Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB. Transcription of carO is induced by light and is deregulated in carotenoid-overproducing mutants. The same regulation pattern is exhibited by carRA and carB; and common conserved DNA elements are found in the three promoters. Heat shock resulted in a modest induction of carO transcription, similar to the one exhibited by carB, confirming a common regulation. Targeted mutagenesis of carO produced no apparent phenotypic modification, including no change in the photoinduction of carotenoid biosynthesis.

The ascomycete Fusarium fujikuroi produces carotenoids by means of the enzymes encoded by three car genes. The enzymes encoded by carRA and carB are responsible of the synthesis of beta-carotene and torulene, respectively, while the product encoded by carT cleaves torulene to produce the acidic xanthophyll neurosporaxanthin. carRA and carB are found in a cluster with a third gene, carO, which codes for an opsin-like protein. However, no information is available on the sequence or chromosomal location of carT, which has been identified only by mutant analysis. Transcription of the three clustered genes is stimulated by light and by mutations in a regulatory gene, leading to overproduction of carotenoids. We have now identified a fourth gene in the car cluster, called carX, which codes for a protein similar to known carotenoid-cleaving oxygenases. carX is transcribed divergently from carRA, and exhibits the same transcriptional pattern as carRA, carB and carO. Targeted deletion of carX resulted in a phenotype characterized by a significant increase in the overall carotenoid content. In the dark, the carX mutants accumulate at least five times more carotenoids than the wild type, and exhibit partial derepression of carRA and carB transcription. The mutants also show more intense pigmentation in the light, but the increase in the carotenoid content relative to the wild type is less than twofold. Under these conditions, the mutants also show a relative increase in the amounts of phytoene and cyclic carotenoids formed, suggesting that CarRA activity is enhanced.

Phytoene synthase, phytoene dehydrogenase and carotene cyclase are three of the four enzyme activities needed to produce the acidic carotenoid neurosporaxanthin from the precursor geranylgeranyl pyrophosphate. In the filamentous fungus Fusarium fujikuroi, these three enzyme activities are encoded by two closely linked genes, carRA and carB, oriented in the same direction in the genome. The two genes are separated by 548 bp and code for two polypeptides of 612 and 541 amino acids, respectively, which are highly similar to the homologous proteins from other filamentous fungi. The ORF of carRA contains a 96-bp insertion that is absent in the other fungal homologues. The 32 additional residues are located in one of the two repeated domains responsible for the cyclase activity in the homologous fungal proteins. We have determined the function of carRA by gene disruption. The resulting mutants were albino and had lost the ability to produce phytoene, as expected from the simultaneous loss of phytoene synthase and carotene cyclase. In the same experiments, we also found transformants in which carB had been deleted. These mutants accumulate phytoene, confirming the function of the gene previously shown by gene-targeted mutagenesis. Expression of carRA and carB is strongly induced by light. Loss of carB or disruption of the carRA ORF led to enhanced expression of the carRA gene, suggesting the existence of a feedback regulatory mechanism.

No abstract