Lutein, zeaxanthin, and meso-zeaxanthin supplementation attenuates inflammatory cytokines and markers of oxidative cardiovascular processes in humans.

BACKGROUND AND AIMS: Systemic inflammation and oxidation are primary contributors to the development of atherosclerosis. Oxidation of low-density lipoprotein (LDL) particles within the vascular endothelium has been hypothesized to be an initial step in the formation of atherosclerotic plaques, with inflammatory cytokines serving as the signaling mechanism for concomitant macrophage activation. Supplementation with the antioxidative macular xanthophylls (lutein [L], zeaxanthin [Z], and meso-zeaxanthin [MZ]) has been shown to aid in the reduction of inflammatory physiologic responses; therefore, we hypothesized that in our study population, supplementation with these xanthophylls would facilitate a systemic reduction in markers of inflammation and cardiovascular lipid oxidation. METHODS AND RESULTS: In this double-blind placebo-controlled supplementation study, participants were randomly allocated to receive the active intervention containing L (10 mg) + MZ (10 mg) + Z (2 mg) or placebo (containing sunflower oil). Serum concentrations of carotenoids (assessed by HPLC), inflammatory cytokines (IL-6, IL-1ß, TNF-α) and oxidized LDL (OxLDL; by solid-phase sandwich ELISA) were measured at baseline and at 6-months. Results showed that over the supplementation period, compared to placebo, the active group demonstrated statistically significant increases in serum concentrations of L, Z, & MZ (p < 0.05), reductions in inflammatory cytokines IL-1ß (p < 0.001) and TNF-α (p = 0.003), as well as a corresponding reduction in serum OxLDL (p = 0.009). CONCLUSIONS: Our data show that L, Z, & MZ supplementation results in decreased serum IL-1ß, TNF-α, and OxLDL. This suggests that these carotenoids are acting systemically to attenuate oxidative lipid products and inflammation, thus reducing their contribution to atherosclerotic plaque formation.

Supplementation With Carotenoids, Omega-3 Fatty Acids, and Vitamin E Has a Positive Effect on the Symptoms and Progression of Alzheimer's Disease.

BACKGROUND: Preliminary work by our center has reported behavior and functional benefits in patients with Alzheimer's disease (AD) following targeted micronutritional supplementation. OBJECTIVE: To build on the existing exploratory research and investigate the impact of these micronutrients on the natural progression of AD in a randomized controlled trial. METHODS: Patients with mild-moderate AD consumed daily 1 g fish oil (of which 500 mg DHA, 150 mg EPA), 22 mg carotenoids (10 mg lutein, 10 mg meso-zeaxanthin, 2 mg zeaxanthin), and 15 mg vitamin E or placebo for 12 months in a double-blind, placebo-controlled, randomized clinical trial. Carotenoids, ω-3FAs, and vitamin E were quantified in blood. Carotenoids were also measured in skin. AD severity was measured using the mini-mental state examination and dementia severity rating scale tools. Behavior, mood, and memory were measured using an informant-based questionnaire. RESULTS: Following 12 months of supplementation, the active group (n = 50) compared to the placebo group (n = 27), demonstrated statistically significant improvements in skin carotenoid measurements, blood carotenoids, ω-3FAs, and vitamin E concentrations (p < 0.05, for all). The active group also performed better in objective measures of AD severity (i.e., memory and mood), with a statistically significant difference reported in the clinical collateral for memory (p < 0.001). CONCLUSION: Exponential increases in the prevalence of AD and its relentless progressive nature is driving the need for interventions that help to ameliorate symptoms and improve quality of life in AD patients. Given the positive outcomes demonstrated in this trial, this combined micronutrient dietary supplement should be considered in the overall management of AD.

Intensive production of the harpacticoid copepod Tigriopus californicus in a zero-effluent 'green water' bioreactor.

Aquaculture is looking for substitutes for fishmeal and fish oil to maintain its continued growth. Zooplankton is the most nutritious option, but its controlled mass production has not yet been achieved. In this context, we have developed a monoalgal 'green water' closed-loop bioreactor with the microalgae Tetraselmis chui that continuously produced the harpacticoid copepod Tigriopus californicus. During 145 days of operation, the 2.2 m3 bioreactor produced 3.9 kg (wet weight) of Tigriopus with (dry weight) 0.79 ± 0.29% eicosapentaenoic acid (EPA), 0.82 ± 0.26% docosahexaenoic acid (DHA), 1.89 ± 0,60% 3S,3'S-astaxanthin and an essential amino acid index (EAAI) of 97% for juvenile Atlantic salmon. The reactor kept the pH stable over the operation time (pH 8.81 ± 0.40 in the algae phase and pH 8.22 ± 2.96 in the zooplankton phase), while constantly removed nitrate (322.6 mg L-1) and phosphate (20.4 mg L-1) from the water. As a result of the stable pH and nutrient removal, the bioreactor achieved zero effluent discharges. The upscaling of monoalgal, closed-loop 'green water' bioreactors could help standardize zooplankton mass production to supply the aquafeeds industry.

Omega-3 fatty acid, carotenoid and vitamin E supplementation improves working memory in older adults: A randomised clinical trial.

BACKGROUND & AIMS: Accumulating evidence suggests that omega-3 fatty acids (ω-3FAs), carotenoids and vitamin E can improve cognitive performance. However, their collective impact on cognition has not yet been investigated in healthy individuals. This study investigated the combined effect of ω-3FA, carotenoid and vitamin E supplementation on the cognitive performance of older adults. METHODS: Cognitively healthy individuals aged ≥65 years consumed daily 1 g fish oil (of which 430 mg docosahexaenoic acid, 90 mg eicosapentaenoic acid), 22 mg carotenoids (10 mg lutein, 10 mg meso-zeaxanthin, 2 mg zeaxanthin) and 15 mg vitamin E or placebo for 24 months in a double-blind, placebo-controlled, randomised clinical trial. RESULTS: Following 24-month supplementation, individuals in the active group (n = 30; aged 69.03 ± 4.41 years; 56.7% female) recorded significantly fewer errors in working memory tasks than individuals receiving placebo (n = 30; aged 69.77 ± 3.74 years; 70% female) (point estimate effect sizes ranged 0.090-0.105). Interestingly, as the cognitive load of the working memory tasks increased, the active group outperformed the placebo group. Statistically significant improvements in tissue carotenoid concentrations, serum xanthophyll carotenoid concentrations and plasma ω-3FA concentrations were also observed in the active group versus placebo (point estimate effect sizes ranged 0.078-0.589). Moreover, the magnitude of change of carotenoid concentrations in tissue, and ω-3FA and carotenoid concentrations in blood were related to the magnitude of change in working memory performance. CONCLUSION: These results support a biologically plausible rationale whereby these nutrients work synergistically, and in a dose-dependent manner, to improve working memory in cognitively healthy older adults. Increasing nutritional intake of carotenoids and ω-3FAs may prove beneficial in reducing cognitive decline and dementia risk in later life. STUDY ID NUMBER: ISRCTN10431469; https://doi.org/10.1186/ISRCTN10431469.

Environmental and Nutritional Determinants of Macular Pigment in a Mexican Population.

Purpose: The carotenoids lutein (L), zeaxanthin (Z), and meso-zeaxanthin deposit at the macula as macular pigment (MP) and provide visual benefits and protection against macular diseases. The present study investigated MP, its nutritional and environmental determinants, and its constituent carotenoids in serum from a Mexican sample, in healthy participants and with metabolic diseases. Additionally, we compared these variables with an Irish sample. Methods: MP was measured in 215 subjects from a rural community in Mexico with dual-wavelength autofluorescence imaging reported as MP optical volume (MPOV). Dietary intake and serum concentrations of L and Z were evaluated. Results: The mean MPOV was 8429 (95% confidence interval, 8060-8797); range. 1171-15,976. The mean L and Z serum concentrations were 0.25 ± 0.15 µmol/L and 0.09 ± 0.04 µmol/L, respectively. The MPOV was positively correlated with L and Z serum concentrations (r = 0.347; P < 0.001 and r = 0.311; P < 0.001, respectively), but not with L + Z dietary estimates. Subjects with daily sunlight exposure of more than 50% were found to have significantly higher MPOV than those with less than 50% (P = 0.005). MPOV and serum concentrations of L and Z were significantly higher in the Mexican sample compared with the Irish sample, but this difference was not reflected in dietary analysis. Conclusions: These new data from a Mexican sample provide evidence of the multifactorial interactions and environmental determinants of MP such as sunlight exposure and dietary patterns. These findings will be essential for future studies in Mexico for eye health, visual function, and ocular pathology.

Omega-3 nutraceuticals, climate change and threats to the environment: The cases of Antarctic krill and Calanus finmarchicus.

The nutraceutical market for EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) is promoting fishing for Euphasia superba (Antarctic krill) in the Southern Ocean and Calanus finmarchicus in Norwegian waters. This industry argues that these species are underexploited, but they are essential in their ecosystems, and climate change is altering their geographical distribution. In this perspective, we advocate the cessation of fishing for these species to produce nutraceuticals with EPA and DHA. We argue that this is possible because, contrary to what this industry promotes, the benefits of these fatty acids only seem significant to specific population groups, and not for the general population. Next, we explain that this is desirable because there is evidence that these fisheries may interact with the impact of climate change. Greener sources of EPA and DHA are already available on the market, and their reasonable use would ease pressure on the Arctic and Antarctic ecosystems.

Stereochemistry of Astaxanthin Biosynthesis in the Marine Harpacticoid Copepod Tigriopus Californicus.

The harpacticoid copepod Tigriopus californicus has been recognized as a model organism for the study of marine pollutants. Furthermore, the nutritional profile of this copepod is of interest to the aquafeed industry. Part of this interest lies in the fact that Tigriopus produces astaxanthin, an essential carotenoid in salmonid aquaculture. Here, we study for the first time the stereochemistry of the astaxanthin produced by this copepod. We cultured T. californicus with different feeding sources and used chiral high-performance liquid chromatography with diode array detection (HPLC-DAD) to determine that T. californicus synthesizes pure 3S,3'S-astaxanthin. Using meso-zeaxanthin as feed, we found that the putative ketolase enzyme from T. californicus can work with ß-rings with either 3R- or 3S-oriented hydroxyl groups. Despite this ability, experiments in the presence of hydroxylated and non-hydroxylated carotenoids suggest that T. californicus prefers to use the latter to produce 3S,3'S-astaxanthin. We suggest that the biochemical tools described in this work can be used to study the mechanistic aspects of the recently identified avian ketolase.

The Impact of Formulation on Lutein, Zeaxanthin, and meso-Zeaxanthin Bioavailability: A Randomised Double-Blind Placebo-Controlled Study.

Lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) have been the focus of research and commercial interest for their applications in human health. Research into formulations to enhance their bioavailability is merited. This 6 month randomised placebo-controlled trial involving 81 healthy volunteers compared the bioavailability of different formulations of free L, Z, and MZ in sunflower or omega-3 oil versus L, Z, and MZ diacetates (Ld, Zd, and MZd) in a micromicellar formulation. Fasting serum carotenoids, macular pigment, and skin carotenoid score were analysed at baseline and 6 months. Serum L, Z, and MZ concentrations increased in all active interventions compared to placebo (p < 0.001 to p = 0.008). The diacetate micromicelle formulation exhibited a significantly higher mean response in serum concentrations of Z and MZ compared to the other active interventions (p = 0.002 to 0.019). A micromicellar formulation with solubilised Z and MZ diacetates is a promising technology advancement that enhances the bioavailability of these carotenoids when compared to traditional carotenoid formulations (ISRCTN clinical trial registration number: ISRCTN18206561).

Targeted Nutritional Intervention for Patients with Mild Cognitive Impairment: The Cognitive impAiRmEnt Study (CARES) Trial 1.

Omega-3 fatty acids (ω-3FAs), carotenoids, and vitamin E are important constituents of a healthy diet. While they are present in brain tissue, studies have shown that these key nutrients are depleted in individuals with mild cognitive impairment (MCI) in comparison to cognitively healthy individuals. Therefore, it is likely that these individuals will benefit from targeted nutritional intervention, given that poor nutrition is one of the many modifiable risk factors for MCI. Evidence to date suggests that these nutritional compounds can work independently to optimize the neurocognitive environment, primarily due to their antioxidant and anti-inflammatory properties. To date, however, no interventional studies have examined the potential synergistic effects of a combination of ω-3FAs, carotenoids and vitamin E on the cognitive function of patients with MCI. Individuals with clinically confirmed MCI consumed an ω-3FA plus carotenoid plus vitamin E formulation or placebo for 12 months. Cognitive performance was determined from tasks that assessed global cognition and episodic memory. Ω-3FAs, carotenoids, and vitamin E were measured in blood. Carotenoid concentrations were also measured in tissue (skin and retina). Individuals consuming the active intervention (n = 6; median [IQR] age 73.5 [69.5-80.5] years; 50% female) exhibited statistically significant improvements (p < 0.05, for all) in tissue carotenoid concentrations, and carotenoid and ω-3FA concentrations in blood. Trends in improvements in episodic memory and global cognition were also observed in this group. In contrast, the placebo group (n = 7; median [IQR] 72 (69.5-75.5) years; 89% female) remained unchanged or worsened for all measurements (p > 0.05). Despite a small sample size, this exploratory study is the first of its kind to identify trends in improved cognitive performance in individuals with MCI following supplementation with ω-3FAs, carotenoids, and vitamin E.

The Role of Nutrition for the Aging Population: Implications for Cognition and Alzheimer's Disease.

Improved life expectancy worldwide has resulted in a significant increase in age-related diseases. Dementia is one of the fastest growing age-related diseases, with 75 million adults globally projected to develop the condition by 2030. Alzheimer's disease (AD) is the most common form of dementia and represents the most significant stage of cognitive decline. With no cure identified to date for AD, focus is being placed on preventative strategies to slow progression, minimize the burden of neurological disease, and promote healthy aging. Accumulating evidence suggests that nutrition (e.g., via fruit, vegetables, fish) is important for optimizing cognition and reducing risk of AD. This review examines the role of nutrition on cognition and AD, with specific emphasis on the Mediterranean diet (MeDi) and key nutritional components of the MeDi, namely xanthophyll carotenoids and omega-3 fatty acids. Given their selective presence in the brain and their ability to attenuate proposed mechanisms involved in AD pathogenesis (namely oxidative damage and inflammation), these nutritional compounds offer potential for optimizing cognition and reducing the risk of AD.

Analysis of Lutein, Zeaxanthin, and Meso-Zeaxanthin in the Organs of Carotenoid-Supplemented Chickens.

The macular carotenoids (i.e., lutein (L), zeaxanthin (Z) and meso-zeaxanthin (MZ)) exhibit anti-inflammatory, antioxidant and optical properties that are believed to support human health and function. Studying the accumulation and distribution of these nutrients in tissues and organs, in addition to the eye, is an important step in understanding how these nutrients might support global human function and health (e.g., heart and brain). Chicken is an appropriate animal model with which to study the accumulation of these carotenoids in organs, as the relevant transport molecules and carotenoid binding proteins for L, Z and MZ are present in both humans and chickens. In this experiment, a sample of 3 chickens that were supplemented with L and MZ diacetate (active group) and a sample of 3 chickens that received a standard diet (control group) were analysed. Both groups were analysed for L, Z and MZ concentrations in the brain, eyes, heart, lung, duodenum/pancreas, jejunum/ileum, kidney and breast tissue. L, Z and MZ were identified in all the organs/tissues analysed from the active group. L and Z were identified in all of the organs/tissues analysed from the control group; while, MZ was identified in the eyes of these animals only. The discovery that MZ is accumulated in the tissues and organs of chickens supplemented with this carotenoid is important, given that it is known that a combination of L, Z and MZ exhibits superior antioxidant capacity when compared to any of these carotenoids in isolation.

Stability of Commercially Available Macular Carotenoid Supplements in Oil and Powder Formulations.

We previously identified that the concentration of zeaxanthin in some commercially available carotenoid supplements did not agree with the product's label claim. The conclusion of this previous work was that more quality assurance was needed to guarantee concordance between actual and declared concentrations of these nutrients i.e., lutein (L) zeaxanthin (Z) and meso-zeaxanthin (MZ) in commercially available supplements. Since this publication, we performed further analyses using different commercially available macular carotenoid supplements. Three capsules from one batch of eight products were analysed at two different time points. The results have been alarming. All of the powder filled products (n = 3) analysed failed to comply with their label claim (L: 19-74%; Z: 57-73%; MZ: 83-97%); however, the oil filled soft gel products (n = 5) met or were above their label claim (L: 98-122%; Z: 117-162%; MZ: 97-319%). We also identified that the carotenoid content of the oil filled capsules were stable over time (e.g., L average percentage change: -1.7%), but the powder filled supplements degraded over time (e.g., L average percentage change: -17.2%). These data are consistent with our previous work, and emphasize the importance of using carotenoid interventions in oil based formulas rather than powder filled formulas.

Quantification of zeaxanthin stereoisomers and lutein in trout flesh using chiral high-performance liquid chromatography-diode array detection.

In our previous work we identified the presence of meso-zeaxanthin [(3R,3'S)-zeaxanthin] in trout flesh and skin (Nolan et al., 2014), but were not able to quantify this carotenoid with the method used at that time. In the present study, we developed a protocol that allows for the quantification of lutein and the three stereoisomers of zeaxanthin [(3R,3'R)-zeaxanthin, meso-zeaxanthin and (3S,3'S)-zeaxanthin] in fish flesh. We tested this protocol in two species of farmed trout (Oncorhynchus mykiss and Salmo Trutta), and we detected and quantified these carotenoids. The concentrations of each carotenoid detected (ranging from 1.18 ± 0.68 ng g-1 flesh for meso-zeaxanthin to 38.72 ± 15.87 ng g-1 flesh for lutein) were highly comparable for the two fish species tested. In conclusion, we report, for the first time, the concentrations of zeaxanthin stereoisomers (including meso-zeaxanthin) and lutein in trout flesh. This work adds further to the knowledge on the presence of these carotenoids in the human food chain.

Assessment of lutein, zeaxanthin and meso-zeaxanthin concentrations in dietary supplements by chiral high-performance liquid chromatography.

We investigated the concordance between actual and declared content of the three macular carotenoids in commercially available supplements aimed at eye health. Three batches of nine products were tested for content of lutein (L), zeaxanthin (Z) and meso-zeaxanthin (MZ) by chiral HPLC-DAD. In every product tested, actual L concentration was close to target, but Z concentration varied greatly (47-248 % of declared concentration), and the L:Z ratio within some supplements was adversely affected in consequence. In six of seven products not declaring MZ, we found this carotenoid, and four of them, using the same L source, contained a concentration of MZ that correlated positively and significantly with measured concentrations of L (r2 = 0.86; P < 0.001). More transparency is needed in terms of concordance between actual and declared concentrations of Z in commercially available formulations, and MZ should be declared in those formulations where it is present.

Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis.

Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7',8' double bonds adjacent to a 3-OH-ß-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-ß-apo-8'-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-ß-apo-8'-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the ß-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

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.

Deviation of the neurosporaxanthin pathway towards beta-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 gamma-carotene and beta-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 zeta-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.

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 (C(40)) into neurosporaxanthin (C(35)). 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 beta-carotene-accumulating cells, indicating a preference for acyclic or monocyclic carotenes. The purified CarT enzyme efficiently cleaved torulene in vitro to produce beta-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.

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi.

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.

A gene of the opsin family in the carotenoid gene cluster of Fusarium fujikuroi.

Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB. Transcription of carO is induced by light and is deregulated in carotenoid-overproducing mutants. The same regulation pattern is exhibited by carRA and carB; and common conserved DNA elements are found in the three promoters. Heat shock resulted in a modest induction of carO transcription, similar to the one exhibited by carB, confirming a common regulation. Targeted mutagenesis of carO produced no apparent phenotypic modification, including no change in the photoinduction of carotenoid biosynthesis.