| Abstrakt: |
Phytases (myo-inositol hexakisphosphate hydrolase) catalyze the hydrolysis of phytate to inorganic phosphate and less phosphorylated myo-inositol derivatives and are widely used as feed additives in animal nutrition. Nevertheless, nowadays, there is a constant search for new phytases and new expression systems for better production of these important enzymes. In this study, we report cloning of two novel bacterial phytases belonging to the different enzyme classes and having different properties in the methylotrophic yeast Pichia pastoris. Sequences of agpPand phyCgenes, encoding histidine acid phytase from Pantoeasp. 3.5.1 and β-propeller phytase from Bacillus ginsengihumiM2.11, respectively, were optimized and chemically synthesized according to the P. pastoriscodon usage bias. The optimized genes were cloned into the yeast vectors pPINK-HC and pPINK-LC under the control of the inducible promoter AOX1 and two different signal peptides—signal sequence of α-amylase gene from Aspergillus nigerand presequence of inulinase gene from Kluyveromyces maxianus. PCR analysis, restriction analysis, and DNA sequencing confirmed correct integration of agpPand phyCgenes into P. pastorisgenome. As a result, recombinant strains of P. pastoriswith codon-optimized bacterial phytase genes (agpPand phyC), encoding histidine acid and β-propeller phytases, integrated into the genome under the alcohol oxidase promoter AOX1 and two different signal peptides, were obtained. Recombinant phytase AgpP was stably expressed and secreted into the culture medium of yeasts, whereas the expression of phyCgene was only confirmed at transcription level. |
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