Metabolic regulation of α-linolenic acid on β-carotene synthesis in Blakeslea trispora revealed by a GC-MS-based metabolomic approach

Abstract

Alpha-linolenic acid (ALA) is known for its ability to promote the production of β-carotene in Blakeslea trispora. However, the mechanism is still poorly understood. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate analysis were used to study mechanisms underlying the regulation effects of ALA on β-carotene synthesis in B. trispora. ALA treatment promoted the biomass of B. trispora and β-carotene production. The maximum β-carotene production 5.344 mg L⁻¹ was realized after 72 h of cultivation in the presence of 50 μL ALA. The intracellular metabolite profiles found upon treatment of the cells with different addition time points of ALA were unique and could be distinguished from the aid of principal component analysis (PCA). Furthermore, partial least-squares-discriminant analysis (PLS-DA) revealed a group classification and pairwise discrimination between the control and ALA treated groups, and 28 differential metabolites with variable importance in the projection (VIP) value greater than 1. The addition of ALA decreased the glycolysis, TCA cycle and fatty acid synthesis. The accumulation of linolenic acid and linoleic acid indicated that ALA was directly absorbed by the fungus and transformed into its own linolenic acid. As a result, the flux from acetyl-CoA to β-carotene synthesis increased. Besides, the addition of ALA increased the level of dissolved oxygen and the production of β-carotene.

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1. Introduction

Carotenoids are a family of natural pigments. Over 700 different kinds of pigments have been reported.¹ Carotenoids can be synthesized by all photosynthetic organisms and some non-photosynthetic organisms, such as bacteria, archaea, protozoa, yeasts, algae and fungi.^2–6 Carotenoids have strong singlet oxygen quenching and antioxidant properties which might protect biological structures from oxidative damage.^7,8 β-Carotene is one of the most widely studied carotenoid and has the highest pro-vitamin A activity among the 50 different carotenoids. It can be converted to vitamin A. β-Carotene has been used as coloring agent for food products, such as soft drinks, margarine, cakes, and baked goods.⁹ It can be produced by Rhodotorula glutinis, Phycomyces blakesleeanus and Blakeslea trispora. Compared with other microorganisms, B. trispora has the advantages of large biomass and high β-carotene content per unit biomass. Therefore, B. trispora is considered as the ideal β-carotene-producing microorganism.

Vegetable oil can promote the production of β-carotene in B. trispora. By adding the vegetable oil, the β-carotene production will be at least 3 times higher than the control. For example, the addition of soybean oil into the synthetic media will enhance the production of β-carotene.^10,11 However, Vereschagina et al. thought that only oils with high content of linoleic, especially linolenic acid, could enhance the formation of lycopene, which is the precursor of β-carotene.¹² We also found that linseed oil, which is rich in α-linolenic acid (ALA), had better effect on promoting β-carotene production in comparison with soybean oil and sunflower oil. Thus, ALA was chosen as a typical stimulator for β-carotene biosynthesis by B. trispora. However, the mechanism how ALA promoted the β-carotene production was unknown. Linolenic acid is a member of poly unsaturated fatty acids (PUFAs) and it is the important component of phospholipids in cell membrane and organelle membrane. It is known that the synthesis of fatty acids and β-carotene requires the common precursor, i.e. acetyl-COA. We speculate ALA can save the consumption of acetyl-COA, and then promote the carotenoid production. We also investigated the transcriptional levels of genes ACC (acetyl CoA carboxylase), carRA, carB and HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) in this study. ACC is the key gene in fatty acid biosynthesis. The genes of carRA, carB and HMGR are key genes in carotenoid biosynthesis pathway (Fig. S1†).

Metabolomics can reflect the intracellular biochemical changes induced by environmental stimulators and be coupled with phenotypic changes to unravel mechanisms underlying stimulation. Gas chromatography (GC)-based metabolomics strategy has been extensively used. Gas chromatography (GC) coupled with mass spectrometry (MS) has been extensively used in metabolomics because of its high separation efficiency and its capacity for accurate identification of hundreds of organic molecules by deconvoluting overlapping chromatographic peaks by the utilization of an AMDIS (Automated Mass Spectra Deconvolution and Identification System).^13–16 For example, Sun et al. and Hu et al. have used this technology to study the effects of trisporic acid and arachidonic acid on B. trispora.^17,18 These previous studies have shown that the metabolomics strategy is a powerful tool and provide a platform to gain insight into the molecular mechanism of microorganism's cellular response to environmental stress factors.

In the present study, a GC-MS-based metabolomics approach and multivariate data analysis were used to investigate the mechanism involved in the promotion of β-carotene production stimulated by ALA at different time points with and without ALA treatment in the B. trispora cell. The deeper understanding of the molecular mechanism underlying ALA promoting the β-carotene biosynthesis will provide us with more information on the metabolism regulation and the construction of industrial strains for improving the β-carotene production.

2. Materials and methods

2.1. Strains and culture conditions

B. trispora ATCC14272, mating type (−) was chosen for the study because (−) strain was the mainly strain to produce β-carotene. The (−) strain was grown on potato dextrose agar (PDA) Petri dishes at 28 °C for 3 days for sporulation. Ten milliliters of sterile distilled water was added to the Petri dish and the spores were collected by scraping the medium surface with bar coater. A spore suspension containing 1.0 × 10⁶ spores per mL was inoculated into 50 mL of liquid medium (consisted of 10 g of glucose, 5 g of yeast extract, 4 g of KH₂PO₄, 0.9 g of K₂HPO₄, 1 g of NH₄Cl, 0.25 g of MgSO₄·7H₂O and 1 L of distilled water).¹⁹ The pH of the medium was adjusted to 6.4 with NaOH (6 mol L⁻¹) and the medium was sterilized at 121 °C for 30 min. The glucose was sterilized alone at 115 °C for 30 min. The flasks were shaken at 220 rpm in a constant temperature oscillator at 28 °C.

2.2. β-carotene extraction and analytical method

After fermentation, the cells were filtered through muslin and dried in a vacuum dryer at 45 °C under 0.08 MPa for 48 h in prior to weighting. The cells were homogenized using mortar-pestle for extraction of β-carotene with petroleum ether until the solution was colorless. β-Carotene was analyzed by high performance liquid chromatography (HPLC) equipped with a Syncronis C18 column (250 mm × 4.6 mm) at 28 °C and acetonitrile : dichloromethane (75 : 25, v/v) with a flow rate of 1.5 mL min⁻¹ as the mobile phase. The absorption of β-carotene was detected at 450 nm. The β-carotene standard product was purchased from Sigma and its concentration was calculated by the standard curve.

2.3. Sampling, quenching, and intracellular metabolites extraction

Compared with the control, 50 μL of ALA (sigma, 98%) was added to the medium at the beginning of fermentation (this concentration of ALA had the best effect on improving the β-carotene production). Samples were taken after 36, 48 and 72 h from the fermentation medium. We harvested five biological replicates to analyze of intracellular metabolites for statistical validation. The mycelia was filtered through muslin and washed with normal saline (0.9%). Water was squeezed quickly from muslin and the mycelia were quenched in liquid nitrogen immediately. Then the mycelia were triturated by mortar and pestle with liquid nitrogen. About 100 mg powder was transferred into 1.5 mL Eppendorf tube, which contained 0.75 mL of cold pure methanol (−40 °C). The mixture was vortexed for 30 seconds and centrifuged at 10, 000 rpm for 10 min at −4 °C. The supernatant was collected into another Eppendorf tube. The aforementioned step was repeated. The supernatant was pooled together and stored at −80 °C for later use.

2.4. Derivatization

Sample derivatization was performed as Roessner et al. with a slight modification. 25 μL of 1 mg mL⁻¹ adonitol dissolved in Milli-Q water was added to each sample as an internal standard to correct for any loss of metabolite during the extraction process.²⁰ The solution of 0.5 mL of supernatant and 25 μL of adonitol was dried in the vacuum centrifuge dryer. Next, 100 μL of 20 mg mL⁻¹ solution of methoxyamine hydrochloride in pyridine was added to protect the carbonyl groups and incubated at 30 °C for 2 h. Subsequently, silylation was carried out by adding 100 μL of MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) with trimethychlorosilane (1%, v/v). The mixture was incubated for 1 h at 30 °C.

