| abstract |
The present application belongs to the technical field of tobacco genetic engineering, and specifically relates to the matter of patent application for GGPPS-directed mutant protein. The three sites 154, 161 and 218 of the existing GGPPS protein are located in the catalytic pocket of the enzyme, and the two sites 209 and 233 are located on the surface of the enzyme molecule; based on these five sites, this application provides a series of GGPPS directed muteins. , including: a series of single-site muteins, double-site muteins, three-site muteins, four-site muteins, and five-site muteins. The inventor used CAST technology to construct a "small but refined" mutant library. Through further screening and based on the needs of directed evolution, the inventor made a detailed analysis of the types of amino acid mutations at specific sites. Preliminary experimental results showed that the synthesis of β-carotene was significantly improved after the amino acid mutation at a specific site, which laid a certain technical foundation for further cultivation of new crop varieties. |