INTRODUCTION

Mycophenolic acid (MPA) is a fungal metabolite that was early introduced by Bartolomeo Gosio in 1893 as an antibiotic against Bacillus anthracis. Moreover, MPA possess antiviral (Barroto et al. 2004), antifungal (Nicoletti et al. 2004), antibacterial (Kavanagh 1947), antitumor (Tressler et al. 1994) and antipsoriasis activities (Muth et al. 1975). Most significantly, MPA is being prescribed as an immunosuppressant in kidney, heart and liver (Geissler & Schlitt 2019) transplantation patients (Geissler & Schlitt 2019) and commercialized under the brands entitled CellCept (mycophenolate mofetil, Roche) and myfortic (mycophenolate sodium, Novartis) (Patil et al. 2012). Mycophenolate, the main combination in both drugs, inhibits IMP dehydrogenase (IMPDH) (Chang et al. 2018). MPA is an uncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH) and therefore inhibits the de novo pathway of guanosine nucleotide synthesis without incorporation to DNA (Danafar & Hamidi 2015, Farazi et al. 1997, Regueira et al. 2011). The T and B lymphocytes are associated with their proliferation on de novo synthesis of purines, whereas other cell types can utilize salvage pathways (Regueira et al. 20119). The MPA has potent cytostatic effects on lymphocytes (Guzara et al. 2016) and accordingly, is the definite cause that why MPA are applied widely as an immunosuppressive pharmaceutical (Danafar & Hamidi 2015, Asfari et al. 2005). The molecular basis of MPA biosynthesis was unknown until recently. The genomic cluster that may be responsible for MPA biosynthesis has been identified in P. Brevicompactum. In this fungus, the cluster consists of eight genes including MPaA (encoding Prenyltransferase), MPaB (encoding a protein with unknown function), MPaC (encoding Polyketide synthase), MPaD (encoding P450 monooxygenase), MPaE (encoding Zn-dependent hydrolase), MPaF (encoding IMP dehydrogenase), MPaG (encoding an O-methyltransferase) and MPaH (encoding an oxidative cleavage enzyme) (Del Cid et al. 2016). By using various techniques, only three out of the eight MPa genes from P. brevicompactum have been experimentally shown to be involved in the MPA biosynthesis. The MPaC is the main gene in this cluster and consequently, mutant strain missing this gene loses its ability of MPA synthesis. The MpaC gene catalyzes the formation of 5-methylorsellinic acid (5-MOA), which is the first step in the MPA biosynthesis (Regueira et al. 2011, Dubus et al. 2002). The MPaD and MPaE genes were biochemically characterized in vivo by heterologous expression in a strain of Aspergillus nidulans which expresses MPaC and produces 5-MOA (Hansen et al. 2012). This biochemical characterization provides the second step in the MPA biosynthesis. The MpaG gene, the putative O-methyl transferase, was biochemically characterized in vitro. The results indicated that MpaG catalyzes the methylation of demethyl mycophenolic acid (DMMPA) (Brennan & Brakeman. 2019) to produce MPA which is the final step in the biosynthesis of MPA (Zhang et al. 2015). Several species of Penicillium specially P. brevicompactum, P. stoloniferum and P. requeforty can produce MPA as a secondary metabolite (Ismaiel & Papenbrock, 2015). The purpose of this article was cloning and sequencing of MPaB and MPaE genes from the new strain of P. brevicompactum. Finally, these sequences were aligned with MPaB and MPaE genes, registered in NCBI GenBank.

MATERIALS AND METHODS

Strains and plasmids

The P. brevicompactum fungus was obtained from the strain collection at the Agriculture Faculty of Urmia University and used as the source of genomic DNA. The plasmid PTG19_T harboring the ampicillin resistance cassette, under the control of the Lac promoter, was used as a template for constructing the gene-targeting manipulation of plasmid DNA and introduction of plasmids into E. coli BL21 by chemical transformation, according to the standard procedures.

Genomic DNA isolation

Genomic DNA was extracted from culture using Plant Genomic DNA Extraction Kit (IBRC cat no. MBK0011) according to the manufacturer’s guideline.  The quality and quantity of the purified genomic DNA were assessed by 1% agarose gel electrophoresis in 1x TBE buffer containing 0.5 µg/mL ethidium bromide (Lee et al. 2012) and spectrophotometer (260/280 nm)Biophotometer (Eppendorf, Germany), respectively.

