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.

Fig. 1. Screening results of three cycles of random mutagenesis (A); saturation mutagenesis at residues V13, S148, and V301 (B); and critical amino acid substitutions by site-directed mutagenesis (C). (A) Twenty representative strains are shown for each sequential mutagenesis step. The average relative lycopene production of the mutants increased progressively with every round of mutagenesis. Strains with the highest relative lycopene production in each round were chosen for further analysis. (B) Twenty representative strains are shown for every position to identify the most advantageous amino acid substitution at these residues. These same color columns represent different residual activities at the same position. (C) Single, double and triple mutation enzymes were constructed to determine which were sufficient and necessary for enhanced MK activity. Every mutant was necessary for MK and worked well with no interference. Wild-type MK was used as the control (OD₄₇₅*).

Fig. 2. Effect of temperature on wild-type and triple-mutant MK activity (A) and stability (B); the effect of pH on wild-type and triple-mutant MK activity (C) and stability (D). (A) The optimal temperature was determined to be 35 °C. When temperature was above 35 °C, the rate of MK (V13D/S148I/V301E) activity decreased faster than wild-type MK; (B) the stability of the triple-mutant MK was improved compared with the wild type. (C) The optimal pH was 7.5 at 25 °C. Mutations did not affect parameters related to pH; (D) pH affected the stability of wild-type and V13D/S148I/V301E MK, although mutations did not change the acid-alkali tolerance of MK.

Fig. 3. Cell growth rates (A) and lycopene production (B) during aerobic fed-batch fermentation of CHL-1 and CHL-2. (A) The cell growth rates of CHL-1 and CHL-2 were similar. (B) Lycopene production of CHL-2 was increased 2.0-fold compared with CHL-1.

Fig. 4. Structural (A) and docking (B) models of wild-type MK. (A) The location of the beneficial mutations are marked in magenta, and the active site catalytic triad residues (Arg-248, Thr-350 and Ala-352) are marked in green. (B) The active site catalytic triad residues and the ATP-binding residues (Lys-12, Asp56, Ser138, Ser-149 and Glu-191) are respectively marked.

Fig. 5. Structural models of the mutations: (A) wild-type V13; (B) V13D; (C) wild-type S148; and (D) S148I. (A and B) The V13D substitution shows Asn-13 forming a salt-bridge with Arg-248, which enlarged the MK catalytic domain. (C and D) The S148I substitution is located in the α-helix lid and improved the stability of the α-helix.