Optimization of IspS ib stability through directed evolution to improve isoprene production.

Enzyme stability is often a limiting factor in the microbial production of high-value-added chemicals and commercial enzymes. A previous study by our research group revealed that the unstable isoprene synthase from Ipomoea batatas (IspS ib ) critically limits isoprene production in engineered Escherichia coli . Directed evolution was, therefore, performed in the present study to improve the thermostability of IspS ib . First, a tripartite protein folding system designated as lac'-IspS ib -'lac, which could couple the stability of IspS ib to antibiotic ampicillin resistance, was successfully constructed for the high-throughput screening of variants. Directed evolution of IspS ib was then performed through two rounds of random mutation and site-saturation mutation, which produced three variants with higher stability: IspS ib N397V A476V , IspS ib N397V A476T , and IspS ib N397V A476C . The subsequent in vitro thermostability test confirmed the increased protein stability. The melting temperatures of the screened variants IspS ib N397V A476V , IspS ib N397V A476T , and IspS ib N397V A476C were 45.1 ± 0.9°C, 46.1 ± 0.7°C, and 47.2 ± 0.3°C, respectively, each of which was higher than the melting temperature of wild-type IspS ib (41.5 ± 0.4°C). The production of isoprene at the shake-flask fermentation level was increased by 1.94-folds, to 1,335 mg/L, when using IspS ib N397V A476T . These findings provide insights into the optimization of the thermostability of terpene synthases, which are key enzymes for isoprenoid production in engineered microorganisms. In addition, the present study would serve as a successful example of improving enzyme stability without requiring detailed structural information or catalytic reaction mechanisms.IMPORTANCEThe poor thermostability of IspS ib critically limits isoprene production in engineered Escherichia coli . A tripartite protein folding system designated as lac'-IspS ib -'lac, which could couple the stability of IspS ib to antibiotic ampicillin resistance, was successfully constructed for the first time. In order to improve the enzyme stability of IspS ib , the directed evolution of IspS ib was performed through error-PCR, and high-throughput screening was realized using the lac'-IspS ib -'lac system. Three positive variants with increased thermostability were obtained. The thermostability test and the melting temperature analysis confirmed the increased stability of the enzyme. The production of isoprene was increased by 1.94-folds, to 1,335 mg/L, using IspS ib N397V A476T . The directed evolution process reported here is also applicable to other terpene synthases key to isoprenoid production.