title: Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector.
abstract: 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.
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Weimin
authors.initials: W
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Taomei
authors.initials: T
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Guohui
authors.initials: G
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Haohua
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Micrbiology, Guangdong Institute of Microbiology Guangzhou, China.
authors.forename: Zilei
authors.initials: Z
authors.lastname: Huang
keywords: Fast TALEN technology; Saccharomyces cerevisiae; bioethanol production; complement; disruption