Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations
Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations
复制标题
DOI：
10.1016/j.bbrc.2004.11.112
复制
发表时间：
2005-01-28
影响因子：
3.1
通讯作者：
Chang, MDT
中科院分区：
文献类型：
--
作者：
Liu, SH;Chou, WI;Chang, MDT
通过对根霉（Rhizopus）葡萄糖淀粉酶（GA）的信号肽（MSP）进行修饰，增加基因拷贝数，并在毕赤酵母（Pichia pastoris）中共表达与分泌囊泡相关的Rab蛋白基因SEC 4，成功地将GA的分泌水平提高了100倍。MSP被设计为含有与酿酒酵母α-交配因子的信号肽的前区融合的小鼠唾液0 β淀粉酶（S8 L）的信号肽，以取代GA的野生型信号肽（WTSP）。与WTSPGA-1相比，含有单拷贝MSPGA基因的巴斯德毕赤酵母MSPGA-1显示GA分泌增加3.6倍。此外，鉴定了携带七个拷贝的MSPGA插入片段的巴斯德毕赤酵母MSPGA-7，并且显示出比WTSPGA-1高56倍的分泌GA。此外，我们发现SEC 4的过表达进一步使每个MSPGA/巴斯德毕赤酵母中GA的分泌水平加倍。总之，当与WTSPGA-1相比时，MSPGA-7-SEC 4克隆显示高达100倍的GA分泌水平。总之，我们已经证明，上述基因操作的组合导致高水平的分泌R。巴斯德毕赤酵母中的赤藓糖醇。(C)2004年爱思唯尔公司All rights reserved.
Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary 0(amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris. (C) 2004 Elsevier Inc. All rights reserved.