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

Author:
Chen, Hailin, Liu, Changqing, Li, Meijie, Zhang, Haibo, Xian, Mo, Liu, Huizhou
Source:
RSC advances 2018 v.8 no.27 pp. 15021-15028
ISSN:
2046-2069
Subject:
Escherichia coli, Saccharomyces cerevisiae, color, directed evolution, enzymes, fermentation, lycopene, medicine, metabolic engineering, mutagenesis, mutants, polymerase chain reaction, screening
Abstract:
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.
Agid:
6183710