Alejandro F. Estrada

The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation.

The fungal proteins of the White Collar photoreceptor family, represented by WC-1 from Neurospora crassa, mediate the control by light of different biochemical and developmental processes, such as carotenogenesis or sporulation. Carotenoid biosynthesis is induced by light in the gibberellin-producing fungus Fusarium fujikuroi. In an attempt to identify the photoreceptor for this response, we cloned the only WC-1-like gene present in the available Fusarium genomes, that we called wcoA. The predicted WcoA polypeptide is highly similar to WC-1 and contains the relevant functional domains of this protein. In contrast to the Neurospora counterpart, wcoA expression is not affected by light. Unexpectedly, targeted wcoA disruptant strains maintain the light-induced carotenogenesis. Furthermore, the wcoA mutants show a drastic reduction of fusarin production in the light, and produce less gibberellins and more bikaverins than the parental strain under nitrogen-limiting conditions. The changes in the production of the different products indicate a key regulatory role for WcoA in secondary metabolism of this fungus. Additionally, the mutants are severely affected in conidiation rates under different culture conditions, indicating a more general regulatory role for this protein.

Regulation by light in Fusarium.

The genus Fusarium stands out as research model for pathogenesis and secondary metabolism. Light stimulates the production of some Fusarium metabolites, such as the carotenoids, and in many species it influences the production of asexual spores and sexual fruiting bodies. As found in other fungi with well-known photoresponses, the Fusarium genomes contain several genes for photoreceptors, among them a set of White Collar (WC) proteins, a cryptochrome, a photolyase, a phytochrome and two presumably photoactive opsins. The mutation of the opsin genes produced no apparent phenotypic alterations, but the loss of the only WC-1 orthologous protein eliminated the photoinduced expression of the photolyase and opsin genes. In contrast to other carotenogenic species, lack of the WC photoreceptor did not impede the light-induced accumulation of carotenoids, but produced alterations in conidiation, animal pathogenicity and nitrogen-regulated secondary metabolism. The regulation and functional role of other Fusarium photoreceptors is currently under investigation.

Identification and biochemical characterization of a novel carotenoid oxygenase: elucidation of the cleavage step in the Fusarium carotenoid pathway

The synthesis of the acidic apo‐carotenoid neurosporaxanthin by the fungus Fusarium fujikuroi depends on four enzyme activities: phytoene synthase and carotene cyclase, encoded by the bifunctional gene carRA, a carotene desaturase, encoded by carB, and a postulated cleaving enzyme converting torulene (C40) into neurosporaxanthin (C35). Based on sequence homology to carotenoid oxygenases, we identified the novel fungal enzyme CarT. Sequencing of the carT allele in a torulene‐accumulating mutant of F. fujikuroi revealed a mutation affecting a highly conserved amino acid, and introduction of a heterologous carT gene in this mutant restored the ability to produce neurosporaxanthin, pointing to CarT as the enzyme responsible for torulene cleavage. Expression of carT in lycopene‐accumulating E. coli cells resulted in the formation of minor amounts of apo‐carotenoids, but no enzymatic activity was observed in β‐carotene‐accumulating cells, indicating a preference for acyclic or monocyclic carotenes. The purified CarT enzyme efficiently cleaved torulene in vitro to produce β‐apo‐4′‐carotenal, the aldehyde corresponding to the acidic neurosporaxanthin, and was also active on other monocyclic synthetic substrates. In agreement with its role in carotenoid biosynthesis, the carT transcript levels are induced by light and upregulated in carotenoid‐overproducing mutants, as already found for other car genes.

Regulation and targeted mutation of opsA, coding for the NOP-1 opsin orthologue in Fusarium fujikuroi.

Opsins are widespread photoreceptor proteins involved in a diversity of light-driven processes in all major taxa that use the apocarotenoid retinal as a light-absorbing prosthetic group. Proteins from the opsin family are also found in filamentous fungi, but no function has been attributed to them. The fungus Fusarium fujikuroi contains two genes for presumptive retinal-binding opsins, which we call carO and opsA, and a gene for an opsin-related protein, called hspO. One report showed that carO is linked and co-regulated with the enzymatic genes of the carotenoid pathway, carRA, carB, and carX, but that its mutation produced no detectable phenotype. Sequence analyses suggest that OpsA, not CarO, is the orthologue of the Neurospora opsin NOP-1. mRNA levels for the three Fusarium opsin genes are induced by heat shock, while those for carO and opsA are induced by light. This photoinduction is lost in mutants of the white collar gene wcoA, which contains much higher carO and opsA mRNA levels than the wild type, indicating a down-regulation of both genes by WcoA. Conversely to carO, opsA mRNA levels are not enhanced in carotenoid-overproducing mutants. Targeted opsA mutants have no discernible external phenotype, but they exhibit a significant decrease in mRNA levels for structural genes of the carotenoid pathway. Similar reductions are produced by mutations in the enzymatic genes carRA and carB, but not in carX, responsible for retinal biosynthesis.

The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway

The accumulation of the apocarotenoid neurosporaxanthin and its carotene precursors explains the orange pigmentation of the Neurospora surface cultures. Neurosporaxanthin biosynthesis requires the activity of the albino gene products (AL‐1, AL‐2 and AL‐3), which yield the precursor torulene. Recently, we identified the carotenoid oxygenase CAO‐2, which cleaves torulene to produce the aldehyde β‐apo‐4′‐carotenal. This revealed a last missing step in Neurospora carotenogenesis, namely the oxidation of the CAO‐2 product to the corresponding acid neurosporaxanthin. The mutant ylo‐1, which exhibits a yellow colour, lacks neurosporaxanthin and accumulates several carotenes, but its biochemical basis is unknown. Based on available genetic data, we identified ylo‐1 in the Neurospora genome, which encodes an enzyme representing a novel subfamily of aldehyde dehydrogenases, and demonstrated that it is responsible for the yellow phenotype, by sequencing and complementation of mutant alleles. In contrast to the precedent structural genes in the carotenoid pathway, light does not induce the synthesis of ylo‐1 mRNA. In vitro incubation of purified YLO‐1 protein with β‐apo‐4′‐carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. We conclude that YLO‐1 completes the set of enzymes needed for the synthesis of this major Neurospora pigment.

Ustilago maydis accumulates β-carotene at levels determined by a retinal-forming carotenoid oxygenase

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for beta-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates beta-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative beta-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of beta-carotene to yield two molecules of retinal. Analyses of beta-carotene and retinal contents in U. maydiscco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the beta-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.

The gene carD encodes the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Neurosporaxanthin (β‐apo‐4′‐carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β‐apo‐4′‐carotenal. On the basis of sequence homology to Neurospora crassa YLO‐1, which mediates the formation of apo‐4′‐lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β‐apo‐4′‐carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid‐overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β‐apo‐4′‐carotenal, its derivative β‐apo‐4′‐carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo‐1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid‐overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established.

Deviation of the neurosporaxanthin pathway towards β-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin‐producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB‐encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid‐overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21‐CarB to perform the fifth desaturation, as implied by the accumulation of γ‐carotene and β‐carotene, which arise through four‐step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five‐step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene‐producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate ζ‐carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.

Neurosporaxanthin Production by Neurospora and Fusarium

The orange pigmentation of the ascomycete fungi Neurospora and Fusarium is mainly due to the accumulation of neurosporaxanthin, a carboxylic apocarotenoid whose possible biotechnological applications have not been investigated. From the discovery of the first enzyme of the biosynthetic pathway in 1989, the prenyltransferase AL-3, to the recent identification of an aldehyde dehydrogenase responsible for the last biosynthetic step, all the enzymes and biochemical reactions needed for neurosporaxanthin biosynthesis in these fungi are already known. Depending on the culture conditions and/or genetic background, Neurospora and Fusarium may produce large quantities of this xanthophyll and minor amounts of other carotenoids. This chapter describes methods for the growth of Neurospora crassa and Fusarium fujikuroi for improved neurosporaxanthin production, the analysis of this xanthophyll, its separation from its carotenoid precursors, and its identification and quantification.

Analysis of al-2 mutations in Neurospora.

The orange pigmentation of the fungus Neurospora crassa is due to the accumulation of the xanthophyll neurosporaxanthin and precursor carotenoids. Two key reactions in the synthesis of these pigments, the formation of phytoene from geranylgeranyl pyrophosphate and the introduction of β cycles in desaturated carotenoid products, are catalyzed by two domains of a bifunctional protein, encoded by the gene al-2. We have determined the sequence of nine al-2 mutant alleles and analyzed the carotenoid content in the corresponding strains. One of the mutants is reddish and it is mutated in the cyclase domain of the protein, and the remaining eight mutants are albino and harbor different mutations on the phytoene synthase (PS) domain. Some of the mutations are expected to produce truncated polypeptides. A strain lacking most of the PS domain contained trace amounts of a carotenoid-like pigment, tentatively identified as the squalene desaturation product diapolycopene. In support, trace amounts of this compound were also found in a knock-out mutant for gene al-2, but not in that for gene al-1, coding for the carotene desaturase. The cyclase activity of the AL-2 enzyme from two albino mutants was investigated by heterologous expression in an appropriately engineered E. coli strain. One of the AL-2 enzymes, predictably with only 20% of the PS domain, showed full cyclase activity, suggesting functional independence of both domains. However, the second mutant showed no cyclase activity, indicating that some alterations in the phytoene synthase segment affect the cyclase domain. Expression experiments showed a diminished photoinduction of al-2 transcripts in the al-2 mutants compared to the wild type strain, suggesting a synergic effect between reduced expression and impaired enzymatic activities in the generation of their albino phenotypes.