期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 142, 期 -, 页码 -出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2020.109675
关键词
Transcription factor; Gal4; Directed evolution; Lycopene; Saccharomyces cerevisiae
资金
- China Postdoctoral Science Foundation [2020M671614]
- National Natural Science Foundation of China [32001032, 21808196]
- Zhejiang Provincial Natural Science Foundation of China [LY18B060001]
- Introduction Program of High-Level Innovation and Entrepreneurship Talents in Jiangsu Province
- Lvyang Jinfeng Talent Attracting Plan of Yangzhou city
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 induc-ibility 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 trans activation of Gal4. Through combinatorial site-directed mutagenesis, the best variant Gal4T406A/V413A was obtained, which successfully increased the transcription of PGAL-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 PGAL-driven genes and therefore enhanced production of the target metabolite.
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