Bioavailability and provitamin A activity of neurosporaxanthin in mice.

Various species of ascomycete fungi synthesize the carboxylic carotenoid neurosporaxanthin. The unique chemical structure of this xanthophyll reveals that: (1) Its carboxylic end and shorter length increase the polarity of neurosporaxanthin in comparison to other carotenoids, and (2) it contains an unsubstituted ß-ionone ring, conferring the potential to form vitamin A. Previously, neurosporaxanthin production was optimized in Fusarium fujikuroi, which allowed us to characterize its antioxidant properties in in vitro assays. In this study, we assessed the bioavailability of neurosporaxanthin compared to other provitamin A carotenoids in mice and examined whether it can be cleaved by the two carotenoid-cleaving enzymes: ß-carotene-oxygenase 1 (BCO1) and 2 (BCO2). Using Bco1-/-Bco2-/- mice, we report that neurosporaxanthin displays greater bioavailability than ß-carotene and ß-cryptoxanthin, as evidenced by higher accumulation and decreased fecal elimination. Enzymatic assays with purified BCO1 and BCO2, together with feeding studies in wild-type, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- mice, revealed that neurosporaxanthin is a substrate for either carotenoid-cleaving enzyme. Wild-type mice fed neurosporaxanthin displayed comparable amounts of vitamin A to those fed ß-carotene. Together, our study unveils neurosporaxanthin as a highly bioavailable fungal carotenoid with provitamin A activity, highlighting its potential as a novel food additive.

Neurosporaxanthin Overproduction by Fusarium fujikuroi and Evaluation of Its Antioxidant Properties.

Neurosporaxanthin (NX) is a carboxylic carotenoid produced by some filamentous fungi, including species of the genera Neurospora and Fusarium. NX biosynthetic genes and their regulation have been thoroughly investigated in Fusarium fujikuroi, an industrial fungus used for gibberellin production. In this species, carotenoid-overproducing mutants, affected in the regulatory gene carS, exhibit an upregulated expression of the NX pathway. Based on former data on a stimulatory effect of nitrogen starvation on carotenoid biosynthesis, we developed culture conditions with carS mutants allowing the production of deep-pigmented mycelia. With this method, we obtained samples with ca. 8 mg NX/g dry mass, in turn the highest concentration for this carotenoid described so far. NX-rich extracts obtained from these samples were used in parallel with carS-complemented NX-poor extracts obtained under the same conditions, to check the antioxidant properties of this carotenoid in in vitro assays. NX-rich extracts exhibited higher antioxidant capacity than NX-poor extracts, either when considering their quenching activity against [O2(1g)] in organic solvent (singlet oxygen absorption capacity (SOAC) assays) or their scavenging activity against different free radicals in aqueous solution and in liposomes. These results make NX a promising carotenoid as a possible feed or food additive, and encourage further studies on its chemical properties.

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium.

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expression.

Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium.

BACKGROUND: The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. RESULTS: The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. CONCLUSIONS: The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.

Carotenoid Biosynthesis in Fusarium.

Many fungi of the genus Fusarium stand out for the complexity of their secondary metabolism. Individual species may differ in their metabolic capacities, but they usually share the ability to synthesize carotenoids, a family of hydrophobic terpenoid pigments widely distributed in nature. Early studies on carotenoid biosynthesis in Fusariumaquaeductuum have been recently extended in Fusarium fujikuroi and Fusarium oxysporum, well-known biotechnological and phytopathogenic models, respectively. The major Fusarium carotenoid is neurosporaxanthin, a carboxylic xanthophyll synthesized from geranylgeranyl pyrophosphate through the activity of four enzymes, encoded by the genes carRA, carB, carT and carD. These fungi produce also minor amounts of ß-carotene, which may be cleaved by the CarX oxygenase to produce retinal, the rhodopsin's chromophore. The genes needed to produce retinal are organized in a gene cluster with a rhodopsin gene, while other carotenoid genes are not linked. In the investigated Fusarium species, the synthesis of carotenoids is induced by light through the transcriptional induction of the structural genes. In some species, deep-pigmented mutants with up-regulated expression of these genes are affected in the regulatory gene carS. The molecular mechanisms underlying the control by light and by the CarS protein are currently under investigation.