Neurosporaxanthin overproduction by Fusarium fujikuroi and evaluation of its antioxidant properties

antioxidants Article Neurosporaxanthin Overproduction by Fusarium fujikuroi and Evaluation of Its Antioxidant Properties Obdulia Parra-Rivero 1, Marcelo Paes de Barros 2,3 , María del Mar Prado 1, José-Vicente Gil 2,4 , Dámaso Hornero-Méndez 5, Lorenzo Zacarías 2, María J. Rodrigo 2, M. Carmen Limón1and Javier Avalos 1,* 1 Department of Genetics, Faculty of Biology, University of Seville, 41012 Seville, Spain; [email protected] (O.P.-R.); [email protected] (M.d.M.P.); [email protected] (M.C.L.) 2Department of Food Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), 46980 Valencia, Spain; [email protected] (M.P.d.B.); [email protected] (J.-V.G.); [email protected] (L.Z.); [email protected] (M.J.R.) 3Interdisciplinary Program in Health Sciences, Institute of Physical Activity Sciences and Sports (ICAFE), Cruzeiro do Sul University, Rua Galvão Bueno 868, São Paulo SP 01506-000, Brazil 4Food Technology Area, Faculty of Pharmacy, University of Valencia, Burjassot, 46100 Valencia, Spain 5 Department of Food Phytochemistry, Instituto de la Grasa (IG-CSIC), 41013 Seville, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-954-557-110 Received: 21 May 2020; Accepted: 12 June 2020; Published: 16 June 2020   Abstract: 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 [O 2 ( 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. Keywords: carotenoids; xanthophylls; free radicals; oxidative stress; fungi; quenching; liposomes 1. Introduction Carotenoids are universally produced by photosynthetic species, but they are also synthesized by heterotrophic organisms, such as many bacteria and fungi [ 1 ]. Biosynthetic pathways for carotenoids are less diverse in fungi than in autotrophic species. In most of the cases investigated, it consists of the synthesis of β -carotene or some xanthophylls, such as astaxanthin, torularhodin and neurosporaxanthin [ 2 ]. Some fungi, such as Blakeslea trispora and Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) are biotechnological sources for the production of β -carotene and astaxanthin, respectively [ 3 ], but experimental facilities have made other fungi more suitable organisms to investigate carotenoid metabolism. Two of the best-known fungal models for carotenogenesis, Antioxidants 2020,9, 528; doi:10.3390/antiox9060528 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 528 2 of 17 Neurospora crassa and Fusarium fujikuroi, produce neurosporaxanthin (NX), a peculiar carboxylated carotenoid. This xanthophyll was described in N. crassa as an acidic pigment, found in a mixture with other carotenoids, and was later identified as the 35-C carboxylic apocarotenoid β -apo-4 0 -carotenoic acid [ 4 ]. NX provides a typical orange pigmentation to the N. crassa cultures, because of its accumulation in the airborne spores, the conidia, and a wealth of information has been generated over several decades on NX production in this fungus [ 5 ]. The genetic analyses of albino N. crassa mutants led to the identification of the first genes involved in this biosynthetic pathway, opening the investigation of their orthologs in other fungi [2,6]. NX was later also found in Fusarium species, such as F. aquaeductuum [ 7 ], F. fujikuroi [ 8 ] or F. oxysporum [ 9 ]. NX is not an abundant carotenoid in nature; in addition to Neurospora and Fusarium, it has been found in very few fungi, such as Verticillium [ 10 , 11 ] and Podospora [ 12 ]. F. fujikuroi is a fungus amenable for biotechnologists [ 13 ], and it is well-known as the industrial source of gibberellins, growth-promoting plant hormones. In recent years, F. fujikuroi has become also a reference model in the investigation of NX biosynthesis, and much information has been accumulated on the biochemistry and genetics of the pathway [ 14 ]. As in N. crassa, all the genes and enzymes needed to produce NX have been characterized in F. fujikuroi, and clear orthologs are found in the genomes of other Fusarium species. The intermediates identified in the carotenoid mixtures in F. fujikuroi are consistent with the biosynthetic pathway depicted in Supplementary Material (Figure S1). The first enzyme specifically involved in NX synthesis is phytoene synthase, whose enzymatic activity resides in the carboxy region (A domain) of the bifunctional polypeptide CarRA. Phytoene synthase condensates two geranylgeranyl diphosphate units to produce 15-cis-phytoene. This colorless precursor is subject of five desaturations by the enzyme CarB [ 15 ], and the cyclization of one end of the molecule by the cyclase domain of CarRA (R domain). The activities of CarRA and CarB lead to the formation of torulene, which is cleaved by the carotenoid oxygenase CarT to produce the apocarotenoid β -apo-4 0 -carotenal [ 16 ], which is finally converted to NX by the aldehyde dehydrogenase CarD [ 17 ]. In F. fujikuroi, a side branch of the pathway leads to retinal through the activity of a second carotenoid oxygenase, CarX. This enzyme cleaves symmetrically β -carotene, derived from a second cyclization on γ -carotene by CarRA (Figure S1). Retinal is