Cloning and Characterization of SpcrtR and Its Expression in Escherichia Coli for Zeaxanthin Production

Abstract Zeaxanthin is produced by a series of enzyme catalytic reactions. β-carotene hydroxylase is a key rate-limiting enzyme that catalyzes the conversion of β-carotene to zeaxanthin. The purpose of this study was to clone and express the Spirulina platensis β-carotene hydroxylase gene (SpcrtR) in E. coli. SpcrtR was amplified using specific primers and cloned in vector pGEX-6p-1, the SpcrtR protein (35kDa) was expressed from pGEX-6p-1-SpcrtR in E. coli. Gene expression was analyzed by SDS-PAGE and western blotting. The accumulation of carotenoids was detected by high-performance liquid chromatography (HPLC). SDS-PAGE and western blotting analysis confirmed that SpcrtR protein (35kDa) was expressed in E. coli expression system. Further, the HPLC results demonstrated that SpcrtR can partially catalyze β-carotene to produce zeaxanthin in E. coli. Zeaxanthin and β-carotene yields in recombinant E.coli were 85.71% (±0.4%) and 14.7% (±0.6%), respectively. The results in our study verified that SpCRTR enzyme partially catalyzes the substrate β-carotene to form zeaxanthin. Overall, SpCRTR provides a new choice for enzymatic zeaxanthin production.