Secretory overexpression of the endoglucanase by Saccharomyces cerevisiae via CRISPR-δ-integration and multiple promoter shuffling
Secretory overexpression of the endoglucanase by Saccharomyces cerevisiae via CRISPR-δ-integration and multiple promoter shuffling
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DOI：
10.1016/j.enzmictec.2018.10.014
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发表时间：
2019-02-01
影响因子：
3.4
通讯作者：
Ogino, Hiroyasu
中科院分区：
文献类型：
--
作者：
Sasaki, Yuka;Mitsui, Ryosuke;Ogino, Hiroyasu
酿酒酵母细胞工厂可以生产各种重组蛋白；因此，需要有效的重组蛋白生产技术。在本研究中，为了在酿酒酵母中建立高效的重组蛋白生产技术，测试了重组蛋白内切葡聚糖酶 II (TrEG) 的分泌生产，我们开发了 2 种新的 TrEG 生产方法，通过成簇规则间隔短回文重复 (CRISPR)-delta 整合以及多个启动子改组，其中涉及 CRISPR 系统对 delta 序列的预分解和随后的 delta 整合以及TrEG 分别与各种启动子缀合和随后的 delta 整合。此外，还检查了同时使用 CRISPR-delta 整合和多个启动子改组方法。 CRISPR-delta整合方法有利于提高整合的TrEG拷贝数及其活性，多启动子改组方法也有利于增强TrEG的转录水平及其活性。此外，同时使用 CRISPR-delta 整合和多个启动子改组方法是最有用的。本发明构建的表达TrEG的转化体YPH499/24CP的羧甲基纤维素酶活性达到559U/L，比常规YEp型载体构建的转化体的羧甲基纤维素酶活性提高了17.3倍。总体而言，同时使用 CRISPR-delta 整合和多个启动子改组对于重组蛋白生产非常有用且易于应用。
Various recombinant proteins can be produced by the yeast Saccharomyces cerevisiae cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in S. cerevisiae, the secretory production of recombinant protein endoglucanase II (TrEG) was tested, We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR)-delta-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the delta-sequence by the CRISPR system and subsequent delta-integration as well as the conjugation of TrEG with various promoters and subsequent delta-integration, respectively. Moreover, simultaneous use of the CRISPR-delta-integration and multiple promoter shuffling methods was also examined. The CRISPR-delta-integration method was effective for improvement of the integrated TrEG copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of TrEG and its activity. Furthermore, simultaneous use of CRISPR-delta-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the TrEG expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-delta-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.