presumably used as chromophore by F. fujikuroi rhodopsins CarO [ 18 , 19 ] and OpsA [ 20 , 21 ]. Illuminationisamajorstimulatingfactorin Fusarium carotenogenesis, andthecontrol ofexpression of the carotenoid genes by light has received detailed attention [ 22 ]. Carotenoid production is much lower without illumination, however mutants overproducing carotenoids are easily identified even in the dark because of their deep orange pigmentation. [ 9 ] These mutants, generically called carS, have been described in F. fujikuroi [ 23 ] and F. oxysporum [ 9 ]. They accumulate large amounts of carotenoids under all tested culture conditions, and contain larger amounts of transcripts for the genes of the carotenoid pathway [ 14 ]. Carotenoid production of the carS mutants is further enhanced under nitrogen starvation, a regulation that also acts at transcription level [ 24 ]. These mutants hold mutations in the gene carS, coding for a protein of the RING finger family, and reintroduction of a wild carS allele restores their normal carotenoid regulation [ 9 , 25 ]. In addition to being a negative regulator of carotenoid biosynthesis, CarS is involved in the control of a large set of Fusarium genes, many of them also regulated by light [26]. The molecular mechanism by which CarS controls carotenogenesis remains to be elucidated, but irrespective of their molecular basis, the carS mutants are very useful for the development of carotenoid overproducing conditions. Indirect evidences suggest a role for carotenoids in the defense against oxidative stress in fungi, both as scavengers of free radicals and quenchers of singlet oxygen [ 27 ]. This is probably also the case for NX in Fusarium and Neurospora, as indicates the induction of NX synthesis in F. aquaeductuum and N. crassa in the presence of hydrogen peroxide [ 7 , 28 ]. Moreover, mutants of the superoxide dismutase gene sod-1 of N. crassa accumulated more carotenoids than the wild type, following illumination [ 29 ]. However, no reports are available on the biochemical and biological properties of NX. The antioxidant activity of dietary carotenoids or carotenoid-enriched extracts from foods or plants has been extensively evaluated. It has been reported that carotenoids exert a high quenching Antioxidants 2020,9, 528 3 of 17 efficiency against singlet oxygen, [O 2 ( 1∆g )] [ 30 ]. Among others, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing ability of plasma (FRAP) methodologies are frequently used for measuring in vitro antioxidant activity of biological extracts. The DPPH assay is a radical scavenging methodology based on electron donations of antioxidants to neutralize relatively stable DPPH • radicals, whilst FRAP procedure measures the capability of the antioxidants in the extract for reducing ferric ion (Fe 3+ )-ligand complex to ferrous (Fe 2+ ) complex in acidic media [ 31 ]. In the last years, a new assay, singlet oxygen absorption capacity (SOAC), was developed to specifically determine the [O 2 ( 1∆g )] quenching capacity of an extract or a pure compound, and the high activity of carotenoids or carotenoids-enriched extracts capturing this reactive oxidizing intermediate was reported [ 32 – 35 ]. On the other hand, the use of liposomes in antioxidant analysis includes important structural aspects of carotenoids in membranes (localization, organization and orientation) that most of the in vitro antioxidant assays lacks [ 36 ]. Within liposomes, it is possible to reproduce some of the carotenoid-phospholipid interactions that occur in natural biological membranes, adding structural features of carotenoids to their inherent scavenging and quenching properties derived from the polyene chains. This study aims to investigate the antioxidant properties of NX. For this purpose, we have settled suitable overproduction conditions of this xanthophyll by a carS mutant of F. fujikuroi, and we have studied, by using different in vitro assays and liposomes, the antioxidant activity of cell extracts from the NX-enriched mutant, compared with those from the same strain to which the wild carS gene has been introduced, resulting in a very low NX content. 2. Materials and Methods 2.1. Strains and Culture Conditions The wild strains IMI58289 and FKMC1995 of Fusarium fujikuroi come from the Commonwealth Mycological Institute (Kew, UK) and the Kansas State University Collection (Manhattan, KS, USA), respectively. The carS mutants SG22 and SG39 were obtained from IMI58289 by chemical mutagenesis [ 23 ] and SF134 from FKMC1995 by the same procedure [ 17 ]. SG256 was derived from SG39 by reintroduction of a wild allele of the carS gene [26]. To improve carotenoid production, experiments were started in low-N medium, originally 10% ICI medium [ 37 ], containing 80 g/L D(+)-glucose, 0.48 g/L NH 4 NO 3 , 5 g/L KH 2 PO 4 , 1 g/L MgSO 4· 7H 2 O, and microelements. Changes in the composition, such as replacement of glucose by sucrose, changes in the nitrogen source, or reduction of KH 2 PO 4 content are described in the text. The optimized medium used to obtain mycelial samples for antioxidant analysis contained 80 g/L sucrose, 0.5 g/L NH 4 NO 3 , 2.4 g/L