This is the first report identifying carotenoids produced by the fungus and characterizing carotenoid biosynthesis genes in the fungus. GzCarRA exhibits high sequence similarity to CarRA of F. fujikuroi (Linnemannstöns et al., 2002) and Al-2 of N. crassa (Arrach et al., 2002). These genes encode a bifunctional enzyme with both phytoene synthase and carotene cyclase activity. Our study showed that ΔgzcarRA does not produce phytoene, suggesting that GzCarRA is required for phytoene synthesis, and the high sequence similarity between GzCarRA and CarRA suggests

that GzCarRA also has cartotene cyclase activity. GzCarB is highly similar to the CarB gene of F. fujikuroi (Linnemannstöns et al., 2002), and Al-1 of N. crassa (Schmidhauser et al., 1990). Al-1 synthesizes 3,4-didehydrolycopene by introducing double bonds to the phytoene substrate via phytofluene, ɛ-carotene, neurosporene, and lycopene. The major products Apoptosis inhibitor of this enzyme are 3,4-didehydrolycopene and lycopene. Hydroxychloroquine concentration γ-Carotene is not the substrate of Al-1, suggesting that torulene is synthesized from 3,4-didehydrolycopene (Hausmann & Sandmann, 2000). In our study, ΔgzcarB accumulated phytoene, indicating that GzCarB also plays a role in the dehydrogenation of

phytoene. Therefore, we deduced that GzCarB is a phytoene dehydrogenase that catalyzes the formation of 3,4-didehydrolycopene and lycopene (Fig. 4). GzCarX and GzCarO show high similarity to carotenoid cleavage oxygenase CarX (Thewes et al., 2005) and opsin-like protein CarO (Prado et al., 2004), respectively, from F. fujikuroi. CarX expressed in Escherichia coli synthesizes retinal from β-carotene, γ-carotene, β-apo-8′-carotenal, and torulene, indicating that the function of CarX is in retinal biosynthesis (Prado-Cabrero et al., 2007b). Opsins are a class of retinal-binding proteins with seven transmembrane helical domains. In this study,

G. zeae did not produce retinal and neither ΔgzcarX nor ΔgzcarO affected neurosporaxanthin and torulene production, suggesting that both genes are not functional in the fungus. GzCarT is highly similar to CarT in F. fujikuroi. CarT functions as a torulene oxygenase, given its catalysis of the conversion of torulene into β-apo-4′-carotenal in vitro and the accumulation of torulene by the CarT null mutant of F. fujikuroi (Prado-Cabrero et Fludarabine cell line al., 2007a). As expected, ΔgzcarT also accumulated torulene and produced no neurosporaxanthin, suggesting that GzCarT is a torulene oxygenase. Based on our results, we propose the following neurosporaxanthin biosynthetic pathway in G. zeae (Fig. 4). Torulene is first synthesized by GzCarRA and GzCarB. The colorless carotenoid phytoene is synthesized from two molecules of GGPP by GzCarRA. GzCarRA is a bifunctional enzyme that contains two domains: one catalyzing phytoene synthesis and the other catalyzing the formation of β-ionone rings.