Gene amplification

The MPa genes were amplified by PCR using genomic DNA as template. The specific primers were designed using Gene Runner software according to the P. brevicompactum IBT23078 sequence database under HQ731031.1 accession number, according to the available nucleotide sequences on the NCBI GenBank.

The specific primers for MPaB and MPaE genesamplification were as follows:

MPaBFWD: 5^´-ATG TCTTTGCCTTTGCCTCCAG-3^´,

MPaBREV:5^´ -CTAATGGAAGGGACATTTCCCCGT–3MPaEFWD: 5´-ATGATCAAATCGCAAACGGTCATC-3´

MPaEREV: 5^´-TTACTTCTGTCCTTCTATG GAATTCTC AATATC-3^´

PCR amplification was performed in a 25 μL total volume reaction containing 100 ng of template DNA, 10 μM for each primer, 2 mM Mg²⁺, 200 μM of each dNTP, 1x PCR buffer and 2.5 unit of Taq DNA polymerase. The following conditions were used for the amplification: hot start at 94 °C (5 min), followed by 30 cycles of denaturation at 94 °C (1 min), annealing at 58 °C (1 min) and extension at 72 °C (2 min). The PCR products were analyzed by electrophoresis in 1% agarose gel in 0.5 X TBE buffer and visualized by ethidium bromide staining on UV transilluminator. The PCR product was purified from agarose gel by high pure PCR product Gel Purification Kit (Yekta Tajhiz cat no.YT9027) according to the manufacturer’s guideline.

MPaB and MPaE genes cloning

DNA bands were sliced under the long-wave ultraviolet (UV) light and recovered by purification kit, subsequently, the purified PCR products were ligated into PTG19_T cloning vector by T/A cloning strategy according to the manufacturer’s instructions (Vivantis, USA). This reaction was performed in a single tube contained: PTG19-T, ligation buffer, PCR product, T4-DNA ligase and sterile Milli-Q water. The ligation product was kept at 16 °C for 16 h.

Transformation and recombinant plasmid isolation

Escherichia coli BL21 competent cells were prepared and the recombinant vectors were transformed into the competent E. coli BL21 cells. The amount of 100 μl of chemo competent cells were added to 5 μl of ligation products. The cells were transformed by the process of heat-shocking. After recovery at 37 °C for 1 h in 100 μl Luria-Bertani medium (LB) without antibiotics, transformed cells were spread on the LB agar plates containing 100 μg/ml ampicillin (Lapointe et al. 2016). The agar plates were incubated overnight at 37 °C. The bacterial clones harboring recombinant plasmid DNA were screened based on their colony PCR. The PCR was used for fidelity verification of E. coli BL21 transformants. In this stage, the number of 5 colonies was selected and individual colonies subjected directly to the PCR master mix using Thermo Scientific reagents. Finally, positive PCR colonies were cultured in LB broth containing ampicillin and used for recombinant DNA extraction. The plasmids were purified using the miniprep, Plasmid Mini Kit (Yekta tajhiz cat no. YT9001). 

Nucleotide sequences analysis

After the selection of positive screened colonies using colony-PCR, the purified plasmids were subjected to sequencing (Macrogen, South Korea). The obtained nucleotide sequences were analyzed by homology search and alignment with other MPaB and MPaE genes using Basic Local Alignment Search Tool (BLAST) and Clustal W software, respectively.

RESULTS

Amplification of MPaB and MPaE genes

The genomic DNA of P. brevicompactum was extracted and MPaB and MPaE genes were amplified with the resulting fragments of 1477 and 780 bp, respectively, compared to the 100 bp DNA ladder (Fig. 1). 

Confirmation of cloning

Among selected colonies on agar plates, some colonies showed amplified fragments of MPaB and MPaE genes (Fig. 2) on 1% agarose gel electrophoresis.

Fig. 1. a. Gel electrophoresis for detection of PCR products of MPaB, lane 1 shows 100 bp DNA ladder. Lanes 2 and 3 show amplified fragments by PCR using the specific primers for MPaB which correspond to 1477 nt. b. Gel electrophoresis for detection of PCR products of MPaE, lane 1 shows 100 bp DNA ladder. Lanes 2 and 3 show amplified fragments by PCR using the specific primers for MPaE which correspond to 780 nt.

