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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 Δ ADH2  to complement the  ADH2  gene ( ADH2  +  As2.4). LC–MS and GC were employed to detect ethanol yields in the native As2.4, Δ ADH2  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.

Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector