Análisis funcional de enzimas de la carotenogénesis y fotoproteinas en hongos

Resumen

My research subject is the metabolism of carotenoids in fungi and its regulation. Mostly, I worked with the model fungus Fusarium fujikuroi (Gibberella fujikuroi, mating group C), known because of its ability to produce gibberellins (growth-promoting plant hormones). As many other fungi, this fungus produces carotenoids. The final product of its pathway is neurosporaxanthin, a xanthophyl apocarotenoid. The whole Fusarium carotenoid biosynthetic pathway has been identified by my group in the University of Seville. It involves three genes, carRA, carB and carT. The latter one is a novel carotenoid oxygenase, whose activity was formerly identified in collaboration with the laboratory of Dr Salim Al-babili (University of Freiburg, Germany). carRA and carB are organized in a cluster together with carX, coding for a second carotenoid oxygenase, and carO, coding for an opsin-like protein. The opsins are light-absorbing proteins that use retinal, an apocarotenoid, as light-harvesting prostetic group. Recent results of the group showed that retinal is made by the carotenoid oxygenase encoded by the gene carX. The five car genes (carRA, carB, carX, carO and carT) are subject of a common transcriptional regulation: their mRNA levels are induced by light and are derepressed in the dark in carotenoid overproduction mutants. Only the last step of the pathway was already unknown, that it's the conversion of ß apo-4'-carotenal into neurosporaxanthin One of the aims of my work is complete the whole pathway in F. fujikuroi, for this purpose we were to Neurospora crassa, another Ascomyces that accumulates neurosporaxanthin like end-product and which carotenoids pathway is well known and shares most of the steps with the one of F. fujikuroi. In Neurospora there is a mutant in the carotenoid pathway named yellow (ylo-1), we characterized this mutation based in the hypothesis that this gene is responsible of the last step in the pathway. The gene ylo 1 from Neurospora crassa encodes an aldehyde dehydrogenase with a transmembrane domain, infrequent in this protein family, we proceed to analyze the protein activity in vitro, and we saw that YLO-1 converts ß apo-4'-carotenal into ß-apo-4'-carotenoic acid or neurosporaxanthin, end-product of the carotenoid pathway in this fungus. Also, it achieves the same reaction on apo-4'-lycopenal, the uncycled version of this substrate, to produce apo-4'-lycopenoic acid. We observed that the chemical analysis of the carotenoids accumulated by the ylo-1 mutant and the cyclase-defective al-2 mutants under optimal conditions for neurosporaxanthin biosynthesis led to the identification of three novel apocarotenoids: apo-4'-lycopenal, apo-4'-lycopenol, and apo-4'-lycopenoic acid. This is the first time these compounds have been found in living matter. According with this data we proposed a new order of late reactions in the N. crassa carotenoid pathway. In agreement with this order, the CAO-2 oxygenase carries out the oxidative cleavage of the pentasaturared precursor 3,4-didehydrolycopene to produce apo-4'-lycopenal, which is oxidized by the aldehyde dehydrogenase YLO-1 to yield apo-4'-lycopenoic acid, which is finally cycled by AL-2 to produce neurosporaxanthin. F. fujikuroi has an ortologue protein to ylo-1, one of the objectives of my current work is to demonstrate that this protein fulfil the last step in F. fujikuroi. Other point of my current work has been mostly dedicated to the investigation of the regulation by light of the car genes and the role of the opsins in Fusarium photobiology. The work concentrated in the functional analysis of the Fusarium protein orthologous to the WC-1 of Neurospora, the best known photoreceptor reported in fungi, responsible of photocarotenogenesis in this fungus. Proteins similar to WC-1 have been recently identified as photoreceptors in other fungi. We cloned the F. fujikuroi wc-1 counterpart (called wcoA), determined the sequence, studied its transcription and generated targeted mutants. In contrast to what was found in Neurospora and other fungi, wcoA is constitutively expressed in F. fujikuroi and its targeted mutations demonstrates that it is not responsible of the carotenoid photoinduction. We saw that the protein WcoA, is not the major photoreceptor for carotenogenesis in this fungus. However, it is the photoreceptor responsible for the photoinduction of mRNA levels of the opsin genes carO y opsA. Additionally, WcoA represses the expression of these genes in the dark. The wcoA mutants show different alterations in the synthesis of gibberellins, bikaverins and fusarins, as well as in mycelial conidiation or hidrophobicity. Thus, the WcoA protein plays a role in the mechanisms that control secondary metabolism and other cellular processes in F. fujikuroi, specially those associated to regulation by nitrogen availability. Others possible candidates to be the mayor photoreceptors of the carotenogenesis are the PPO (predictable photoactive opsin), opsins that contain the conserved lysine residue essential for retinal binding, in this PPO the retinal binding and photoreactivity have not been demonstrated. Fusarium has two PPO, carO and opsA , and one ORP (opsin related protein) presumably lack light-dependent functions.The targeted mutation of gene carO produces no detectable phenotypic changes in F. fujikuroi under laboratory conditions. So we studied the possibility of opsA like carotenoid photoreceptor, like carO mutation of the opsA gene do no produce any severe phenotype under laboratory conditions, we saw that the regulatory pattern of gene opsA differs from that of gene carO. In contrast to carO, opsA mRNA levels are detectable in the dark, exhibit a weak induction in the light and do not show significant alterations in carotenoid overproducing mutants. So another aim of my current work is to find the photoreceptor of the carotenogenesis in F. fujikuroi. F. fujikuroi has others candidates to be photoreceptor like Cryptochromes, we are studying if one of the two predicted Cryptochromes of F. verticillioides (the closer Fusarium sp. already sequenced to F. fujikuroi) is the main carotenogenesis photoreceptor of F. fujikuroi. Recently, I started a new research project on the analysis of the carotenoid metabolism in the basidiomycete fungus Ustilago maydis. I have confirmed that this fungus accumulates carotenoids (mainly beta-carotene), and, in addition to the genes required for its synthesis. We already investigated the gene cco1 of Ustilago maydis, a carotenoid oxygenase orthologous to carX of F. fujikuroi. The Cco1 protein produces in vitro retinal from ß-carotene, its gene expression inversely correlates with ß-carotene concentration in vivo ant the cco1 mutants lack retinal. Thus, the function of the Cco1 in this fungus is retinal biosynthesis. We found three opsin genes in the U. maydis genome and we saw that they exhibit different regulatory patterns. Two of them are induced by light. The cco1 mutants, presumably devoid of photoactive opsins, show no detectable phenotypic alterations except an increase in their ß carotene content. I have found the presence in its genome of genes that should act on beta-carotene, either to introduce oxidated radicals, or to cleave this or other carotenoids to produce apocarotenoid products of unknown biological relevance. As a starting approach, I intend to investigate the enzymatic activities of the products of these genes on beta-carotene or other carotenoids.