Fig. 2. Agarose gel displaying the results of colony PCRs to check for positive insertion events in transformation assays. Lane 1 shows 100 bp DNA ladder. Lane 2 shows PCR amplification with the MPaB specific primers to check for positive cloning events of MPaB gene (1477 nt). Lane 3 shows PCR amplification with the MPaE specific primers to check for positive cloning events of MPaE gene (780 nt).

Alignment of MPaB and MPaE sequences

Various sequences of the MPaB and MPaE genes have been recorded in the GenBank, obtained by different sequencing methods. The names, accession numbers and other information are given (Table 1). Furthermore, the alignment of these recorded genes was performed (Fig. 3, Fig 4).

Table 1. Various sequences of the MPaB and MPaE gene recorded in the GenBank.

Fig. 3. Alignment of amino acid sequences of the most recognized MPaB proteins registered in GenBank which their implications are listed in Table 1.

Fig. 4. Alignment of amino acid sequences of the most recognized MPaE proteins registered in the GenBank which their implications are listed in table 1.

Fig. 5. Partial nucleotide sequence of the MPaB (up) and MPaE (down) gene sequencing diagram. Peaks represent position of nucleotides.

MPaB and MPaE nucleotide sequencing

The partial sequences of MPaB and MPaEwere shown (Fig. 5). The obtained sequence was analyzed using BLAST and Clustal W software programs. Sequence alignment was performed at both the nucleotide (Fig. 6 and 7) and amino acid levels (Fig. 8, and 9). These sequences were compared and aligned with MPaB and MPaE sequences in P. brevicompactum IBT23078. The results showed differences at both nucleotide and amino acid levels. According to the results of the current study, new strain of P. brevicompactum has been discovered.

The complete sequence of MPA genes from P. brevicompactum IBT23078 has already been published (Regueira et al.2011). The present study provides two homologues for MPaB and MPaE genes from MPA gene cluster. Together with the IBT23078 previously available sequences, we extended the genetic knowledge of P. brevicompactum, by performing a comparative analysis of another strain gene sequences. The MPaB amplification produced amplicons of 1477 nt and the MpaE PCR amplification generated amplicons of 780 nt with the same length compared to the MPaB and MpaE genes recorded in GenBank. The subcloned MPaB and MPaE genes were aligned with MPaB and MPaE from the IBT23078 recorded in the GenBank which several differences were observed over the entire length of the both genes. The sequence similarity analyses showed that the amplified products were 95.15 and 98.85 (%) identical in amino acid residues compared to the MPaB and MPaE from P. brevicompactum IBT23078. Although the bioinformatics analysis of the MPaB and MPaE has not designated catalytic tasks of these deduced proteins, future functional analyses will reveal their roles in relation to MPA biosynthesis (Regueira et al. 2011). This is the first comparative analysis of MPaB and MPaE genes from MPA gene cluster at the nucleotide and amino acid levels. These comparisons provide definitive evidence for classifying the members of this species. The identification of alternative MPaB and MPaE genes in P. brevicompactum strains may accelerate further research on MPA biosynthesis.

DISCUSSION

The discovery of the MPA gene cluster has provided new insights into the biosynthesis of MPA, a very powerful important immunosuppressive drug, used in preventing acute rejection in liver transplantation (Hao e al, 2008). To better understand the MPA biosynthesis, identification of the gene cluster responsible for its production has great importance and identification of involved genes in the cluster by sequencing would result in the identification of the complete gene cluster.

Fig. 6. Nucleotide alignment of MPaB and MPaB accession number: HQ731031.1 from P. brevicompactum IBT23078. Asterisks represent identical nucleotides.

Fig. 7. Nucleotide alignment of MPaE and MPaE accession number: HQ731031.1 from P. brevicompactum IBT23078. Asterisks represent identical nucleotides.

Fig. 8. Amino acid alignment of MPaB and MPaB accession number: HQ731031.1from P. brevicompactum IBT23078. Different amino acids are in bold and underlined. Asterisks represent identical amino acids between two sequences.

Fig. 9. Amino acid alignment of MPaE and MPaE accession number: HQ731031.1from P. brevicompactum IBT23078. Different amino acids are in bold and underlined. Asterisks represent identical amino acids between two sequences.