Improvement Of The Phytase Production Strain #74

Phytase (myo-inositol hexakisphosphate phosphohydrolases) can hydrolyze phytate to lower myo- inositol phosphates and inorganic phosphate and has extensive applications in animal nutrition, human nutrition and synthesis of lower inositol phosphates. One of the main objectives of phytase industrial production is to further improve the protein expression level of the phytase production strain and reduce its production costs.Using the commercial phytase strain #74 as the host strain, which has the highest expression level at present, through the coexpression of protein disulfide isomerase and Vitreoscilla hemoglobin(VHb), and increasing the appA-m gene copy number, we aimed at further improving the phytase production level and decreasing the costs. Besides, the cell surface display of the phytase on P. pastoris was also tried.According to the reported gene sequence (GenBank accession number AJ302014) of disulfide isomerase (PDI) from Pichia pastoris, the PDI encoding gene was cloned and then ligated into the vector pPICZA. Plasmid pPICZA-pdi was then transformed into the competent cell of P. pastoris #74. Among the 600 positive clones, P. pastoris #49 had the highest phytase activity of 16,582 U/mL in a 5 L fermenter, 20% more than that of P. pastoris #74. Western blot result revealed that the PDI was functionally expressed in P. pastoris #49 with the molecular mass of about 65 kD, resulting in the improvement of phytase production. Using the E. coli phytase gene appA-m which has been modified according to the codon bias of P. pastoris, the expression vector pPIC9K-appA-m was constructed and transformed into P. pastoris #49 for multicopy integration. Among the 300 clones, P. pastoris #79 showed the highest phytase activity of 17,434 U/mL, 10% higher than that of P. pastoris #49 and 34% higher than that of P. pastoris #74.According to the reported sequence (EMBL accession numbers Y13397 and AF008245), the ADH2 promoter of 600 bp was obtained from Pichia stipitis. Moreover, based on the Vitreoscilla hemoglobin sequence (GenBank accession number M27601), the vgb gene was modified and synthesized according to the codon bias of P. pastoris, without changing the amino acid sequence of VHb. The PsADH2- promoter and the vgb-m gene were fused through overlap PCR. The P. pastoris expression vector pPIC9K-PsADH2-vgb-m was then constructed and transformed into P. pastoris #74. Under oxygen limitation, P. pastoris #15 showed the highest phytase activity of 249 U/mL, three times higher than that of P. pastoris #74.Besides, we also tried the cell surface display of E. coli phytase. Based on the published sequence ofα-agglutinin (Genbank accession number X16861) from Saccharomyces cereviase, AGα1 encoding gene was obtained by PCR and expression vector pPIC9K-appA-m-AGα1 was constructed, and then transformed into P. pastoris GS115. Phytase activity was detected on the yeast cell surface.