Cloning and characterization of the geranylgeranyl diphosphate synthase (GGPS) responsible for carotenoid biosynthesis in Pyropia umbilicalis

Abstract

Carotenoid metabolism in red algae is not well understood. Geranylgeranyl diphosphate (GGPP), synthesized by GGPP synthase (GGPS), is a precursor for the biosynthesis of many biologically important metabolites, including carotenoids and chlorophylls. GGPSs have been functionally characterized in many organisms, but not in species of the primitive red algal order Bangiales. Here, we cloned and characterized the gene encoding GGPS (PuGGPS) in Pyropia umbilicalis (Bangiales). PuGGPS encodes a protein of 345 amino acids with an N-terminal transit peptide. The catalytic activity of PuGGPS for the production of GGPP was verified by a color complementation assay in Escherichia coli and subsequent high-performance liquid chromatography analysis. Homology modeling of PuGGPS showed that its tertiary structure resembles that of other known GGPSs and that this structure allows for the precise docking of the enzymatic product of PuGGPS, GGPP. When leafy thalli of P. umbilicalis were treated with norflurazon, an inhibitor of the key carotenoid metabolism enzyme phytoene desaturase, the expression of PuGGPS increased by twofold compared with that of the control in the first 2 h, suggesting a prompt response to metabolic perturbation. Prolonged norflurazon treatment failed to increase PuGGPS expression. Sequence analysis showed that PuGGPS shares seven conserved motifs with other previously identified GGPSs from different organisms, including two aspartate-rich GGPS signature motifs. Phylogenetic analysis also indicated that PuGGPS is a member of the type II GGPSs found in eubacteria and plants.