, and SDS-PAGE identification of recombinant EcPNP
To induce EcPNP expression, Pichia pastoris GS115 transformants were inoculated in BMGY medium at 30°C with vigorous shaking (220 rpm), and then transferred to BMMY and incubated at 30°C with vigorous shaking (220 rpm) up to 96 h. Methanol (0.5-1%, v/v) was supplied to the culture medium once a day [23].
Extraction of the enzyme was carried out by destroying the yeast cells using a benchtop two-stage high pressure homogenizer (APV - 2000, SPX Flow, USA). The device can treat liquid samples at pressures up to 2000 bar. The two-stage (two times 1200 bar) was used after spin pellets for 1 min at 14.000 x g at 4°C and resuspend pellets in breaking buffer (Tris-Cl (1 M, pH 8.0) – 10mM, EDTA (0.5 M, pH 8.0) – 1mM, Triton X-100 - 2% (v/v), SDS (10%) - 1% (w/v), NaCl (5 M) - 100 mM, PMSF - 2mM) for the treatment of yeast suspensions. The suspension temperature at the exit of the homogenizer did not exceed 40°C. Treated samples were cooled to 15°C immediately at the outlet of the device using a water-ice bath. After that, the yeast culture liquid medium was added to the homogenate.
Homogenate was precipitated using gradient precipitation with 25%-65% ammonium sulphate to obtain primary purified purine nucleoside phosphorylase. The prepared solution in 65% ammonium sulphate was incubated for 4 h at 40C. The fraction containing the enzyme was separated by centrifugation at 14.000 x g at 4°C for 30 min. The collected pellets (enzyme fraction) were dissolved in BS (150 mM NaCl, 5.2 mM Na2HPO4, 1.7mM KH2PO4), pH 7.4.
The molecular weight was determined with 10% PAGE electrophoresis in the presence of Na dodecyl sulphate (SDS) according to the Laemmle method [24]. The protein concentration was determined by the Lowry method, using bovine serum albumin (BSA) as the standard [25].

Determination of EcPNP expression 
The expression of EcPNP in Pichia pastoris yeast cells were determined by Purine Nucleoside Phosphorylase Activity Fluorometric Assay Kit (ab204706, Abcam PLC, UK) [26]. 

Enzymatic hydrolysis of inosine with obtained EcPNP
Reaction mixture contained 3.5 mM KH2PO4, pH 8.0, 10 mM 9-beta-D-ribofuranosylhypoxanthine (99%, Sigma-Aldrich, Cat. I4125-100G) and 5 µl EcPNP fraction (total protein cons. 15.8 mg/ml). The reaction mixtures were incubated at 50°C up to 5 h. Substrate and product quantities were determined using HPLC.

HPLC detection
The quantitative determination of the products was performed by HPLC analysis using a Shim-pack GIST C18 5 µm, 4.6 × 150 mm column (Shimadzu) under the following conditions: flow gradient (100-90% 10 mM KH2PO4 in H2O and 0-10% MeCN) for 30 min, flow rate 0.5 mL/min, detection PDA at 254 nm. The retention times for the substrates and products are: 9-beta-D-ribofuranosylhypoxanthine (Inosine) ~ 20.41 min; 6-hydroxypurine (hypoxanthine) ~ 16.31 min.

Designing, cloning and transformation of the recombinant pPICZαA-PNP 
Plasmid DNA pPICZαA-PNP (4256 bp) was created on the basis of the transfer vector pPICZαA (3593 bp), which was designed for intracellular expression of proteins in the yeast Pichia pastoris. Figure 1 shows the genetic maps of the cloned plasmid DNA pPICZαA-PNP.
The obtained clones were screened by PCR. Figure 2 shows the result of PCR analysis of recombinant clones. According to the results of PCR, the selected clones contain DNA fragments (gene) with a molecular weight of 720 bp (Figure 2). Thus, PCR indicated the presence of an insert (the PNP-deoD gene) in the recombinant plasmid.
Figure 3 shows a comparative restriction analysis of the recombinant plasmid pPICZαA-PNP and the original pPICZαA vector at a single restriction site of FastDigest SacI. After electrophoresis in 0.8% agarose gel, linear DNA was detected, the mass of which corresponds to theoretical calculations according to the physical maps of plasmids. At the same time, the difference between the sizes of the two plasmids was evident.
Linearized vector plasmid with FastDigest SacI was transformed into Pichia pastoris GS115 yeast using a Electroporator 2510 and the yeast transformants was grown in YPD selection medium containing the antibiotic Zeocin™ (100 μg/mL).

cDNA (gene) sequencing 
Nucleotide sequencing by Sanger was used to verify cloned E. coli strain RKMUz - 221 PNPase. The results showed that the purine nucleoside phosphorylase gene has 98.47% sequence similarity with Escherichia coli strain BM28 (GenBank accession no. CP10280.1). There were eight different mutations discovered in PNP of E. coli strain RKMUz – 221 and all of which were single substitutions (T>G, G>A, G>A, C>T, T>G, T>C, C>A and C>T) (Figure 4).

