β-1,3-1,4葡聚糖酶基因双拷贝工程菌株的构建与发酵研究  

Construction and Fermentation of Two-copy Engineered Yeast of β-1, 3-1, 4-glucanase

作　　者：裴红蕾[1] 陈轶群[1] 吕俊楠[1] 杨雯涵[1] 李志民[1] 曹云鹤[1] 

机构地区：[1]中国农业大学动物科技学院,动物营养学国家重点实验室,北京100193

出　　处：《中国畜牧杂志》2013年第3期64-68,共5页Chinese Journal of Animal Science

基　　金：国家科技支撑计划项目(2013BAD10B01、2011BAD26B02-2)

摘　　要：本试验旨在研究β-1,3-1,4葡聚糖酶双拷贝基因毕赤酵母工程菌株的构建及发酵。首先,通过对质粒pGAP-glu-opt进行PCR扩增获得密码子优化的β-1,3-1,4葡聚糖酶基因glu-opt,用EcoR I和Not I双酶切后与毕赤酵母表达载体pPIC9K连接,获得重组质粒p9K-glu-opt。该重组质粒经Sac I线性化后转化毕赤酵母GS115菌株,利用不含组氨酸的MDS平板和摇瓶培养,筛选重组菌株,命名为GS115/9K-glu-opt。制备GS115/9K-glu-opt感受态细胞,再用线性化的重组质粒pPIC-glu-opt二次转化,在含有抗生素Zeocin的YPDS平板上筛选glu-opt基因双拷贝重组菌株GS115/2xglu-opt。双拷贝重组菌在10 L发酵罐中进行高密度发酵培养及甲醇诱导表达,β-1,3-1,4葡聚糖酶最高酶活达到21 600 U/mL,约为单拷贝工程菌株X33/pPIC-glu-opt发酵活力的1.44倍;蛋白表达量达8.4 g/L,约为X33/pPIC-glu-opt的1.68倍。This study was described the construction and fermentation of two copy strains of/3-1, 3-1, 4 glucanase. First of all, The codon-optimized/3-1, 3-1, 4 glucanase （glu-opt） sequence of pGAP-glu-opt was amplified by PCR. After double-digestion with EcoRI and Not I, the DNA fragment was inserted into the vector pPIC9K to be the recombinant plasmid p9K-glu-opt. Then the recombinant plasmid pPIC-glu-opt was completely linearized by Sacl and transformed into P. pastoris GS115 by electrotransformation. After cultivation on MDS plates without histidine and screened in shaker flask, the one copy strains GSll5/9K-glu-opt was got. Moreover, the linearized recombinant plasmid pPIC-glu-opt was transformed into GSll5/9Kglu-opt and cultured on YPDS plates with antibiotic Zeocin. After cultivation, the two copy strains GS115/2xglu-opt was got. Finally, in a 10-L fermentor, by methanol induction for 72 h, the/3 -1, 3-1, 4-glucanase was overexpressed with a yield of 21,600 U/mL , about 1. 44 times of X33/ pPIC-glu-opt; the protein was amounted to 8.4 g/L, about 1.68 times of X33/pPIC-glu-opt.

关 键 词：β-1 3—1 4葡聚糖酶 双拷贝 毕赤酵母 发酵 

分 类 号：S816.7[农业科学—饲料科学]