2.5. GC-MS analysis

GC-MS analysis was performed on an Agilent 7890-5975C GC-MS solution system (Agilent, USA) coupled to a DB-5 capillary column, 30 m × 250 μm (internal diameter) × 0.25 μm (film thickness). The MS was operated in scan mode (mass range 50–550 a.m.u. at 2 s per scan), solvent delay: 4 min. The injector temperature was 300 °C, and the carrier gas was helium at a constant flow rate of 1 mL min⁻¹. The oven temperature was initially held at 80 °C for 1 min. Thereafter the temperature was raised with a gradient of 10 °C min⁻¹ until it reached 160 °C, and then the temperature increased to 300 °C at a rate of 5 °C min⁻¹, which was held for 6 min. The ion source and ion source surface temperature were set at 200 °C and 280 °C, respectively. The injection volume was 1 μL, and the split ratio was 20 : 1. Full scan mode was used at a rate of 20 scans per s and mass scan range was 50–550 m/z. Metabolites in samples were identified by m/z compared with NIST mass spectral library.

2.6. Multivariate data analysis

Peak areas of each sample were normalized to that of internal standard adonitol on the same chromatograph. The SIMCA-P 10.0 package was used to analyze the multivariate data. Partial least squares discriminant analysis (PLS-DA) was applied to the data analysis after mean-centering and Par-scaling. R²X (cum) and Q² (cum) were used to evaluate the fit degree and the predictive ability of the models (Table S1†). Independent-samples T-test was used to test whether these metabolites had statistical significance by SPSS 19.0 (SPSS inc, USA).

2.7. Enzyme assays

The cell mass was washed with physiological saline (0.86% NaCl). A certain weight of wet cells was ground into powder with liquid nitrogen. According to the weight and volume ratio of 1 to 9, 10% of homogenate was made with physiological saline. Next, the 10% of homogenate was diluted into different concentration with physiological saline. The best sampling concentration was determined and centrifuged at 2500 rpm for 10 min at 4 °C. The supernatant was used for determining SOD and CAT enzyme activity. All the reagents were purchased from Nanjing Jiancheng Bioengineering Institute. The determination of enzyme activity was performed according to the instruction book of the reagent kits.

2.8. Real time PCR analysis

Samples were taken after 36, 39, 42 and 48 h fermentation and they were freezed in liquid nitrogen immediately and stored at −80 °C for use. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, USA). The cDNA synthesis reaction was performed using TaKaRa primescript™ RT master mix according to the manufacturer's instructions. The relative quantification was performed using the SYBR Green Realtime PCR Master Mix (TOYOBO) on a Mastercycler Realplex2 cycler (Eppendorf, Hamburg, Germany). The real-time PCR primers are presented in Table S2.† The real-time PCR cycling conditions were as follows: 95 °C for 5 min followed by 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. Measurements were performed in triplicate. The transcriptional levels of ACC, carRA, carB and HMGR genes obtained by real-time PCR were normalized to the tef1 gene and presented as relative to expression of the corresponding group without ALA treatment (value = 1) using the comparative method of Livak and Schmittgen.²¹

3. Results

3.1. Effects of ALA on the morphology, β-carotene production and cell growth

ALA was added to the fermentation medium at the beginning of the fermentation. After the addition, the medium color changed into milk white. Small pellets were formed by spores after 24 h inoculation because ALA decreased the cell membrane viscosity and increased the cell membrane fluidity. However, the spores in the control groups grew adhering to flask wall and then formed clumps. Therefore, the biomass was greatly improved with ALA treatment, and the biomass was more than 3 times higher than that of the control at 36 h (Fig. 1). With ALA treatment, while the biomass in the control began to decrease at 48 h, the biomass was kept constant from 48 h to72 h. The addition of ALA caused a 93.8% increase in β-carotene production in B. trispora. At the same time, the production of β-carotene reached the highest value at 5.344 mg L⁻¹.

Fig. 1 Biomass (a) and β-carotene content (b) with and without ALA treated. The error bar indicates the SD of three biological replicates.

3.2. Effects of ALA on the activity of antioxidant enzymes

We determined the SOD and CAT enzyme activities in order to explain the increase of dissolved oxygen after adding ALA. Compared with the control, ALA treated groups had higher SOD and CAT enzyme activities before 48 h (Fig. 2). Moreover, the two enzyme activities with ALA treatment had significant difference compared with the control at 36 h. The increased SOD and CAT enzyme activities showed that the dissolved oxygen really increased.

Fig. 2 The specific activity of SOD (a) and CAT (b) during β-carotene production by B. trispora. The error bar indicates the SD of three biological replicates.

3.3. Effects of ALA on the intracellular metabolite profiles of B. trispora

Metabolomics was used to investigate and establish the differences in β-carotene accumulation with and without ALA treatment on a metabolic level. Pellets can strongly influence metabolite production. In some instances, pellets are a prerequisite for successful production of secondary metabolites.²² The changes in intermediates would help us to explain the cellular metabolism accounting for ALA treatment. More than 200 peaks were selected and 60 compounds were identified as fatty acids, saccharides, organic acids, polyols, etc. The similarity of other peaks was lower than 75% and they were not identified as metabolites. Differences between the samples could be detected in the PLS scores plots (Fig. 3a, c and e). The major metabolic perturbations that cause these discriminations were identified from the line plots of the X-loadings of the first component of the PLS-DA models (Fig. 3b, d and f). The metabolite with a VIP (variable importance in the projection) value greater than 1 was seemed to have a significant contribution to separate the two groups by the models.²³ On the basis of parameter VIP > 1 and P-value < 0.05 by the T-test, 28 important differential metabolites and 22 compounds including carbohydrates (glucose), fatty acids (hexadecanoic acid, octadecanoic acid, 9,12-octadecanoic acid, linolenic acid and tetracosanoic acid), TCA cycle intermediates (malate, citrate), amino acids (Ala, Gly, Thr, Pro, Phe, α-aminoadipic acid, His, Lys and Tyr), and other compounds (phosphoric acid, glycerol, myo-inositol, adenosine and ergosterol) were identified (Table S3†). Compared with the control, the glucose content was higher with ALA treatment. The linolenic acid and linoleic acid contents were even 7 times and 3.8 times higher than the control, respectively. What's more, the content of TCA cycle intermediates, such as citrate, succinate, fumarate and malate, they all decreased. Besides, the amino acids (i.e. Lys, Pro and Thr) produced by the TCA cycle at the same time decreased at 36 and 48 h except for histidine (Fig. 4).

Fig. 3 PLS-DA model plots for the control group (red square) without ALA treatment versus ALA treatment group (black square) at each time point. Cross-validated scores scatter plots of the pairwise comparison between the control versus ALA-treated cells at (a) 36, (c) 48, and (e) 72 h, respectively. Loading line plots of pairwise comparison between the control versus ALA-treated cells at (b) 36, (d) 48, and (f) 72 h, respectively.

Fig. 4 Metabolic changes induced by ALA mapped onto metabolic pathways during the three different growth phases on 36, 48 and 72 h. Colored symbols represent an increase, decrease, or no change in the level of the metabolite at each time point, as compared to the control. The differential metabolites were marked in purple.

3.4. Quantitative analysis of transcriptional levels of ACC, carRA, carB and HMGR

For genes HMGR, carRA and carB, the maximum mRNA levels were obtained at 36 h after ALA addition when the transcriptional level of these genes had a great difference compared with the control (Fig. 5). Then the transcriptional level of these genes reduced gradually, but these genes were still up-regulated compared with the control. There was no difference in the gene transcription level compared with the control at 48 h. As the key gene in fatty acid biosynthesis, the gene ACC was significantly down-regulated at 42 and 48 h compared with the control. This result was consistent with our speculation that ALA can save the consumption of acetyl-COA to synthesis of fatty acids, and then promote the carotenoid production.

