A new producer of a recombinant aflatoxin-degrading enzyme obtained via heterologous expression in Pichia pastoris

Contamination of food and feed with mycotoxins causes significant economic losses in the food and feed industry and poses a serious threat to the human health and animal life because of mutagenic, carcinogenic and other disruptive properties of these secondary metabolites of fungi. Enzymatic degradation of mycotoxins represents an efficient and environmentally safe alternative to the chemical decontamination of agricultural and food products. In this study, a synthetic adtz gene encoding ADTZ, an aflatoxin-degrading oxidase from Armillaria tabescens , was integrated into the genome of a Pichia pastoris GS115 strain under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter. To amplify the adtz gene, oligonucleotide sequences were constructed with specific restriction sites HindIII and NotI added to the 5' end. The adtz gene-containing pPIG-ADTZ plasmid obtained with the use of the pPIG-1 vector was linearized by digestion with restriction endonuclease ApaI, followed by transforming the cells of P. pastoris recipient strain GS115 by electroporation. The transformed yeast cell were selected on YPD medium with an antibiotic. PCR amplification, restriction analysis and Sanger sequencing confirmed insertion of the target gene. As a result, 54 transformed clones containing the target gene were obtained, and the most productive clone secreting the recombinant ADTZ-14 (2.1 mg/ml of the total extracellular protein) was selected. Recombinant ADTZ represented a monomeric protein (78±3 kDa) possessing a high affinity to aflatoxin B1 (AFB1). Saving the functional properties of the recombinant protein was shown using experiments on assessment of its ability to degrade AFB1 during short-time and prolonged incubation. The obtained protein was able to degrade AFB1 by 14 % after a 2-h incubation at 40 °С; after 72 and 120 h of incubation at 30 °С, the content of AFB1 in ADTZ-14 culture liquid (CL) reduced by 50 and 80 %, respectively, compared to content in CL of non-transformed control GS115. These data suggest a quite high biotechnological potential of a new recombinant ADTZ preparation in relation to the decontamination of agricultural products contaminated with AFB1. Thus, the earlier developed expression system intended to increase the copy number of heterologous genes in Pichia pastoris was first used to obtain a recombinant protein able to degrade AFB1. Using this approach, we transformed yeast cells with the pPIG-ADTZ plasmid and obtained 154 recombinant clones of P. pastoris , 77 % of which contained the target sequence of the adtz gene. Productivity of the best transformant (clone ADTZ-14) was 2.1 mg of protein per 1 ml of culture liquid, and about half of the pool of the extracellular proteins fell to the share of recombinant ADTZ able to degrade 80 % of AFB1 incubated in cell-free culture broth at 30 °С and pH 7.0.