Screening Of Pichia Pastoris Transformed With Phytase PhyA Gene And Research On Culture Medium For Engineering Yeast

The phytase phyA gene recombinant plasmid pPIC9K-PhyA was introduced into Pichia pastoris GS115 strain by electroporation after being linearnized with Bglll. Compared the transformation condition, it was better to adopt the Bio-Rad, under the condition of 2.0 kilo-volt,one pulser. which had the higher transformantion rate than that reported under the condition of 1.0 kilo-volt ,50uF, 200 O with BTX genePulser .Then, 15 recombinants were screened. They grew quickly in MD plate, but slowly in MM plate, which proved their phenotypes of methanol utilization were Muts. The 15 recombinants all showed the measurable phytase activities. Particularly, the transformant PP-NP-5 showed the highest expression, when it was cultured in BMGY substrate and induced in BMMY substrate, its phytase activity in 36 hours reached 52041u/ml, which was 101 times of that of the original strain A.niger N25, 513.4u/ml, 45% higher than that of transformant reported by Hongning Wang, 2.47 times higher than 15000u/ml reported by Bin Yao, and 1.00 times higher than 26000u/ml of the recombinant KM71, reported by Yanming Han, which meant the phytase gene was high-secretedly expressed.Further more, PP-NP-5 strain was identified at DNA level .The result of Dot-blotting and PCR analysis showed the gene phyA was transmitted into Pichia pastoris GS115 strain. Southern blotting analysis showed phyA gene was intergrated into the chromosome genome through gene replacement event (double-crossover events) at the AOX1 locus with one copy of phyA gene.The malt was made of Pichia recombinant substrate material firstly in the world. As the culture medium in growth phase for PP-NP-5, the malt wort was prepared with malt and water according to the ratio of 1:6. And, as the culture medium in inducing phase, the malt extract liquid was made at high temperature with the same ratio. When cells were concentrated and non-concentrated in the second phase, the phytase activities were 22598 u/ml, 12720 u/ml respectively. The results showed the substrate cost could be decreased 198.78 times and 64.59 times respectively. It proved that malt wort was suitable for growth of the engineering Pichia, and high temperature malt extract liquid for phytase inducing.