Directed evolution of the transcription factor Gal4 for development of an improved transcriptional regulation system...

As a well-characterized eukaryotic DNA binding transcription factor, Gal4 has been extensively employed to construct controllable gene expression systems in Saccharomyces cerevisiae. The problem of insufficient inducibility arises in the constructs with multiples genes under control of GAL promoters due to the low expression level and activity of the native Gal4. In order to obtain improved transcriptional regulation systems for multi-gene pathways, Gal4 mutants with improved activity were created by directed evolution. During the preliminary screening, five positive Gal4 variants were isolated based on the lycopene-indicated high-throughput screening method. Analysis of the mutation sites revealed that the majority of positive mutations are localized in the middle homology region with unspecified function, suggesting an important role of this domain in transactivation of Gal4. Through combinatorial site-directed mutagenesis, the best variant Gal4^T406A/V413A was obtained, which successfully increased the transcription of P_GAL-driven lycopene pathway genes and led to 48 % higher lycopene accumulation relative to the wild-type Gal4. This study demonstrates the viability of modifying Gal4 activity by directed evolution for elevated expression of P_GAL-driven genes and therefore enhanced production of the target metabolite.