KH2PO4,1g/L MgSO4·7H2O, and microelements from 10% ICI medium. Mycelia were incubated in 250 mL of culture medium in 500-mL Erlenmeyer flasks. The flasks were inoculated with 10 6 conidia and grown at 30 ◦ C in the dark on an orbital shaker at 150 rpm. Conidia for inoculation of IMI58289 strains were obtained from mycelia grown for one week on EG agar medium [ 26 ] under light at 22 ◦ C. Conidia were collected in water by scrubbing the surface of the cultures with a sterile spatula, filtered through borosilicate filters VitraPOR Filter-Crucible, ROBU ® (Glasfilter-Geräte GmbH, Hattert, Germany) and counted in a haemocytometer (Bürker chamber, Blau Brand, Germany). To check growth of liquid cultures in flasks, 10-mL samples were filtered through Whatman paper and mycelial biomass was dried and weighed. Samples were also examined under microscope for the presence of conidia. When detected, they were counted in a haemocytometer. 2.2. Analyses of Carotenoids Mycelial samples were frozen at − 80 ◦ C and freeze-dried for 24 h. The dry samples were weighed and ground with sea sand in a mortar. Carotenoids were extracted with acetone up to bleaching of the samples, and dissolved in acetone as required, depending on the carotenoid concentration. Neutral and polar carotenoids (NX) were determined by the subtraction protocol, Antioxidants 2020,9, 528 4 of 17 as described [ 38 ]. Spectrophotometric measurements were recorded from 350 to 650 nm in a Shimadzu UV spectrophotometer 1800. In the case of the presence of bikaverins, they were precipitated by addition of 1:10 of 10 M NaOH to the acetone extract [ 39 ]. The total carotenoid content of SG256 was estimated using the extinction coefficient (E 1%, 1 cm) of the most abundant carotenoid, γ -carotene [ 7 ]. Total carotenoid contents in NX-rich samples were estimated using a global extinction coefficient of 2000, except for the analyses carried out in parallel for SG22 and SG39, in which their high NX contents were taken into account to use a global extinction coefficient of 1800. NX concentration in the SG39 and SG256 samples was estimated combining information from the areas of the peaks in the HPLC chromatograms and the spectrophotometric spectra from crude mycelial samples, using a NX extinction coefficient of 1715. Proportions of peak areas in chromatograms displayed in Results for SG39 were 88.7% for peak 1 (NX), 2.4% for peak 2 (torulene), 2.5% for peak 3 ( γ -carotene), 1.3% for peak 4 ( ζ -carotene) and 5.1% for peak 5 ( β -carotene). Percentages in the SG256 chromatogram were 10.6% for peak 1 (NX), 81.8% for peak 3 ( γ -carotene) and 7.6% for peak 5 ( β -carotene). Accordingly, 88.7% and 10.6% of the crude spectra were attributed to NX in the respective SG39 and SG256 extracts. Following the calculations, NX concentrations were estimated as 8.3 mg/g dry weight in SG39 and 0.003 mg/g dry weight in SG256. HPLC analyses were achieved as described [ 40 ], using a Waters 2695 HPLC equipment, fitted with a reversed-phase C18 column Mediterranea SEA18, 3 µ m, 20 × 0.46 cm (Teknokroma, Barcelona, Spain), a Waters2998 photodiode array detector, andcontrolledwith Empower2software (Waters Cromatograf í a, S.A., Barcelona, Spain). 2.3. Antioxidant Assays The antioxidant activity of carotenoid extracts from SG256 (colorless, with trace level of NX) and SG39 (NX-enriched) strains was evaluated in this study by their: (i) reaction with the 2,2-diphenyl- 1-picrylhydrazyl radical (DPPH • ) (DPPH assay); (ii) ferric-reducing activity in aqueous solution (FRAP assay); (iii) quenching activity against singlet oxygen [O 2 ( 1∆g )] generated in organic solvent (SOAC assay); and (iv) scavenging activity against hydroxyl radicals (HO•) in liposomes. The chemicals for these assays were obtained from Sigma-Aldrich (Barcelona, Spain), except the fluorescent probe C 11 -BODIPY 581/591 (Invitrogen, Thermo Scientific, Madrid, Spain), 1,4-naphthalene endoperoxide (EP) (InvitroTech, Kyoto, Japan), β -apo-8 0 -carotenal (Extrasynthese, Genay, France) and liquid chromatography grade solvents – n-hexane, chloroform, ethyl acetate, methanol, and ethanol – purchased from Merck Co. (Darmstadt, Germany). Graphics of liposome assays were plotted with software OriginPro 2016, 64-bit, Sr-2.5 (OriginLab Coorporation, Northampton, MA, U.S.A.). 2.3.1. DPPH and FRAP Assays Freeze-dried mycelia of SG256 (colorless with trace level of NX) and SG39 (NX-enriched) were extracted with 10 mL of 80% methanol or methanol and immediately used for DPPH and FRAP assays, respectively. For 2-2 diphenyl-1-picrylhydrazyl (DPPH • ) determinations, a 100 mM DPPH • methanolic solution was prepared, and 290 µ L were mixed with 10 µ L of the fungal extract and allowed to stand for 30 min at 27 ◦ C in complete darkness. Absorbance was measured at 515 nm and compared to the absorbance obtained for the control (blank without samples). DPPH • scavenging capacity was expressed as inhibition percentages by the formula: % DPPH•scavenging capacity =[(515nmAcontrol −515nmAsample)/515nmAcontrol]×100 FRAP reagent was prepared by mixing 300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tripyridyl-s- triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl 3 .6H 2 O in a ratio of 10:1:1 (by volume). The methanolic fugal extracts were appropriately diluted in methanol:water 8:1. The FRAP reagent (260 µ L) was mixed Antioxidants 2020,9, 528 5 of 17 with 40 µ L of the diluted extracts and incubated for 30 min at 37 ◦ C, and then absorbance at 593 nm was read and compared to the absorbance obtained for the control (FRAP reagent, with methanol:water 8:1). In both DPPH and FRAP assays, the reaction mix was prepared in a 96-well polystyrene microtiter plate (Fisher Scientific, USA) and absorbance measurements were carried out using a SPECTROstar Omega reader (BMG Labtech, Offenburg, Germany). 2.3.2. Quenching Activity The quenching activity of fungal carotenoid extracts was determined by the Singlet Oxygen Absorption Capacity Assay (SOAC), with some modifications [ 32 , 33 ]. Briefly, freeze-dried mycelial SG256 (colorless with trace level of NX) and SG39 (NX-enriched) were extracted with 5 mL of acetone. Equivalent volume aliquots of SG256 and SG39 acetone extracts were diluted 1:8 in cooled ethanol/chloroform/water mixture (50:50:1, v/v/v), and immediately used for assay. In each well of a 96-wellquartzmicroplateequippedwithlid(Espectrosil2000)andPTFEsealingfoil(Hellma, Muellheim, Germany) an aliquot of 15 µ L was mixed with 150 µ L of 0.10 mM 2,5-diphenyl-3,4-benzofuran (DPBF) and 75 µ L of 1.5 mM 1,4-naphthalene endoperoxide (EP). DPBF was used as an indicator of the [O 2 ( 1∆g )] quenching capacity, whereas EP generates [O 2 ( 1∆g )] by thermal decomposition in organic solvent. Absorbance changes of DPBF at 413 nm were monitored for 60 min at 35 ◦ C in a microplate SPECTROstar Omega reader. A standard solution of α -tocopherol ( α toc) was used to calculate relative SOAC values as: [(t1/2sample −t1/2blank)/(t1/2αtoc −t1/2blank αtoc)] ×[(αtoc, g/L)/(sample, g/L)] The estimated concentration of NX in the final reaction mixture of SG39 samples was 21 µ M, while in SG256 it was not detectable. For comparative purposes, a standard solution of β -carotene at final concentration of 26 µM in the reaction mixture was evaluated. 2.3.3. Scavenging Activity in Liposomes Standard solutions of β -carotene ( β C 273.3 µ g/mL) and β -apo-8 0 -carotenal (ApoC 283.3 µ g/mL), chemical structures included in the scheme depicted in Supplementary Material (Figure S1), were used for comparison with carotenoid extracts from SG256 (colorless) and SG39 (NX-enriched) strains, stored under N 2 atmosphere at − 80 ◦ C and protected from light to avoid oxidation. All solutions and extracts were prepared in chloroform (CHCl 3 ). NX concentration in the extracts of SG39 and SG256, determined spectrophotometrically, were 25.2 µg/mL and undetectable, respectively. For preparation of unilamellar liposomes, egg-yolk phosphatidylcholine (EYPC) was selected for 1.5 mM liposome preparation, since EYPC is a major phospholipid component of biological membranes and contains a significant percentage of polyunsaturated fatty acids (PUFAs), which are the main targets for oxidative damage by free radicals. Standard and fungal carotenoids in CHCl 3 were previously mixed with EYPC solutions (also in CHCl 3 ) to reach a final concentration of 10 µ M in the reaction mixture (carotenoid/phospholipid molar ratio <1.0%, to avoid carotenoid aggregation within liposomes). Control liposomes lack carotenoids. Chloroform was subsequently removed from lipid-carotenoid solutions by vacuum in a round-bottom flask adapted to a rotatory evaporator apparatus working at a low speed and moderate heating ( ≤ 30 ◦ C), to allow the formation of a homogeneous dried lipid film. The lipid-carotenoid film was stored overnight at − 20 ◦ C, in the dark, after flushing N2to avoid oxidation. In the next day, multilamellar liposomes were prepared by adding 50 mM phosphate saline buffer (PBS), pH 7.5, then agitated in vortex for 5 min, and exposed for 10 min in an ultrasound bath, at room temperature. After lipid-carotenoid film hydration, 25 µ L of the free radical-sensitive probe C 11 -BODIPY 581/591 stock solution, in methanol, were added to multilamellar liposomes, followed by additional 5 min vortex and 10 min ultrasound bath, to reach a final concentration of 10 µ M. After that, unilamellar liposomes were prepared by extrusion through 100 nm-pore polycarbonate membranes Antioxidants 2020,9, 528 6 of 17 (MilliPore, Burlington, MA, USA), at 37 ◦ C, in a mini-extruder device (Avanti Lipids. Co., Alabaster, AL, USA). After 15 passes, a clean, transparent and homogeneous unilamellar 1.5 mM EYPC liposomes suspension was obtained and immediately used in the oxidation assays. Reactivehydroxylradicals(HO • )wereproducedinaqueous solution, inthe presenceof unilamellar liposomes, by mixing 25 mM H 2 O 2 with 1.5 mM Fe 2+ :6 mM EDTA (Fenton reaction) in 50 mM PBS buffer, pH 7.5, 37 ◦ C. Liposome oxidation was monitored for 170 min by the fluorescence decay at 600 nm of the pre-loaded C 11 -BODIPY 581/591 probe in liposomes, as aforementioned [ 41 ]. Areas above curves (AAC) were integrated from time zero to 170 min (t 0 and t 170 , respectively) to quantify the extension of membrane oxidation in liposomes. 