| 摘要： |
Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine. Metabolic engineering in microbial hosts has shown that mevalonate kinase (MK, EC2.7.1.366) is one of the rate-limiting enzymes in the lycopene synthetic pathway. In this study, a directed evolution strategy in Escherichia coli was used to optimize the activity of Saccharomyces cerevisiae MK. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: V13D, S148I, and V301E. V13D was near the MK catalytic center, in the -sheet that forms a salt-bridge with nearby Arg-248. S148I was located in the -helix lid and improved the stability of the -helix. V301E may increase MK folding by influencing its secondary structure. The Km (RS)-mevalonate of purified mutant MK decreased by 74% compared with the Km (RS)-mevalonate of the wild-type MK, and the Kcat (RS)-mevalonate was improved by 26% compared with wild type. Fermentation experiments revealed that lycopene production of the mutant MK increased 2.4-fold compared with wild-type MK. |
