Functional identification of al-3 from Neurospora crassa as the gene for geranylgeranyl pyrophosphate synthase by complementation with crt genes, in vitro characterization of the gene product and mutant analysis

Abstract

In this work the Neurospora crassa al-3 gene function was determined. Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods. This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity. The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine. Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora. Carotenoid accumulation was observed in E. coli transformants when the N. crassa al-3 gene substitutes the GGPP synthase gene ( crtE) in the carotenogenic crt cluster. Cell-free studies with E. coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenyl-pyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate.