毕赤氏酵母醇氧化酶-2基因启动子突变体的分离和鉴定  

Isolation and Characterization of P_(AOX2) Mutant in Pichia pastoris

作　　者：戴秀玉[1] 王韫恂[1] 周坚[1] 王忆琴[1] 

机构地区：[1]中国科学院微生物研究所,北京100080

出　　处：《Acta Genetica Sinica》2000年第7期641-646,共6页

基　　金：中国科学院微生物研究所所长基金

摘　　要：巴斯德毕赤氏酵母表达系统已被广泛用于生产外源蛋白的奇主菌。利用该系统将外源基因整合交换到染色体上时，AOX1基因被破坏，使表达菌的甲醇利用缓慢，给发酵生产造成一定影响。在不改变现有表达系统前提下，从AOX1功能缺陷菌株分离出Mut+自发突变体。通过突变体在甲醇培养基中生长曲线的测定，HSA表达产物的聚丙烯酰胺凝胶电泳检测，证明突变体的甲醇利用能力和蛋白表达比原始菌株大大提高；突变体AOX2基因上游区域经PCR扩增进行全序列测定，结果表明在PAOX2阻遏蛋白结合区域－255和－529两个碱基发生改变，该突变改善了AOX1基因缺陷功能，有利于外源基因的高表达。Spontaneous Mut+ mutants of P. pastoris AOX1-defective expression strain have been isolated, they were identified as phenotypically utilized methanol to grow as wild type. The results obtained from measuring growth curve when cultivated in medium in which methanol as a sole carbon souree and detecting HSA protein on SDS-PAGE confirmed that the mutants have increased ability to utilize methanol and express foreign HSA gene product. The promoter region of AOX2 gene from the mutants has been cloned by PCR amplification, and the DNA fragment is 1022bp in size. Sequencing analysis showed that there are two point mutations at positions of -529 and -255 from the translation initiation codon respectively. The mutations improved AOX-1 defective function and facilitate the foreign gene for higher expression.

关 键 词：毕赤氏酵母 AOX2基因启动子 自发突变 外源蛋白 

分 类 号：Q933[生物学—微生物学]