Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene |
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| Authors: | Hailin Chen Changqing Liu Meijie Li Haibo Zhang Mo Xian Huizhou Liu |
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| Affiliation: | CAS Key Laboratory of Bio-based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101 People''s Republic of China.; Sino-Danish College, University of Chinese Academy of Sciences, No. 19 (A) Yuquan Road, Beijing 100049 People''s Republic of China |
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| 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.Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine. |
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