3. Results 3.1. Development of Conditions for Improved NX Production In a previous study, it was found that carotenoid production of carS mutants was about 4–5 times higher in a low-N than in a high-N medium [ 24 ], indicating that nitrogen is a negative regulator of the pathway. Since the carS mutants accumulate carotenoids constantly during cultivation [ 23 ], incubations of up to five weeks were carried out in low-N medium using the carS mutant SG22, obtained from the wild strain IMI58289 [ 23 ]. Previous experiments of the research group have used another wild strain, FKMC1995, which exhibits a better sporulation than IMI58289 on solid medium. The carS mutant SF134, previously obtained from this strain [ 17 ], was cultured in parallel with SG22. Both strains hold mutations in conserved residues of the carS gene [25]. The results showed an increase in carotenoid content during the first three weeks of culture (Figure 1a), reaching approximately 6 mg of carotenoids/g dry mass. The biomass kept growing, however, for 4 weeks (Figure 1b). Carotenoid production was lower in the mutant SF134, which also exhibited less biomass in the cultures (Figure 1b). This last result may be due to the dedication of resources to the production of conidia, which were detected in the SF134 cultures, but not in those of SG22 (Figure 1c). Most carotenoids accumulated in these culture conditions were polar, basically NX, as indicated the absorption spectrum of the samples. Not only the amount of carotenoid, but also the proportion of NX were higher in SG22 than in SF134. The medium used for this experiment contains glucose as a carbon source. Previous results showed that sucrose is a more efficient carbon source for the synthesis of other metabolites, such as bikaverin [42]. Therefore, experiments were performed in parallel with the two strains using sucrose instead of glucose. The results were similar to those obtained in glucose, except that the production of the SG22 strain was more efficient with sucrose (Figure 1d). Therefore, sucrose was used hereafter as a carbon source. The nitrogen source of the low-N medium was NH 4 NO 3 , and its concentration, 0.48 g/L, was low enough to be the limiting element, and therefore the first to run out during cultivation. Further experiments tested with different concentrations of NH 4 NO 3 showed that the production of carotenoids decreased when exceeding 1 g/L of NH 4 NO 3 (Figure 2a). Below 2 g/L, the biomass was proportionally lower, as less nitrogen was used (Figure 2b), as expected to be the limiting factor for growth. Although the highest amounts of total carotenoids were obtained with 1 g/L, the proportion of NX was lower, presumably due to lower aeration. Taking mycelial growth and NX production into account, we chose 0.48 g/L as an adequate NH 4 NO 3 concentration. Since NH 4 NO 3 provides two atoms of nitrogen per molecule, NaNO 3 and NH 4 Cl were also tested in equimolar amounts of N at 0.48 g/L of NH 4 NO 3 . Lower growth and carotenoid yields were observed with these nitrogen sources, and even a much lower proportion of NX in the case of NaNO3. Antioxidants 2020,9, 528 7 of 17 Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 17 Figure 1. Effect of carbon source on carotenoid production, growth and conidiation in low-N medium. Time course of carotenoid production (a,d), growth (b), and conidia concentration (c) in flask cultures of the carS mutants SG22 and SF134 in low-N medium with glucose or with the same amount of sucrose as carbon source. The data show the average and standard deviations from two determinations. The medium used for this experiment contains glucose as a carbon source. Previous results showed that sucrose is a more efficient carbon source for the synthesis of other metabolites, such as bikaverin [42]. Therefore, experiments were performed in parallel with the two strains using sucrose instead of glucose. The results were similar to those obtained in glucose, except that the production of the SG22 strain was more efficient with sucrose (Figure 1d). Therefore, sucrose was used hereafter as a carbon source. The nitrogen source of the low-N medium was NH 4 NO 3 , and its concentration, 0.48 g/L, was low enough to be the limiting element, and therefore the first to run out during cultivation. Further experiments tested with different concentrations of NH 4 NO 3 showed that the production of carotenoids decreased when exceeding 1 g/L of NH 4 NO 3 (Figure 2a). Below 2 g/L, the biomass was proportionally lower, as less nitrogen was used (Figure 2b), as expected to be the limiting factor for growth. Although the highest amounts of total carotenoids were obtained with 1 g/L, the proportion of NX was lower, presumably due to lower aeration. Taking mycelial growth and NX production into account, we chose 0.48 g/L as an adequate NH 4 NO 3 concentration. Since NH 4 NO 3 provides two atoms of nitrogen per molecule, NaNO 3 and NH 4 Cl were also tested in equimolar amounts of N at 0.48 g/L of NH 4 NO 3 . Lower growth and carotenoid yields were observed with these nitrogen sources, and even a much lower proportion of NX in the case of NaNO 3 . Figure 1. Effect of carbon source on carotenoid production, growth and conidiation in low-N medium. Time course of carotenoid production ( a , d ), growth ( b ), and conidia concentration ( c ) in flask cultures of the carS mutants SG22 and SF134 in low-N medium with glucose or with the same amount of sucrose as carbon source. The data show the average and standard deviations from two determinations. Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 17 Figure 2. Carotenoid production and growth in the mutant SG22 under different culture conditions. (a,b) Effect of nitrogen on concentrations of neutral (blue lower bars) and polar (orange upper bars) carotenoids and mycelial mass in the cultures. The strain was cultured in low-N medium with 80 g/L sucrose as carbon source and with the indicated nitrogen sources. The three bars on the right correspond to 0.48 g/L of NH 4 NO 3 , 1 g/L of NaNO 3 , and 0.64 g/L of ClNH 4 , respectively. (c) Effect of phosphate on carotenoid content and growth. Low-N medium contained sucrose instead of glucose and the indicated KH 2 PO 4 concentration. The data show the average and standard deviations from two determinations. The culture medium contains a high concentration of phosphate (5 g/L of KH 2 PO 4 ). Information on phosphate dependence for carotenoid production in Fusarium is missing, and therefore we tested lower concentrations of this compound (Figure 2c). To facilitate calculations, we decided to use hereafter 0.5 g/L NH 4 NO 3 as nitrogen source. The results showed growth inhibition only at phosphate concentrations below 0.5 g/L, indicating that phosphate is in excess in culture medium. Nevertheless, the concentration of carotenoids dropped below 2.4 g/L of phosphate, and therefore this was considered a suitable phosphate concentration for carotenoid production purposes. 3.2. Obtaining of NX-Enriched Extracts for Antioxidant Assays To check the antioxidant properties of NX, we took advantage of the high NX content of extracts of carS mutants of F. fujikuroi grown in Low-N medium with 80 g/L sucrose, 0.5 g/LNH 4 NO 3 and 0.5 g/L of KH 2 PO 4 (optimized medium). The available carS mutants were obtained by mutagenesis, and they may carry other secondary mutations. In a recent study about the effect of the carS mutation on the Fusarium transcriptome, a different carS mutant, SG39, was used in parallel with a complemented SG39 with the wild allele, called SG256, with low carotenoid content [26]. To test if SG39 was equally efficient under our conditions for high NX production, SG22 and SG39 and SG256 were grown in optimized medium. The results indicated that SG39 had even higher carotenoid production capacity than SG22, with similar crude spectra, but with carotenoid levels reaching about 9 mg/g dry mass (Figure 3a). In contrast, as expected from the complementation of the carS mutation in SG39, SG256 cultures contained very low amounts of carotenoids, ca. 0.018 mg/g dry mass, that is, ca. 400-fold less than the original SG39 mutant. Moreover, the absorption spectrum of SG256 biomass suggested the presence of minor amounts of yellowish carotenoids, with shorter absorption maxima than NX (inner spectrum in Figure 3a). Figure 2. Carotenoid production and growth in the mutant SG22 under different culture conditions. ( a , b ) Effect of nitrogen on concentrations of neutral (blue lower bars) and polar (orange upper bars) carotenoids and mycelial mass in the cultures. The strain was cultured in low-N medium with 80 g/L sucrose as carbon source and with the indicated nitrogen sources. The three bars on the right correspond to 0.48 g/L of NH 4 NO 3 , 1 g/L of NaNO 3 , and 0.64 g/L of ClNH 4 , respectively. ( c ) Effect of phosphate on carotenoid content and growth. Low-N medium contained sucrose instead of glucose and the indicated KH 2 PO 4 concentration. The data show the average and standard deviations from two determinations. Antioxidants 2020,9, 528 8 of 17 The culture medium contains a high concentration of phosphate (5 g/L of KH 2 PO 4 ). Information on phosphate dependence for carotenoid production in Fusarium is missing, and therefore we tested lower concentrations of this compound (Figure 2c). To facilitate calculations, we decided to use hereafter 0.5 g/L NH 4 NO 3 as nitrogen source. The results showed growth inhibition only at phosphate concentrations below 0.5 g/L, indicating that phosphate is in excess in culture medium. Nevertheless, the concentration of carotenoids dropped below 2.4 g/L of phosphate, and therefore this was considered a suitable phosphate concentration for carotenoid production purposes. 