Fig. 5 Time courses of ACC, carRA, carB and HMGR transcripts in B. trispora after ALA addition. All results were standardized to tef1 mRNA steady-state levels. All results were the ratio of the ALA-treated group to the corresponding control group. The values are means of three independent experiments. * means significantly different (P < 0.05) and ** means great significantly different (P < 0.01).

4. Discussion

In the present study, the addition of ALA caused a great increase of biomass and β-carotene production in B. trispora. The levels of glucose in ALA-treated groups were higher than that of the control (Fig. 4). This result implied that the glucose consumption was less with ALA treatment. However, the biomass was over 3 times higher than the control at 36 h. This phenomenon could be explained from the following aspects. On the one hand, the addition of ALA made the spore form small pellets, which facilitated the transfers of oxygen and nutrient absorption and utilization.²⁴ B. trispora is a strictly aerobic microorganism. It was known that the spore formed small pellets rather than clumps with ALA treatment. The morphology of pellets increased the dissolved oxygen and it would be beneficial to the growth of B. trispora. On the other hand, with ALA treatment, the fungus had a large amount of linolenic acid and linoleic acid and their content were 7 times and 3.8 times respectively higher than the control. Our results agreed with the results in that the addition of exogenous lipids allowed an increase of total biomass and resulted in a higher production of microbial lipids.^12,25 We also found that the content of glycerol with ALA treatment only decreased at 36 h while its content showed no difference compared with the control at 48 and 72 h (Fig. 4). Glycerol, as a supplementary carbon source to glucose, was investigated in batch cultures for β-carotene production by B. trispora. According to Mantzouridou et al., the greater amount of glycerol was added, the more lipid accumulation was formed.²⁶ The main fraction of total lipids is expected to be in the form of TAGs in B. trispora.²⁷ Cellular TAGs represent the energy storage lipids and also the site where lipophilic carotenoids are deposited. Above all, glycerol is closely related with lipid accumulation. We speculated that glycerol and fatty acids were involved in the formation of membrane lipids.

Besides the biomass, the β-carotene content also increased by adding ALA in B. trispora. It was known that the spore formed small pellets rather than clumps with ALA treatment. The morphology of pellets increased the dissolved oxygen. The dissolved oxygen increase would induce oxidative stress increase. Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), hydroxyl radicals (HO˙), and superoxide radicals (O₂˙⁻), are formed by the fungus in the course of normal metabolic activity. Certain levels of ROS are important for physiological functions such as cell wall biosynthesis and cell growth. However, the excessive ROS was harmful. The aerobic organisms possess both enzyme systems (SOD and CAT etc.) and nonenzymatic antioxidants (ascorbic acid, glutathione, proline, trehalose, polyols, tocopherols, carotenoids and melanins) to defend the organism from exceeding oxidative stress.²⁸ They act as radical scavengers capable to remove oxidants from the cell. Oxidative stress would lead to the cessation of cell growth, and it could also provoke morphological changes, making the cells adapted to environmental changes as well as the decrease in intracellular oxidants.²⁹ From Fig. 2, we could find that the specific activity of SOD and CAT really increased especially at 36 and 48 h. This also meant the increase of dissolved oxygen and then induced oxidative stress increase. Therefore, the fungus would be induced to synthesize more β-carotene to protect the cell from oxygen damage. From the point of view of gene transcription, the genes HMGR, carRA and carB reached the maximum mRNA levels at 36 h, too. Although these genes' transcriptional level gradually reduced after 36 h, but they still up-regulated compared with the control. The results strongly suggested that ALA was a new stimulator for carotene production in B. trispora.

Carotenoid is one of the components of the cell membrane. β-Carotene molecules are randomly distributed in the hydrophobic interior of the lipid bilayer without preferred, well-defined orientation. This structure tended to increase membrane fluidity.³⁰ Fatty acids were also the components of the cell membrane. In B. trispora, the amount of unsaturated fatty acids, including linolenic acid and linoleic acid, was higher than other non-fatty acid components. The high content of unsaturated fatty acid made the cell membrane fluidity increase and allowed more β-carotene molecules to accumulate deeper into the interior of the bilayer.³¹ Thus, the large amount of unsaturated fatty acids promoted the carotenoid production.³² We found that linolenic acid and linoleic acid content with ALA treatment were respectively 7 times and 3.8 times higher than the control. However, hexadecanoic acid and octadecanoic acid content were almost as same as the control (Fig. 4). From the point of view of ACC transcriptional level, the mRNA level was no difference at 36 and 39 h, while decreased significantly from 42 h. Therefore, based on the above analysis, we speculated that the most of linolenic acid was not de novo synthesis and ALA might be directly absorbed by the fungus and transformed into its own linolenic acid and other fatty acids. It was known that fatty acids and β-carotene synthesis had the common precursor, such as acetyl-CoA. The TCA cycle provides NADPH and acetyl-CoA to synthesize fatty acids. Compared with the control, the content of TCA cycle intermediates, such as citrate, succinate, fumarate and malate, they all decreased. Besides, the amino acids (i.e. Lys, Pro and Thr) produced by the TCA cycle at the same time decreased at 36 and 48 h (Fig. 4). Because the fungus absorbed ALA to form its own fatty acid and they didn't have to use glucose de novo synthesis, TCA cycle didn't need to provide much more acetyl-CoA for fatty acid synthesis. Therefore, based on the above analysis we hold that the addition of ALA induced the decrease of TCA cycle flux and enabled more acetyl-CoA to synthesize β-carotene. Besides, we also found that the content of ergosterol was lower than the control with ALA-treatment (Fig. 4). Ergosterol is a special component of the yeast and fungal cell membrane, which has the same function as the cholesterol in the animal cell membranes. Cholesterol and carotenoids competed for the incorporation into bilayers. Only membranes with low cholesterol concentration allowed carotenoids to be incorporated into membranes effectively.³³ Therefore, if the ergosterol content is high in the cell membrane, the β-carotene content will decrease. Sun et al. used ergosterol biosynthesis inhibitors ketoconazole to limited ergosterol accumulation and enhanced the lycopene production.³⁴ Thus, ALA might have an inhibitory effect on ergosterol synthesis and then promoted β-carotene accumulation in the cell membrane.

Among these amino acids, the content of histidine with ALA treatment was much higher than the control at 36 and 48 h. Histidine has the ability to scavenge both the hydroxyl radical and singlet oxygen in many studies.^35,36 Histidine is generally recognized as the most active amino acid for scavenging singlet oxygen.³⁷ It is known the lipid-soluble antioxidants β-carotene and lycopene also play a role in scavenging singlet oxygen. Their mechanisms are different in that carotenoids are physical quenchers of singlet oxygen while the hydrophilic scavenger histidine interacts chemically with singlet oxygen. Based on the above proof, we supposed the increased content of histidine was related with its antioxidant property in B. trispora. Histidine might be another antioxidant compound in the cell of B. trispora. It has been reported that proline has antioxidant properties, too. Proline defended mycelium cells of Colletotrichum trifolii from the action of UV radiation, heat and salt stresses, and H₂O₂, preventing apoptosis.³⁸ Moreover, phenylalanine and tyrosine play a major role in cellular redox sensing and the antioxidant response by oxidative modification of an amino acid side chain.³⁹ In short, many amino acids had a close relation with the antioxidant activities.

In summary, ALA caused the spore to form small pellets and increased the dissolved oxygen which induced the increase of oxidative stress. Thus, the fungus will produce more β-carotene to sustain the stability of cell membrane. We employed metabolomics approach to study the metabolic profile at different time points in B. trispora and gained a better understanding of the changes of intracellular metabolites after ALA treatment. With ALA treatment, the content of linolenic acid was much higher than the control. This indicted that most of the linolenic acid was not de novo synthesis and this saved more acetyl-COA to synthesize β-carotene. Thus, the increased dissolved oxygen and the decreased consumption of acetyl-COA were the reason for the β-carotene increase by adding ALA. The knowledge gained from this study could prompt us in the future work to increase carotenoid production by metabolic engineering in microorganisms, for example, to down-regulate the TCA cycle and amino acid synthesis and increase the unsaturated fatty acid production such as linolenic acid.

