Multipoint Mutation And Expression In Pichia Pastoris Of Phytase Gene (phyA)

By using long-distance inverse PCR, Arg(cga) and Arg(cgc) in the expression fragment of phytase phyA gene were mutated synonymously to Arg(aga),which is a bias code of yeastThe mutated gene phyAm-4 was cloned into pUC18 vector, and the four mutated sites were confirmed by sequencing.The expression plasmid pPIC9k-phyAm-4 was constructed and transformed into GS115 strain. Positive clones,of which the chromosomes were integrated with phyA gene,were identified by the phenotype and PCR.The phytase activity of PP-NPm-4-2 with codons optimized reached 133800 u/ml in BMGY/BMMY culture medium after being induced with 36h, which was the 1.10 times higher than PP-NPm-8,and The phytase activity of PP-NPm-4-2 with codons optimized reached 136900u/ml in malt wort culture medium after being induced with 36h, which was the 1.87 times higher than PP-NPm-8.SDS-PAGE analysis suggust that the expression product could be secreted and over-expressed, the size of enzyme protein was 70.15KD. Southern blotting analysis to the one yeast transformants showed that phyA gene was intergrated into the chromosome genome. Additionally, The genetic stability of transformant is quite good.On these bases, to construct high yield phytase gene engineered microbial strains for the industrialized production of phytase...