Mutants of the carotene cyclase domain of al-2 from Neurospora crassa.

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.

A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces.

Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.