Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector.
Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector.
复制标题
使用新型重组 TALEN 载体高效破坏 ADH2 基因提高酿酒酵母的乙醇产量
DOI：
10.3389/fmicb.2016.01067
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发表时间：
2016
影响因子：
5.2
通讯作者：
Huang Z
中科院分区：
文献类型：
--
作者：
Ye W;Zhang W;Liu T;Tan G;Li H;Huang Z
生物乙醇在能源供应和经济发展中的作用日益重要。然而，生物乙醇产量低，缺乏高效的遗传操作方法，限制了其应用。本研究将一种新型的转录激活因子样效应核酸酶（TALEN）载体电穿孔到酿酒酵母As 2. 4中，对乙醇脱氢酶基因ADH 2和潮霉素抗性基因hyg进行测序。利用抗FLAG单克隆抗体进行的Western blot分析证明TALE蛋白在As2.4菌株中成功表达。qPCR和测序证实了17 bp靶基因的准确敲除，效率为80%。将TALEN载体和ADH 2 PCR产物电穿孔到Δ ADH 2中以互补ADH 2基因（ADH 2 + As 2. 4）。采用LC-MS和GC检测天然As2.4、Δ ADH 2 As2.4和ADH 2 + As2.4菌株中的乙醇产率。结果表明，通过对As2.4中ADH 2的破坏，乙醇产量提高了52.4 ± 5.3%。ADH 2 + As2.4的生物乙醇产量与天然As2.4的生物乙醇产量几乎相同。这项研究是第一次报道在S。酿酒酵母采用快速TALEN技术，提高生物乙醇产量。本工作为S.以高效、特异的方式对酿酒酵母进行发酵，从而促进了酿酒酵母生物乙醇产量的提高。酿酒酵母的代谢工程。
Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2+ As2.4). LC–MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2+ As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2+ As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering.