IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.