3.2. Obtaining of NX-Enriched Extracts for Antioxidant Assays To check the antioxidant properties of NX, we took advantage of the high NX content of extracts of carS mutants of F. fujikuroi grown in Low-N medium with 80 g/L sucrose, 0.5 g/LNH 4 NO 3 and 0.5 g/L of KH 2 PO 4 (optimized medium). The available carS mutants were obtained by mutagenesis, and they may carry other secondary mutations. In a recent study about the effect of the carS mutation on the Fusarium transcriptome, a different carS mutant, SG39, was used in parallel with a complemented SG39 with the wild allele, called SG256, with low carotenoid content [ 26 ]. To test if SG39 was equally efficient under our conditions for high NX production, SG22 and SG39 and SG256 were grown in optimized medium. The results indicated that SG39 had even higher carotenoid production capacity than SG22, with similar crude spectra, but with carotenoid levels reaching about 9 mg/g dry mass (Figure 3a). In contrast, as expected from the complementation of the carS mutation in SG39, SG256 cultures contained very low amounts of carotenoids, ca. 0.018 mg/g dry mass, that is, ca. 400-fold less than the original SG39 mutant. Moreover, the absorption spectrum of SG256 biomass suggested the presence of minor amounts of yellowish carotenoids, with shorter absorption maxima than NX (inner spectrum in Figure 3a). Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 17 Figure 3. Analysis of carotenoids in the SG22, SG39 and SG256 mycelial samples. (a) Absorption spectra of the cell extracts of the indicated strains. No absorption was detected for SG256 at the scale used for SG39 and SG22. The spectrum of a concentrated SG256 sample is shown in the left inner graph. Wavelength of spectrum peaks are indicated (nm). Carotenoid concentrations in SG22 and SG39 mycelia are represented in the right inner graph. Estimated neurosporaxanthin (NX) content in SG39 is indicated by the paler band shown behind. (b) Chromatogram of the HPLC separation of the carotenoids accumulated by the SG39 and SG256 strains of F. fujikuroi (detection wavelength at 450 nm). UV/Visible spectrum of the major peak is shown in the inner box. Relative areas of labeled peaks for SG39 and SG256 are indicated in Materials and Methods. B: bikaverin peak and spectrum. For comparison, the chromatogram of a sample from SG39 grown under standard culture conditions (DG minimal medium) is also shown [14]. The asterisk corresponds to an unknown carotenoid. HPLC analysis of the carotenoids accumulated by SG39 confirmed that NX is the major carotenoid under these culture conditions, whereas the NX precursors were found at very low concentrations (Figure 3b, upper chromatogram). The combination of spectrophotometric and HPLC data allowed us to estimate that about 89% of carotenoid absorption corresponds to NX (details in Materials and Methods), resulting in about 8.3 mg NX/g dry mass. Among the minor carotenoids, β-carotene predominated in SG39 extracts. This carotenoid pattern contrasts with previous analysis from the same strain growing on DG minimal medium, routinely used in our laboratory as standard culture conditions, which contains higher amounts of NX precursors (Figure 3b, second chromatogram). As expected from its crude spectrum, the carS-complemented strain SG256, contains only traces of carotenoids (Figure 3b, lower chromatogram), with γ-carotene as the major carotenoid and a very low NX content, estimated as 0.003 mg/g dry mass. A peak at a low retention time in the SG256 chromatogram, labeled as B, has an absorption spectrum (inner box) consistent with bikaverin [42]. This polyketide pigment is expected to be also present in SG39, but its occurrence is masked by the 400-fold higher concentration of carotenoids in this strain. As a result of the drastic differences in their NX content, extracts from SG39 and SG256 cultures obtained under these conditions were further used for antioxidant assays. 3.3. Antioxidant Assays The antioxidant activity of SG39 and SG256 extracts was initially determined by measuring their quenching activity against [O 2 ( 1 Δ g )] (SOAC assay, Figure 4a). Later, the scavenging activities of fungal extracts were measured by DPPH• and FRAP methodologies in methanolic:aqueous solutions (Figure 4b,c, respectively), and by scavenging HO• radicals in liposomes (Figure 5). Figure 3. Analysis of carotenoids in the SG22, SG39 and SG256 mycelial samples. ( a ) Absorption spectra of the cell extracts of the indicated strains. No absorption was detected for SG256 at the scale used for SG39 and SG22. The spectrum of a concentrated SG256 sample is shown in the left inner graph. Wavelength of spectrum peaks are indicated (nm). Carotenoid concentrations in SG22 and SG39 mycelia are represented in the right inner graph. Estimated neurosporaxanthin (NX) content in SG39 is indicated by the paler band shown behind. ( b ) Chromatogram of the HPLC separation of the carotenoids accumulated by the SG39 and SG256 strains of F. fujikuroi (detection wavelength at 450 nm). UV/Visible spectrum of the major peak is shown in the inner box. Relative areas of labeled peaks for SG39 and SG256 are indicated in Materials and Methods. B: bikaverin peak and spectrum. For comparison, the chromatogram of a sample from SG39 grown under standard culture conditions (DG minimal medium) is also shown [14]. The asterisk corresponds to an unknown carotenoid. Antioxidants 2020,9, 528 9 of 17 HPLC analysis of the carotenoids accumulated by SG39 confirmed that NX is the major carotenoid under these culture conditions, whereas the NX precursors were found at very low concentrations (Figure 3b, upper