Acknowledgements

The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (No. 21376017 and No. 21176018). The authors thank Mohamed Reda Zahi for revising this paper.

References

1. G. Britton, S. Liaaen-Jensen and H. Pfander, Carotenoids handbook, Birkhauser, Basel, Switzerland, 2004.
2. A. M. Yurkov, M. M. Vustin, B. V. Tyaglov, I. A. Maksimova and S. P. Sineokiy, Pigmented basidiomycetous yeasts are a promising source of carotenoids and ubiquinone Q10, Microbiology, 2008, 77, 1–6.
3. A. A. E. R. El-Banna, A. M. A. El-Razek and A. R. El-Mahdy, Isolation, identification and screening of carotenoid-producing strains of Rhodotorula glutinis, Food Nutr. Sci., 2012, 3, 627–633.
4. J. A. Del Campo, M. García-González and M. G. Guerrero, Outdoor cultivation of microalgae for carotenoid production: Current state and perspectives, Appl. Microbiol. Biotechnol., 2007, 74, 1163–1174.
5. V. Kuzina and E. Cerda-Olmedo, Ubiquinone and carotene production in the Mucorales Blakeslea and Phycomyces, Appl. Microbiol. Biotechnol., 2007, 76, 991–999.
6. K. Nanou, T. Roukas and E. Papadakis, Improved production of carotenes from synthetic medium by Blakeslea trispora in a bubble column reactor, Biochem. Eng. J., 2012, 67, 203–207.
7. P. Di Mascio, S. Kaiser and H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys., 1989, 274, 532–538.
8. P. Di Mascio, M. E. Murphy and H. Sies, Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols, Am. J. Clin. Nutr., 1991, 53, 194S–200S.
9. S. W. Kim, W. T. Sco and Y. H. Park, Enhanced synthesis of trisporic acids and β-carotene production in Blakeslea trispora by addition of non-ionic surfactant, Span 20, J. Ferment. Bioeng., 1997, 84, 330–332.
10. A. Ciegler, M. Arnold and R. F. Anderson, Microbiological production of carotenoids. V. Effect of lipids and related substances on production of beta-carotene, J. Agric. Food Chem., 1959, 7, 98–101.
11. S. M. Choudhari, L. Ananthanarayan and R. S. Singhal, Use of metabolic stimulators and inhibitors for enhanced production of β-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896, Bioresour. Technol., 2008, 99, 3166–3173.
12. O. A. Vereschagina, A. S. Memorskaya and V. M. Tereshina, The Role of Exogenous Lipids in Lycopene Synthesis in the Mucoraceous Fungus Blakeslea trispora, Microbiology, 2010, 79, 593–601.
13. S. G. Villas-Bôas, S. Mas, M. Åkesson, J. Smedsgaard and J. Nielsen, Mass spectrometry in metabolome analysis, Mass Spectrom. Rev., 2005, 24, 613–646.
14. U. Roessner, C. Wagner, J. Kopka, R. N. Trethewey and L. Willmitzer, Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass Spectrometry, Plant J., 2000, 23, 131–142.
15. J. M. Halket, A. Przyborowska, S. E. Stein, W. G. Mallard, S. Down and R. A. Chalmers, Deconvolution gas chromatography/mass spectrometry of urinary organic acids – potential pattern recognition and automated identification of metabolic disorders, Rapid Commun. Mass Spectrom., 1999, 13, 279–284.
16. S. E. Stein, An integrated method for spectrum extraction and compound identification from gas chromatography/mass spectrometry, J. Am. Soc. Mass Spectrom., 1999, 10, 770–781.
17. J. Sun, H. Li and Q. P. Yuan, Metabolic regulation of trisporic acid on Blakeslea trispora revealed by a GC-MS-based metabolomic approach, PLoS One, 2012, 7(9), e46110.
18. X. M. Hu, H. Li, P. W. Tang, J. Sun, Q. P. Yuan and C. F Li, GC-MS-based metabolomics study of the responses to arachidonic acid in Blakeslea trispora, Fungal Genet. Biol., 2013, 57, 33–41.
