人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母系统中的高效表达

Overexpression of Artificial Synthetic Gene ofAspergillus niger NRRL3135 Phytase in Pichia pastoris

本文以黑曲霉 (Aspergillusniger)NRRL3135菌株植酸酶基因为对象 ,通过基因人工合成的方法去除了该基因的内含子与信号肽编码序列 ,换用在毕赤酵母 (Pichiapastoris)中使用频率较高的密码子以优化其表达。该人工合成植酸酶基因 (PhyA as)以N端融合的方式正确插入到毕赤酵母表达载体pPICZαA。通过电击将重组表达载体整合入酵母染色体DNA中得到重组转化子。SDS PAGE结果与表达产物酶学性质研究表明植酸酶得到分泌表达 ,且与天然产物性质基本一致。筛选得若干株高产基因工程菌 ,其中SPAN Ⅲ菌株达到了在摇床培养条件下 ,每毫升发酵液产生 16 5 0 0 0u植酸酶的水平 。

The phytase gene of \%Aspergillus niger\% NRRL 3135 was modified with a deletion of intron and signal coding sequence.Then,according to the codon preference of \%Pichia pastoris,\%modified \%phyA\% gene was artificially synthesized and cloned into expression vector of pPICZαA.The recombinant plasmid was transformed into chromosome of \%Pichia pastoris\% X\|33 strain by electroporation.The results of SDS\|PAGE and enzymatic kinetic analysis proved that the recombinant phytase was secreted into culture medium with nearly same character of natural phytase.After screening for high level productive yeast strains,a strain named SPAN\|Ⅲ produced recombinant phytase with 165000u/mL under the condition of shake cultivation.It will satisfy the demand for industrialized production in some degree.