| 摘要： |
Background: Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity. Therefore, it is important to prepare an excellent IDI by protein engineering. Results: Directed evolution strategy (error-prone PCR) was utilized to optimize the activity of Saccharomyces cerevisiae IDI. 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: L141H, Y195F, and W256C. L141H, located near the active pocket inside the tertiary structure of IDI, formed a hydrogen bond with nearby beta-phosphates of isopentenylpyrophosphate (IPP). Phe-195 and Cys-256 were nonpolar amino acids and located near the hydrophobic group of IPP, enlarging the hydrophobic scope, and the active pocket indirectly. Purified IDI was characterized and the result showed that the K-m of mutant IDI decreased by 10% compared with K-m of the parent IDI, and K-cat was 28% fold improved compared to that of the original IDI. Results of a fermentation experiment revealed that mutant IDI had a 1.8-fold increased lycopene production and a 2.1-fold increased yield capacity compared to wild-type IDI. Conclusion: We prepared an engineered variant of IDI with improved catalytic activity by combining random and site directed mutagenesis. The best mutants produced by this approach enhanced catalytic activity while also displaying improved stability in pH, enhanced thermostability and longer half-life. Importantly, the mutant IDI could play an important role in fed-batch fermentation, being an effective and attractive biocatalyst for the production of biochemicals. |