chromatogram). The combination of spectrophotometric and HPLC data allowed us to estimate that about 89% of carotenoid absorption corresponds to NX (details in Materials and Methods), resulting in about 8.3 mg NX/g dry mass. Among the minor carotenoids, β -carotene predominated in SG39 extracts. This carotenoid pattern contrasts with previous analysis from the same strain growing on DG minimal medium, routinely used in our laboratory as standard culture conditions, which contains higher amounts of NX precursors (Figure 3b, second chromatogram). As expected from its crude spectrum, the carS-complemented strain SG256, contains only traces of carotenoids (Figure 3b, lower chromatogram), with γ -carotene as the major carotenoid and a very low NX content, estimated as 0.003 mg/g dry mass. A peak at a low retention time in the SG256 chromatogram, labeled as B, has an absorption spectrum (inner box) consistent with bikaverin [ 42 ]. This polyketide pigment is expected to be also present in SG39, but its occurrence is masked by the 400-fold higher concentration of carotenoids in this strain. As a result of the drastic differences in their NX content, extracts from SG39 and SG256 cultures obtained under these conditions were further used for antioxidant assays. 3.3. Antioxidant Assays The antioxidant activity of SG39 and SG256 extracts was initially determined by measuring their quenching activity against [O 2 ( 1∆g )] (SOAC assay, Figure 4a). Later, the scavenging activities of fungal extracts were measured by DPPH • and FRAP methodologies in methanolic:aqueous solutions (Figure 4b,c, respectively), and by scavenging HO • radicals in liposomes (Figure 5). Altogether, SG39 extracts showed significant higher antioxidant activity than SG256 extracts: 6-fold higher DPPH • scavenging activity, 2-fold higher FRAP capacity and comparable SOAC scores with β -carotene, whereas no SOAC activity was detected in SG256 extracts. Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 17 Altogether, SG39 extracts showed significant higher antioxidant activity than SG256 extracts: 6-fold higher DPPH• scavenging activity, 2-fold higher FRAP capacity and comparable SOAC scores with β-carotene, whereas no SOAC activity was detected in SG256 extracts. Figure 4. Antioxidant activities of extracts from SG39 (NX-enriched) and SG256 (NX-poor control) fungal strains against: (a) singlet oxygen [O 2 ( 1 Δ g )], by quenching activity in singlet oxygen absorption capacity (SOAC) assay; (b) 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) radical in aqueous solution; and (c) Fe 2+ -mediated free radicals in aqueous solution, by ferric reducing ability of plasma (FRAP) assay. Abbreviations: 256, SG256; 39, SG39; βC, β-carotene; n.d.: not detectable; A.U.: arbitrary units. Data are presented as mean and ± standard deviation, (x ± SD, n ≥ 3). *p<0.05 (statistical analysis performed with the t-Student’s test at significance level of 5%). Control experiments were carried out to characterize the oxidation pattern of C 11 -BODIPY 581/591 in EYPC liposomes under 25 mM H 2 O 2 + 1.5 mM Fe 2+ treatment (Supplementary Material 2, Figure S2). As shown in SM2, the liposomes are promptly oxidized by Fe 2+ addition, although a slight spontaneous oxidation is also evident in the presence or absence of 25 mM H 2 O 2 , both in the absence of the redox catalyst Fe 2+ . As additional controls, no differences were observed between any carotenoid:liposome system and EYPC liposomes (without carotenoids or fungal extracts) in the absence of Fe 2+ to trigger the Fenton reaction (data not shown). The index of membrane oxidation was determined by the fluorescence decay of C 11 -BODIPY 581/591 probe (Figure 5a) in 1.5 mM EYPC liposomes containing either 10 µM β-carotene (PCL: βC), 10 µM β-apo-8′-carotenal (PCL:ApoC), or 10 µM NX-enriched SG39 (compared to SG256) extracts by HO• radicals formed from the Fenton reaction (25 mM H 2 O 2 + 1.5 mM Fe 2+ :6 mM EDTA) in 50 mM PBS buffer, pH 7.5. The extension of membrane oxidation was quantified from t 0 to t 170 , as the area above curves of Figure 5a in all liposomal systems (Figure 5b). NX-rich SG39 extracts prevented liposome oxidation triggered by HO• radicals in aqueous solution, whereas PCL:βC (−27%), PCL:ApoC (−19%), and SG256 extracts (−30%) partially inhibited the progression of membrane oxidation, as depicted in Figure 5b. Figure 4. Antioxidant activities of extracts from SG39 (NX-enriched) and SG256 (NX-poor control) fungal strains against: ( a ) singlet oxygen [O 2 ( 1∆g )], by quenching activity in singlet oxygen absorption capacity (SOAC) assay; ( b ) 2,2-diphenyl-1-picrylhydrazyl radical (DPPH • ) radical in aqueous solution; and ( c ) Fe 2+ -mediated free radicals in aqueous solution, by ferric reducing ability of plasma (FRAP) assay. Abbreviations: 256, SG256; 39, SG39; β C, β -carotene; n.d.: not detectable; A.U.: arbitrary units. Data are presented as mean and ± standard deviation, (x ± SD, n ≥ 3). * p<0.05 (statistical analysis performed with the t-Student’s test at significance level of 5%). Antioxidants 2020,9, 528 16 of 17 43. 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