Article pubs.acs.org/JAFC
Sesamol Enhances Cell Growth and the Biosynthesis and Accumulation of Docosahexaenoic Acid in the Microalga Crypthecodinium cohnii Bin Liu,†,§ Jin Liu,§,# Peipei Sun,† Xiaonian Ma,§ Yue Jiang,⊥ and Feng Chen*,§,Δ †
School of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, China Institute for Food and Bioresource Engineering, College of Engineering, Peking University, Beijing 100871, China # Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, Maryland 21202, United States ⊥ School of Food Science, Jiangnan University, Wuxi 214122, China Δ Singapore−Peking University Research Centre for a Sustainable Low-Carbon Future, CREATE Tower, Singapore 138602 §
S Supporting Information *
ABSTRACT: Sesamol is a strong antioxidant phenolic compound found in sesame seed. It possesses the ability to scavenge intracellular reactive oxygen species (ROS) and to inhibit malic enzyme activity and NADPH supply, resulting possibly in cell proliferation and alteration in the fatty acid composition. In the present study, the effect of sesamol on the growth and accumulation of docosahexaenoic acid (DHA) was investigated in the marine microalga Crypthecodinium cohnii, a prolific producer of DHA. C. cohnii showed a great decrease in the intracellular ROS level with the addition of sesamol. In contrast, the biomass concentration, DHA content (% of total fatty acids), and DHA productivity were significantly increased by 44.20, 11.25, and 20.00%, respectively (P < 0.01). Taken together, this work represents the first report of employing sesamol for enhanced production of DHA by C. cohnii, providing valuable insights into this alga for future biotechnological applications. KEYWORDS: Crypthecodinium cohnii, docosahexaenoic acid, microalgae, reactive oxygen species, sesamol
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INTRODUCTION
Docosahexaenoic acid (DHA, 22:6n-3), a very long chain polyunsaturated fatty acid, has attracted much attention as a food ingredient because of its broad beneficial effects on human health. DHA is an important component of the membranes of human nervous, visual, and reproductive tissues and is regarded as essential for the neurological development of infants.1−3 In addition, DHA plays an important role in alleviating cardiovascular diseases, hypertension, diabetes, and neuropsychiatric disorders.2−4 Currently, DHA is mainly produced from fish oils, but fish oils have certain disadvantages such as scarcity, unpleasant odor, and quality concerns. Efforts to explore microalgae as an alternative source of DHA have been made, such as screening of new strains, strain improvement by mutation, and culture condition optimization.5−7 Crypthecodinium cohnii, a flagellated marine microalga, has been considered as a prolific producer of DHA.8,9 The DHA biosynthetic pathway in C. cohnii, however, remains elusive.10,11 Conventionally, DHA is synthesized via a serial elongation and desaturation of oleic acid (C18:1), which is aerobic and requires NADPH-dependent acyl desaturases. In some organisms, DHA can be synthesized via an alternative pathway (Figure 1), which is catalyzed by a multifunctional complex resembling polyketide synthases (PKS) and requires no oxygen or NADPH-dependent desaturases.12,13 It has been suggested that C. cohnii might utilize the PKS route for the biosynthesis and accumulation of DHA.10,14,15 © XXXX American Chemical Society
Figure 1. Possible pathways of DHA biosynthesis in C. cohnii: FAS, conventional fatty acid synthase (FAS) route; PKS, polyketide synthase (PKS) route. In both routes, DHA is synthesized from acety-CoA and malonyl-CoA with the reducing power of NADPH. By comparison, about 46% less NADPH is required in the PKS route. Malic enzyme (ME), which can be inhibited by sesamol, is one of the most important NADPH sources in oleaginous microorganism. EL, elongase; DS, desaturase.