19. J. Wöstemeyer, Strain-dependent variation in ribosomal DNA arrangement in Absidia glauca, Eur. J. Biochem., 1985, 146, 443–448.
20. U. Roessner, A. Luedemann, D. Brust, O. Fiehn, T. Linke, L. Willmitzer and A. R. Fernie, Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems, Plant Cell, 2001, 13, 11–29.
21. K. J. Livak and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method, Methods, 2001, 25, 402–408.
22. S. Braun and S. E. Vecht-lifshitz, Mycelial morphology and metabolite production, Trends Biotechnol., 1991, 9, 63–68.
23. S. S. Szeto, S. N. Reinke, B. D. Sykes and B. D. Lemire, Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through ¹H NMR-based metabolic footprinting, J. Proteome Res., 2010, 9, 6729–6739.
24. Y. Zhou, J. Du and G. T. Tsao, Mycelial pellet formation by Rhizopus oryzae ATCC 20344, Appl. Biochem. Biotechnol., 2000, 84, 779–789.
25. F. Mantzouridou, M. Z. Tsimidou and T. Roukas, Performance of crude olive pomace oil and soybean oil during Carotenoid Production by Blakeslea trispora in submerged fermentation, J. Agric. Food Chem., 2006, 54, 2575–2581.
26. F. Mantzouridou, E. Naziri and M. Tsimidou, Industrial glycerol as a supplementary carbon source in the production of β-Carotene by Blakeslea trispora, J. Agric. Food Chem., 2008, 56, 2668–2675.
27. V. M. Tereshina, A. S. Memorskaya and E. P. Feofilova, Lipid composition of cells of heterothallic strains in the developmental cycle of Blakeslea trispora, Appl. Biochem. Microbiol., 2005, 41, 394–398.
28. D. J. Jamieson, Oxidative stress responses of the yeast Saccharomyces cerevisiae, Yeast, 1998, 14, 1511–1527.
29. N. N. Gessler, A. A. Aver'yanov and T. A. Belozerskaya, Reactive oxygen species in regulation of fungal development, Biochemistry, 2007, 72, 1091–1109.
30. J. Gabrielska and W. I. Gruszecki, Zeaxanthin (dihydroxy-b-carotene) but not β-carotene rigidifies lipid membranes: a H-NMR study of carotenoid-egg phosphatidylcholine liposomes, Biochim. Biophys. Acta, 1996, 1285, 167–217.
31. A. V. Popova and A. S. Andreeva, Carotenoid-lipid interactions, Adv. Planar Lipid Bilayers Liposomes, 2013, 17, 215–236.
32. J. J. L. Lee, L. W. Chen, J. H. Shi, A. Trzcinski and W. N. Chen, Metabolomic profiling of Rhodosporidium toruloides grown on glycerol for carotenoid production during different growth phases, J. Agric. Food Chem., 2014, 62, 10203–10209.
33. S. Carmen, J. Robert and A. D. Horst, Competitive carotenoid and cholesterol incorporation into liposomes: effects on membrane phase transition, fluidity, polarity and anisotropy, Chem. Phys. Lipids, 2000, 106, 79–88.
34. Y. Sun, Q. P. Yuan and F. Vriesekoop, Effect of two ergosterol biosynthesis inhibitors on lycopene production by Blakeslea trispora, Process Biochem., 2007, 42, 1460–1464.
35. A. M. Wad and H. N. Tucker, Antioxidant characteristics of L-histidine, J. Nutr. Biochem., 1998, 9, 308–315.
36. I. B. C. Matheson and J. Le, Chemical reaction rates of amino acids with singlet oxygen, Photochem. Photobiol., 1979, 29, 279–881.
37. C. S. Foote and R. W. Denny, Chemistry of singlet oxygen. VII. Quenching by β-carotene, J. Am. Chem. Soc., 1968, 90, 6233–6235.
38. C. Chen and M. B. Dickman, Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 3459–3464.
39. P. J. Winyard, C. J. Mooday and C. Jacob, Oxidative activation of antioxidant defence, Trends Biochem. Sci., 2005, 30, 453–461.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra08748a

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