Dataset Information

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

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 K _{m (RS)-mevalonate} of purified mutant MK decreased by 74% compared with the K _{m (RS)-mevalonate} of the wild-type MK, and the K _{cat (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.

SUBMITTER: Chen H

PROVIDER: S-EPMC9080002 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Chen Hailin H Liu Changqing C Li Meijie M Zhang Haibo H Xian Mo M Liu Huizhou H

RSC advances 20180420 27

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 ...[more]

PMID: 35541305

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Project description:BackgroundDuring transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli.Methodology/principal findingsThe mutability index (MI) was developed by Wright et al. to estimate the relative transcription-associated mutability of bases per transcription event. Using the most stable fold of each ssDNA that have an average length n, MI was defined as (the number of folds in which the base is unpaired)/nx(highest -DeltaG of all n folds in which the base is unpaired), where DeltaG is the free energy. The MI values show a significant correlation with mutation data under stress but not with spontaneous mutations in E. coli. Protein sequence diversity is preferred under stress but not under favorable conditions. Therefore, we evaluated the selection pressure on MI in terms of the protein sequence diversity for all the protein-coding sequences in E. coli. The distributions of the MI values were lower at bases that could be substituted with each of the other three bases without affecting the amino acid sequence than at bases that could not be so substituted. Start codons had lower distributions of MI values than did nonstart codons.Conclusions/significanceOur results suggest that the majority of protein-coding sequences have evolved to promote protein sequence diversity and to reduce gene knockout under stress. Consequently, transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis under stress. Nonrandom transcription-associated mutagenesis under stress should improve the survival of E. coli.

Dataset Information

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

Publications

Directed evolution of mevalonate kinase in <i>Escherichia coli</i> by random mutagenesis for improved lycopene.

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