In past decades, efforts have been made to improve the production of DHA by C. cohnii, which can be achieved by enhancing either cell growth or intracellular DHA proportion.8,16 As indicated, DHA production by C. cohnii could be improved by the application of certain elicitors, for example, ndodecane, acetic acid, and ethanol.17−19 Intracellular DHA content of C. cohnii was improved significantly by the addition of n-dodecane in the medium.17 Both acetic acid and ethanol enhanced final cell density and DHA production.18,19 It has Received: March 20, 2015 Revised: May 25, 2015 Accepted: May 28, 2015
A
DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
250 °C for 3 min. FAMEs were identified by NIST 11 mass spectral library (NIST/EPA/NIH mass spectral library, 2011 edition). FAMEs were quantified by using a standard FAMEs mix (Sigma-Aldrich) with heptadecanoic acid as the internal standard. The response factors of representative fatty acids related to the internal standard are shown in Table S1 of the Supporting Information. Determination of Enzyme Activities. The cells were harvested by centrifugation at 3000g for 5 min. The pellet was resuspended in extraction buffer (containing 100 mM KH2PO4/KOH (pH 7.5), 20% (v/v) glycerol, 1 mM benzamidine·HCl, and 1 mM DTT) and ground with liquid nitrogen. The disrupted cell suspension was centrifuged at 12000g for 10 min at 4 °C, and the supernatant was used for enzyme analysis. The activities of NADP+-dependent malic enzyme (ME), NADP+-dependent isocitrate dehydrogenase (ICDH), and glucose-6phosphate dehydrogenase (G-6-PDH) were determined using continuous spectrophotometric assays following the increase of NADPH at 340 nm. All assays were carried out at 25 °C. ME, ICDH, and G-6-PDH were assayed as described by Hsu and Lardy,26 Wynn and Ratledge,24 and Langdon,27 respectively. The enzyme activity was defined as the reducing amount of NADP+ (nmol) catalyzed by the enzyme solution with 1 mg of protein in 1 min (nmol min−1 (mg protein)−1). The protein concentrations of enzyme solutions were determined according to the method of Bradford,28 with BSA as a standard. Determination of Intracellular Reactive Oxygen Species. The intracellular ROS levels in collected cells were determined using the ROS assay kit (Beyotime Institute of Biotechnology, China) according to the instructions. The 2′,7′-dichlorofluorescein (DCF) fluorescence intensity of 106 cells/mL was detected by the fluorescent microplate reader Fluoroskan Ascent (Thermo Fisher, USA) with the excitation and emission wavelengths at 485 and 535 nm, respectively. Statistical Analysis. The data of all the measurements were obtained from three repetitions and analyzed using a one-way analysis of variance (ANOVA) with subsequent post hoc multiple-comparison LSD tests, which were calculated by using SPSS Statistics 20 software. Statistically significant values were defined as P < 0.05.
been reported that antioxidants, such as vitamin C, hydrogen sulfide, ginsenoside, and sesamol, have great potential to enhance the proliferation capacity of a broad range of cells, likely due to their high ability to scavenge intracellular reactive oxygen species (ROS).20−22 Sesamol (3,4-methylenedioxyphenol) is a natural phenolic compound found in sesame oil. In addition to its potent antioxidation activity, sesamol is able to inhibit malic enzyme, which is a major source of NADPH in many oleaginous microorganisms.23,24 C. cohnii synthesizes saturated and monounsaturated fatty acids via a NADPH-dependent fatty acid synthase complex of enzymes. The application of sesamol attenuates the intracellular production of NADPH by inhibiting malic enzyme and thus may decrease the accumulation of these de novo synthesized fatty acids leading to the buildup of fatty acid precursors in C. cohnii. The precursors, on the other hand, maybe redirected to the biosynthesis of DHA via the desaturase-independent PKS pathway, resulting in the possible increase in DHA content. To validate this hypothesis for improved DHA production by C. cohnii, a set of sesamol concentrations were tested. Our work represents, to the best of our knowledge, the first effort to utilize the natural phenolic compound sesamol for enhanced DHA production by C. cohnii and may provide alternative directions to lower the production cost in future industrial applications.
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MATERIALS AND METHODS
