Optimization of IspSib stability through directed evolution to improve isoprene production

Abstract

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.

Keywords: biosensor; directed evolution; enzyme stability; isoprene production; isoprene synthase; isoprenoid.