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Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene

Chen, Hailin, Liu, Changqing, Li, Meijie, Zhang, Haibo, Xian, Mo, Liu, Huizhou
RSC advances 2018 v.8 no.27 pp. 15021-15028
Escherichia coli, Saccharomyces cerevisiae, color, directed evolution, enzymes, fermentation, lycopene, medicine, metabolic engineering, mutagenesis, mutants, polymerase chain reaction, screening
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 β-sheet that forms a salt-bridge with nearby Arg-248. S148I was located in the α-helix lid and improved the stability of the α-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.