Microalga and Culture Conditions. C. cohnii (ATCC 30556) was maintained heterotrophically in liquid By+ medium (pH 6.5) with 5 g/L glucose at 16 °C and subcultured every month. By+ medium contained (per liter) 15 g of sea salt (Sigma), 1 g of yeast extract (Oxoid), and 1 g of tryptone (Oxoid). The inocula were prepared by culturing the microalga in 500 mL Erlenmeyer flasks with 100 mL of By+ medium at 25 °C for 48 h with orbital shaking at 150 rpm in the dark. The seed of C. cohnii was inoculated to 100 mL of fresh modified By+ medium (containing, per liter, 15 g of sea salt, 0.5 g of yeast extract, 0.5 g of tryptone, and 20 g of glucose) for batch culture in 500 mL Erlenmeyer flasks. The media of all fermentations were supplemented with various concentrations of sesamol (0−2 mM), and each concentration was carried out in triplicate. The pH of By+ medium was adjusted to 6.5 prior to autoclaving. After inoculation, the flasks were incubated at 25 °C in an orbital shaker at 150 rpm in the dark for 96 h. Determination of Residual Glucose Concentration, Cell Dry Weight, and Specific Growth Rate. The cells were centrifuged at 3000g for 5 min. The concentration of glucose in the supernatant was determined according to the method of Miller.25 The pellet was washed with deionized water twice and filtered through a predried filter paper (Whatman GF/C). Then the cells on the disks were dried to constant weight in a vacuum oven at 80 °C. The specific growth rate (μ) in the log phase was calculated by using the equation μ = (ln X2 − ln X1)/(t2 − t1), where X1 and X2 are the cell dry weight concentrations (g/L) at time t1 and t2, respectively. Fatty Acid Analysis. Twenty milligrams of lyophilized algal cells was incubated in a solvent mixture (1 mL of toluene, 2 mL of 1% sulfuric aicd in methanol (v/v), and 0.8 mg of heptadecanoic acid in 0.8 mL of hexane as the internal standard) overnight at 50 °C. The formed fatty acid methyl esters (FAMEs) were then extracted three times with hexane in a reciprocating shaker. The FAMEs were analyzed by using a GC-MS-QP 2010 SE (electron ionization type) gas chromatograph−mass spectrometer (Shimadzu, Japan) and a Stabilwax-DA capillary column (30 m × 0.25 mm × 0.25 μm) (Shimadzu, Japan). Helium was used as the carrier gas. The injection temperature, ion temperature, and interface temperature were set at 250, 200, and 260 °C, respectively. The initial column temperature was set at 150 °C. The column temperature subsequently rose to 200 °C by 10 °C/min and then to 250 °C by 15 °C/min, followed by a hold at
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RESULTS AND DISCUSSION Effect of Sesamol on Cell Growth. In the present study, several sesamol concentrations of 0, 0.5, 1, 1.5, and 2 mM were tested to examine the effect of sesamol on the cell growth of C. cohnii. Compared with the control group (0 mM), the addition of 0.5 mM sesamol in the medium significantly enhanced the growth of C. cohnii, leading to an increase in biomass concentration by 44.20% (Figure 2A). No significant difference in biomass accumulation of C. cohnii was observed when the concentration of sesamol was increased from 0.5 to 1 mM (Figure 2A). Further increase in sesamol concentration to 2 mM, however, severely inhibited algal growth with a biomass concentration of only 0.06 g/L, indicating that high sesamol concentration (>2 mM) is lethal to the growth of C. cohnii (Figure 2A). Our results were in accordance with previous studies in which phenolic compounds promoted cell growth at appropriate concentrations and caused cell apoptosis at higher concentrations (>1 mM).29,30 Notably, in the presence of 1 mM sesamol, there was an obvious growth lag within the first 24 h of cultivation, as evident by the much lower amount of biomass accumulated (Figure 2B). Thereafter, sesamol induced a significantly greater specific growth rate (from 0.041 to 0.067 h−1), leading to a higher final cell density (Figure 2B). Microorganisms tend to accumulate ROS under certain conditions, such as nitrogen depletion.31 The buildup of ROS may have an adverse effect on cell growth and cause oxidative damage to cellular components.32 As indicated by Figure 3B, the intracellular ROS level in C. cohnii supplemented with sesamol was reduced by 63.5% B
DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Effect of sesamol on the growth of C. cohnii: (A) biomass concentration at 96 h of growth with different concentrations of sesamol; (B) cell growth curves (triangles) of C. cohnii and residual glucose concentration (diamonds) in medium growing with (solid) or without (open) 1 mM sesamol. Figure 3. Effect of sesamol on TFA accumulation ROS levels and TFA accumulation in C. cohnii: (A) TFA content as affected by different concentrations of sesamol; (B) time course of TFA accumulation (squares) and intracellular ROS levels (triangles) in C. cohnii growing with (solid) or without 1 mM sesamol (open); (C) cells growing in different concentrations of sesamol stained by Nile red under fluorescence (upper) and white light (lower). Lipids in C. cohnii can be stained by Nile red, and there is a positive correlation between the fluorescence intensity and lipid contents. The scale bar represents 20 μm.
