Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene
Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene
复制标题
通过RNAi介导的内源性高表达基因的基因沉默提高里氏木霉中异源脂肪酶的产量
DOI：
10.1016/j.biortech.2012.01.013
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发表时间：
2012-04-01
影响因子：
11.4
通讯作者：
Dong, Zhi-Yang
中科院分区：
文献类型：
--
作者：
Qin, Li-Na;Cai, Fu-Rong;Dong, Zhi-Yang
以纤维二糖水解酶I基因（cbh 1）为启动子，在里氏木霉（Trichoderma reesei）中表达了黑曲霉（Aspergillus尼日尔）的脂肪酶基因（Lip）。CBHI占T. reesei。通过实时荧光定量PCR、SDS-PAGE和活性测定等方法对重组酶及CBHI基因和蛋白的表达进行分析。结果表明，通过RNAi技术沉默cbh 1基因，可以有效抑制cbh 1基因的表达，降低CBHI可以显著提高异源脂肪酶的产量。CBHI产量下降的重组菌株表现出1.8- 3.2倍的脂肪酶活性比亲本菌株的增加。本研究为提高异源目的基因在T.通过RNA干扰阻止特定内源基因的高表达，(C)2012爱思唯尔有限公司保留所有权利。
A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. (C) 2012 Elsevier Ltd. All rights reserved.