Expression and purification of EcPNP
Fluorometric assay (ab204706, Abcam PLC, UK) was performed to study the expression level of the recombinant enzyme. The highest level of PNP expression (activity) was enriched at the 72 h of cultivation when adding 0.5% - 1.0% methanol to the medium every 24 h. The yeasts were homogenized as described in material and methods. Then homogenate was separated by centrifuging at 14.000 g for 30 min after gradient precipitation with 25%-65% ammonium sulphate. The fraction contains 15.8 mg/ml total proteins. The fraction containing PNP was analyzed with 10% SDS-PAGE electrophoresis by staining with Coomassie G-250 (Figure 5). Figure 5 shows the ≈ 28 kDa band which corresponds to the recombinant enzyme based on its nucleotide sequence. 

The current study described the designing and cloning of the recombinant plasmid pPICZαA-PNP for the expression of the EcPNP of conditionally pathogenic E. coli strain RKMUz-221 in Pichia pastoris yeast and enzymatic activity of recombinant EcPNP. Besides, the study showed the EcPNP in the yeast for the first time. 
Nowadays, Pichia pastoris is most effectively used in the large-scale production of heterologous recombinant proteins [12]. The successful creation of the recombinant pPICZαA-PNP plasmid is an important stage in the expression of the target protein in Pichia pastoris yeast. The obtained clones of pPICZαA-PNP after transformation into E. coli TOP10 were screened by PCR and restriction analysis, which confirmed the presence of the insert (the PNP-deoD gene) in the recombinant plasmid. The recombinant plasmid pPICZαA-PNP isolated from selected clones was then transformed into Pichia pastoris GS115 yeast for intracellular expression of protein. The selection of recombinant Pichia clones is carried out on a medium containing the antibiotic Zeocin™, which ensures the survival of only transformed yeasts [23]. The nucleotide sequencing of the EcPNP gene of E. coli strain RKMUz – 221 showed 98.47% sequence similarity with E. coli strain BM28 [27] and eight different mutations in the PNP, represented as single nucleotide substitutions. It was interesting to see if these mutations would affect the structure and, most importantly, the functional activity of the PNP enzyme, since some mutations may have no effect, while others may alter the enzymatic activity or stability of proteins. The identification of these mutations can provide important information for understanding the genetic diversity of PNP enzymes and their potential applications in biotechnology. 
For the determination of influence of mutations in cDNA and enzymatic activity of the recombinant EcPNP in the stage of expression in Pichia pastoris cells used a fluorometric assay [26]. Fluorometric assay showed that the highest activity of expressed EcPNP was enriched at 72 h of cultivation after induction with methanol (0.5% - 1.0%). The specific activity of recombinant EcPNP was studied by hydrolysis of inosine to hypoxanthine and D-ribose because inosine as a purine type nucleoside is main substrate for purine nucleoside phosphorylase [28]. For this purpose, the recombinant EcPNP containing fraction was separated from the yeast homogenate by gradient precipitation with 25%-65% ammonium sulphate. Because by using gradual changes in ammonium sulfate concentration, different proteins in a mixture can be precipitated sequentially based on their solubility properties [29-30]. In this protein purification stage using gradient ammonium sulfate precipitation, the optimal ammonium sulfate concentration needed for protein precipitation was determined based on the molecular weight of the recombinant protein. The salt concentration was titrated to find the value that resulted in maximum protein recovery while minimizing contamination from other protein impurities. The course of enzymatic hydrolysis of inosine by recombinant EcPNP was analyzed using HPLC. The degree of hydrolysis of the inosine in reaction mixture was quantified by HPLC for 1-5 h. More than 50% of the inosine was hydrolyzed within 5 h under our experimental conditions, which indicates the high hydrolytic activity of the recombinant EcPNP expressed in Pichia. It should be noted that the cell lysate of normal Pichia pastoris GS115 does not exhibit hydrolytic activity (HPLC chromatogram isn’t provided). Thus, the revealed hydrolytic activity of recombinant EcPNP indicates the ability to efficiently catalyze purine nucleosides, such as inosine, and can be used in the development of new antiviral or cytotoxic prodrugs based on modified nucleosides.

A new recombinant pPICZαA-PNP plasmid, consisting of 4256 base pairs, was successfully cloned to express the recombinant PNP of E. coli strain RKMUz-221 in the yeast Pichia pastoris. The molecular weight of the obtained recombinant EcPNP, determined by electrophoresis was approximately 28 kDa. The enzymatic hydrolysis results revealed that the recombinant EcPNP exhibited significant activity by hydrolyzing over 50% of inosine (9-beta-D-ribofuranosylhypoxanthine) within 5 hours. These findings demonstrate the potential of the obtained recombinant EcPNP for generating modified nucleosides. Moreover, the expression of the recombinant enzyme in Pichia pastoris yeast provides a cost-effective and scalable approach for large-scale production. 

The study was carried out within the framework of project F-FA-2021-360, Ministry of Innovative Development of the Republic of Uzbekistan.

ShA and SS provided direction for this research idea; JA and SS: writing – review the manuscript, JA, ShKh, SG and SS: cloning, HPLC, data analysis; AM, AB: synthesis and purification of oligonucleotides (primers); KhN: DNA sequencing; OA, TS, and OY: cultivation of Pichia pastoris strains; FE and KhD: isolation of DNA, protein fractionation, SDS-PAGE. All authors have read and agreed to the published version of the manuscript.

There is no conflict of interest among the authors.