Mycophenolic acid (MPA) is the active ingredient in the most important immunosuppressive pharmaceuticals. It has antifungal, antibacterial, antiviral, anti-psoriasis, and antitumor activities. Therefore, its overproduction in addition to gene expression analysis was our main target. Through this study, we isolated a novel potent mycophenolic acid (MPA) producer strain of the genusPenicilliumfrom the refrigerated Mozzarella cheese and it was identified with the molecular marker ITS andbenAgenes asP. arizonenseHEWt1. Three MPA overproducer mutants were isolated by exposing the wild type to different doses of gamma-rays, and the fermentation conditions for the highest production of MPA were optimized. The results indicated that MPA amounts produced by the mutants MT1, MT2, and MT3 were increased by 2.1, 1.7, and 1.6-fold, respectively, compared with the wild-type. The growth of both mutant and wild-type strains on PD broth, adjusted to pH 6 and incubated at 25 °C for 15 d, were the best conditions for maximum production of MPA. In a silico study, five orthologs genes of MPA biosynthesizing gene clusters inP. brevicompactumwere predicted from the genome ofP. arizonense. Sequencing and bioinformatic analyses proved the presence of five putative genes namelympaA, mpaC, mpaF, mpaG, andmpaHin theP. arizonenseHEWt1 genome. Gene expression analysis by qRT-PCR indicated an increase in the transcription value of all annotated genes in the three mutants over the wild type. A highly significant increase in the gene expression ofmpaC, mpaF, andmpaHwas observed inP. arizonense-MT1 compared with wild-type. These results confirmed the positive correlation of these genes in MPA biosynthesis and are the first report regarding the production of MPA byP. arizonense.Kew word.Mycophenolic acid,Penicillium arizonense, mutagenesis, gene expression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02884-z.