Water treatment membranes embedded with a stable and bactericidal nanodiamond material.

Filtration has emerged as a critical technology to reduce waterborne diseases caused by poor water quality. Filtration technology presents key challenges, such as membrane selectivity, permeability and biofouling. Nanomaterials can offer solutions to these challenges by varying the membranes' mechanical and bactericidal properties. This research uses nanodiamond particles with facile surface functionality and biocompatibility properties that are added to membranes used for filtration treatments. Scanning and transmission electron microscopy (SEM and TEM) and Fourier transform infrared spectroscopy (FTIR) were performed to study the membrane surface. FTIR spectra confirms an increase in oxygen functional groups onto the ultradispersed diamond's (UDD) surface following acid treatment. SEM images show particle deagglomeration of functionalized UDD at the membrane surface. Tensile strength tests were done to measure the UDD mechanical properties and Coliscan membrane filtration characterization was performed to determine the filter effectiveness. Polyether sulfone (PES) and polyvinylidene (PVDF) membranes expressed a change in their yield point when UDD was incorporated into the porous matrix. A significant microorganism reduction was obtained and confirmed using t-test analysis at a 95% level of confidence. UDD-embedded membranes exhibit a significant bactericidal reduction compared to commercial membranes suggesting these membranes have the potential to enhance current membrane filtration systems.

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.

Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus-Plant Interaction.

Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

A RALDH-like enzyme involved in Fusarium verticillioides development.

Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid, an important chordate morphogen. Retinal also occurs in some fungi, such as Fusarium and Ustilago spp., evidenced by the presence of rhodopsins and ß-carotene cleaving, retinal-forming dioxygenases. Based on the assumption that retinoic acid may also be formed in fungi, we searched the Fusarium protein databases for RALDHs homologs, focusing on Fusarium verticillioides. Using crude lysates of Escherichia coli cells expressing the corresponding cDNAs, we checked the capability of best matches to convert retinal into retinoic acid in vitro. Thereby, we identified an aldehyde dehydrogenase, termed CarY, as a retinoic acid-forming enzyme, an activity that was also exerted by purified CarY. Targeted mutation of the carY gene in F. verticillioides resulted in alterations of mycelia development and conidia morphology in agar cultures, and reduced capacity to produce perithecia as a female in sexual crosses. Complementation of the mutant with a wild-type carY allele demonstrated that these alterations are caused by the lackof CarY. However, retinoic acid could not be detected by LC-MS analysis either in the wild type or the complemented carY strain in vivo, making elusive the connection between CarY enzymatic activity and retinoic acid formation in the fungus.

Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions.

Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon-carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.

Biological roles of fungal carotenoids.

Carotenoids are terpenoid pigments widespread in nature, produced by bacteria, fungi, algae and plants. They are also found in animals, which usually obtain them through the diet. Carotenoids in plants provide striking yellow, orange or red colors to fruits and flowers, and play important metabolic and physiological functions, especially relevant in photosynthesis. Their functions are less clear in non-photosynthetic microorganisms. Different fungi produce diverse carotenoids, but the mutants unable to produce them do not exhibit phenotypic alterations in the laboratory, apart of lack of pigmentation. This review summarizes the current knowledge on the functional basis for carotenoid production in fungi. Different lines of evidence support a protective role of carotenoids against oxidative stress and exposure to visible light or UV irradiation. In addition, the carotenoids are intermediary products in the biosynthesis of physiologically active apocarotenoids or derived compounds. This is the case of retinal, obtained from the symmetrical oxidative cleavage of ß-carotene. Retinal is the light-absorbing prosthetic group of the rhodopsins, membrane-bound photoreceptors present also in many fungal species. In Mucorales, ß-carotene is an intermediary in the synthesis of trisporoids, apocarotenoid derivatives that include the sexual hormones the trisporic acids, and they are also presumably used in the synthesis of sporopollenin polymers. In conclusion, fungi have adapted their ability to produce carotenoids for different non-essential functions, related with stress tolerance or with the synthesis of physiologically active by-products.

The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism.

Survival of micro-organisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. We previously described the gene cut-1 of Neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. Here we report on the functional analysis of cutA, the orthologous gene in the phytopathogenic fungus Fusarium fujikuroi. cutA mRNA levels increased transiently after exposure to 0.68 M NaCl and were reduced upon return to normal osmotic conditions; deletion of the gene resulted in a partial reduction in tolerance to osmotic stress. ΔcutA mutants contained much lower intracellular levels of glycerol than the wild-type, and did not exhibit the increase following hyper-osmotic shock expected from the high osmolarity glycerol (HOG) response. cutA is linked and divergently transcribed with the putative glycerol dehydrogenase gene gldB, which showed the same regulation by osmotic shock. The intergenic cutA/gldB regulatory region contains putative stress-response elements conserved in other fungi, and both genes shared other regulatory features, such as induction by heat shock and by illumination. Photoinduction was also observed in the HOG response gene hogA, and was lost in mutants of the white collar gene wcoA. Previous data on glycerol production in Aspergillus spp. and features of the predicted CutA protein lead us to propose that F. fujikuroi produces glycerol from dihydroxyacetone, and that CutA is the enzyme involved in the synthesis of this precursor by dephosphorylation of dihydroxyacetone-3P.

Light-mediated participation of the VIVID-like protein of Fusarium fujikuroi VvdA in pigmentation and development.

The phytopathogen Fusarium fujikuroi is a model fungus for the production of different secondary metabolites. These include the acidic xanthophyll neurosporaxanthin, produced through the activity of the enzymes encoded by the car genes, of which carRA and carB play a major role. Expression of these genes is induced by light but, in contrast to other fungi, the induction is not impaired under continuous illumination in null mutants of the F. fujikuroi wc-1-like gene, wcoA. Therefore, we investigate the role of other blue-light photoreceptors. Here we describe the identification, regulation and targeted mutation of the F. fujikuroi gene vvdA, homologous of the Neurospora crassa vivid (vvd) gene. As found for vvd in N. crassa, expression of vvdA in F. fujikuroi is strongly stimulated by light, an activation that is severely reduced in the wcoA mutants. Deletion of vvdA in F. fujikuroi results in a paler pigmentation under constant light, explained by a reduced carotenoid production, a regulatory effect opposite to the enhanced carotenoid accumulation characteristic of the vvd mutants of N. crassa. No major changes were appreciated in the transcriptional regulation of the car genes following exposure to light, but a noticeable reduction was found upon long-term incubation under constant illumination. Additionally, the vvdA mutants produce less conidia and their colonies exhibit morphological alterations under constant light, such as a more compact development of aerial mycelia and a more solid attachment to the agar surface. The results indicate that VvdA participates in the regulation by light of mycelial development and affects the accumulation of carotenoids but it is not responsible of the transcriptional photoadaptation of the car genes.

The oxygenase CAO-1 of Neurospora crassa is a resveratrol cleavage enzyme.

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving ß-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cß double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

The RNAi Machinery in the Fungus Fusarium fujikuroi Is Not Very Active in Synthetic Medium and Is Related to Transposable Elements.

Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.

Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi.

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Effects of 4 Different Velocity-Based Resistance-Training Programming Models on Physical Performance.

PURPOSE: To examine the effects of 4 programming models (linear [LP], undulating [UP], reverse [RP], and constant [CP]) on physical performance. METHODS: Forty-eight moderately strength-trained men were randomly assigned to LP, UP, RP, and CP groups according to their 1-repetition maximum (1RM) in the full-squat exercise (SQ) and followed an 8-week training intervention using the SQ and monitoring movement velocity for every repetition. All groups trained with similar mean relative intensity (65% 1RM), number of repetitions (240), sets (3), and interset recovery (4 min) throughout the training program. Pretraining and posttraining measurements included, in the SQ, 1RM load, the average velocity attained for all absolute loads common to pretests and posttests (AV), and the average velocity for loads that were moved faster (AV > 1) and slower (AV < 1) than 1 m·s-1 at pretraining tests. Moreover, countermovement jump height and 20-m running sprint time were measured. RESULTS: A significant time effect was found for all variables analyzed (P < .05), except for 20-m running sprint time. Significant group × time interactions were observed for 1RM, AV > 1, and AV (P < .05). After training, all groups attained significant strength gains on 1RM, AV, AV > 1, and AV < 1 (P < .001-.01). LP and RP groups improved their countermovement jump height (P < .01), but no significant changes were observed for UP and CP. No significant improvements were achieved in 20-m running sprint time for any groups. CONCLUSIONS: These different programming models are all suitable for improving physical performance. LP and RP induce similar or greater gains in physical performance than UP and CP.

Functional analysis of the carS gene of Fusarium fujikuroi.

The ascomycete fungus Fusarium fujikuroi is a model system in the investigation of the biosynthesis of some secondary metabolites, such as gibberellins, bikaverin, and carotenoids. Carotenoid-overproducing mutants, generically called carS, are easily obtained in this fungus by standard mutagenesis procedures. Here we report the functional characterization of gene carS, responsible for this mutant phenotype. The identity of the gene was demonstrated through the finding of mutations in six independent carS mutants and by the complementation of one of them. The F. fujikuroi carS gene was able to restore the control of carotenogenesis in a similar deregulated mutant of Fusarium oxysporum, but only partially at the transcription level, indicating an unexpected complexity in the regulation of the pathway. Due to the pleiotropic characteristics of this mutation, which also modifies the production of other secondary metabolites, we did a screening for carS-regulated genes by subtracted cDNA hybridization. The results show that the carS mutation affects the regulation of numerous genes in addition to those of carotenogenesis. The expression of the identified genes was usually enhanced by light, a regulatory effect also exhibited by the carS gene. However, in most cases, their mRNA levels in carS mutants were similar to those of the wild type, suggesting a regulation that affects mRNA availability rather than mRNA synthesis.

Light-dependent functions of the Fusarium fujikuroi CryD DASH cryptochrome in development and secondary metabolism.

DASH (Drosophila, Arabidopsis, Synechocystis, human) cryptochromes (cry-DASHs) constitute a subgroup of the photolyase cryptochrome family with diverse light-sensing roles, found in most taxonomical groups. The genome of Fusarium fujikuroi, a phytopathogenic fungus with a rich secondary metabolism, contains a gene encoding a putative cry-DASH, named CryD. The expression of the cryD gene is induced by light in the wild type, but not in mutants of the "white collar" gene wcoA. Targeted ΔcryD mutants show light-dependent phenotypic alterations, including changes in morphology and pigmentation, which disappear upon reintroduction of a wild-type cryD allele. In addition to microconidia, the colonies of the ΔcryD mutants produced under illumination and nitrogen starvation large septated spores called macroconidia, absent in wild-type colonies. The ΔcryD mutants accumulated similar amounts of carotenoids to the control strain under constant illumination, but produced much larger amounts of bikaverin under nitrogen starvation, indicating a repressing role for CryD in this biosynthetic pathway. Additionally, a moderate photoinduction of gibberellin production was exhibited by the wild type but not by the ΔcryD mutants. The phenotypic alterations of the ΔcryD mutants were only noticeable in the light, as expected from the low expression of cryD in the dark, but did not correlate with mRNA levels for structural genes of the bikaverin or gibberellin biosynthetic pathways, suggesting the participation of CryD in posttranscriptional regulatory mechanisms. This is the first report on the participation of a cry-DASH protein in the regulation of fungal secondary metabolism.

Luminescent graphene quantum dots fabricated by pulsed laser synthesis.

Graphene has been the subject of intense research in recent years due to its unique electrical, optical and mechanical properties. Furthermore, it is expected that quantum dots of graphene would make their way into devices due to their structure and composition which unify graphene and quantum dots properties. Graphene quantum dots (GQDs) are planar nano flakes with a few atomic layers thick and with a higher surface-to-volume ratio than spherical carbon dots (CDs) of the same size. We have developed a pulsed laser synthesis (PLS) method for the synthesis of GQDs that are soluble in water, measure 2-6 nm across, and are about 1-3 layers thick. They show strong intrinsic fluorescence in the visible region. The source of fluorescence can be attributed to various factors, such as: quantum confinement, zigzag edge structure, and surface defects. Confocal microscopy images of bacteria exposed to GQDs show their suitability as biomarkers and nano-probes in high contrast bioimaging.

Fusarium Photoreceptors.

Light is an important modulating signal in fungi. Fusarium species stand out as research models for their phytopathogenic activity and their complex secondary metabolism. This includes the synthesis of carotenoids, whose induction by light is their best known photoregulated process. In these fungi, light also affects other metabolic pathways and developmental stages, such as the formation of conidia. Photoreceptor proteins are essential elements in signal transduction from light. Fusarium genomes contain genes for at least ten photoreceptors: four flavoproteins, one photolyase, two cryptochromes, two rhodopsins, and one phytochrome. Mutations in five of these genes provide information about their functions in light regulation, in which the flavoprotein WcoA, belonging to the White Collar (WC) family, plays a predominant role. Global transcriptomic techniques have opened new perspectives for the study of photoreceptor functions and have recently been used in Fusarium fujikuroi on a WC protein and a cryptochrome from the DASH family. The data showed that the WC protein participates in the transcriptional control of most of the photoregulated genes, as well as of many genes not regulated by light, while the DASH cryptochrome potentially plays a supporting role in the photoinduction of many genes.

HmbC, a Protein of the HMG Family, Participates in the Regulation of Carotenoid Biosynthesis in Fusarium fujikuroi.

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

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.

Genetic basis of carotenoid overproduction in Fusarium oxysporum.

The phytopathogenic fungus Fusarium oxysporum is a model organism in the study of plant-fungus interactions. As other Fusarium species, illuminated cultures of F. oxysporum exhibit an orange pigmentation because of the synthesis of carotenoids, and its genome contains orthologous light-regulated car genes for this biosynthetic pathway. By chemical mutagenesis, we obtained carotenoid overproducing mutants of F. oxysporum, called carS, with upregulated mRNA levels of the car genes. To identify the regulatory gene responsible for this phenotype, a collection of T-DNA insertional mutants obtained by Agrobacterium mediated transformation was screened for carotenoid overproduction. Three candidate transformants exhibited a carS-like phenotype, and two of them contained T-DNA insertions in the same genomic region. The insertions did not affect the integrity of any annotated ORFs, but were linked to a gene coding for a putative RING-finger (RF) protein. Based on its similarity to the RF protein CrgA from the zygomycete Mucor circinelloides, whose mutation results in a similar carotenoid deregulation, this gene (FOXG_09307) was investigated in detail. Its expression was not affected in the transformants, but mutant alleles were found in several carS mutants. A strain carrying a partial FOXG_09307 deletion, fortuitously generated in a targeted transformation experiment, exhibited the carS phenotype. This mutant and a T-DNA insertional mutant holding a 5-bp insertion in FOXG_09307 were complemented with the wild type FOXG_09307 allele. We conclude that this gene is carS, encoding a RF protein involved in down-regulation of F. oxysporum carotenogenesis.