at 24 h of cultivation and remained significantly lower at 48 h, as compared to the sesamol-free cultures. This result is in line with previous works that showed sesamol or other antioxidants have the potential to attenuate intracellular ROS levels, thereby enhancing cell growth.20,21 To check if the increase in the final cell density is related to the consumption of glucose, the residual glucose in the culture medium supplemented or not with sesamol was monitored, and the results are shown in Figure 2B. During the whole culture period of 96 h, no significant difference in glucose concentration was observed between the sesamol-containing and sesamol-free cultures, suggesting that the presence of sesamol had little effect on the glucose consumption rate by C. cohnii. Likely, in the presence of sesamol, the alga may possess a higher growth coefficient based on glucose (0.35 g/g versus 0.27 g/g) and transform more carbon per glucose to algal biomass. Effect of Sesamol on Fatty Acid Accumulation. In contrast to the enhanced biomass production, sesamol exhibited an adverse effect on the accumulation of fatty acids (Figure 3). The total fatty acids (TFA) content per cell dry weight was decreased by 25.24% in the presence of 0.5 mM sesamol. No significant difference was observed in the TFA content between the cultures with 0.5 and 1 mM sesamol; however, the further increase of sesamol concentration to 1.5 mM inhibited TFA accumulation remarkably, and the TFA content accounted for only 46.29% of the control cultures
without sesamol (Figure 3A,C). After staining with the lipophilic stain Nile red, the fluorescence intensity of C. cohnii also showed a decrease in response to sesamol in a concentration-dependent manner (Figure 3C). This decrease in fatty acid content was accompanied by the attenuation of ROS level in C. cohnii (Figure 3B), indicating that sesamol may inhibit fatty acid biosynthesis by the sequestration of the intracellular ROS, which is thought to be positively linked to lipid accumulation.33 Effect of Sesamol on Docosahexaenoic Acid Production. Sesamol exhibited significant effects on not only TFA accumulation but also fatty acid composition (Table 1). The fatty acids in C. cohnii consisted mainly of C14:0, C16:0, C18:0, C18:1, and C22:6, with C22:6 being the most abundant (36.97% of TFA). The addition of sesamol showed little influence on the percentage of saturated fatty acids (C14:0, C16:0, and C18:0). In contrast, there was a significant decrease in C18:1, which was sesamol concentration dependent and C
DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. Effect of Sesamol Concentration on the Fatty Acids Content of C. cohnii ATCC 30556a sesamol concentration fatty acid
0 mM
C14:0 C16:0 C18:0 C18:1 DHA
24.36 25.75 2.92 10.00 36.97
± ± ± ± ±
0.35 0.16 0.06 0.14 0.38
C14:0 C16:0 C18:0 C18:1 DHA TFA TSF Δ/mol
48.83 51.6 5.85 20.04 74.07 200.38 53.03 2.32
± ± ± ± ± ±
2.20 1.82 0.29 0.37 1.60 6.23
0.5 mM
1 mM
Fatty Acid Proportion (% of TFA) 21.32 ± 0.23 21.23 ± 0.66 25.24 ± 0.27 26.16 ± 1.27 2.84 ± 0.38 3.62 ± 1.43 9.48 ± 0.21* 8.97 ± 0.28** 41.13 ± 0.23** 40.02 ± 2.27* Fatty Acid Content (mg/g Cell Dry Weight) 31.96 ± 0.50 30.28 ± 1.85 37.83 ± 0.39 37.26 ± 1.33 4.25 ± 0.55 5.13 ± 1.91 14.21 ± 0.34 12.79 ± 0.77 61.66 ± 0.69 57.05 ± 4.14 149.92 ± 0.85 142.52 ± 4.20 49.40 51.01 2.56 2.49
1.5 mM 22.01 28.4 3.71 8.52 37.36
± ± ± ± ±
0.36 0.07 0.08 0.07*** 0.44
20.42 26.33 3.44 7.90 34.62 92.70 54.12 2.33
± ± ± ± ± ±
1.88 1.92 0.33 0.67 2.21 7.01
Cells of C. cohnii were collected at 96 h for FA analysis. TSF, total saturated fatty acids; Δ/mol, degree of unsaturation. Values were calculated according to ref 36. Δ/mol = [1.0 (% monoene) + 2.0 (% diene) + 3.0 (% triene)]/100. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
a
dropped from 10.00 to 8.50% in the presence of 1.5 mM sesamol (Table 1). On the other hand, DHA percentage of TFA was increased significantly by sesamol and reached 41.13% in the presence of 0.5 mM sesamol, which was 11.25% higher than the control cultures without sesamol. Notably, the sesamol-mediated enhancement in DHA percentage was observed during the whole culture period (Figure 4B). Our
results were in good agreement with those found in cultures of Mucor circinelloides and Nannochloropsis sp. that the C18:1 content (%TFA) decreased significantly in the presence of sesamol.23,34 From the weight content point of view, the presence of sesamol attenuated the content of all fatty acids, but DHA was much less affected as compared to other fatty acids (Table 1). Notably, because of the considerable increase in biomass, a final DHA yield of 0.23 g/L was obtained in the presence of 1 mM sesamol, still significantly higher than the control cultures (Figure 4B). In terms of DHA productivity, it was enhanced significantly by sesamol at concentrations of 0.5− 1 mM, but dropped remarkably when sesamol reached 1.5 mM (Figure 4A). Effects of Sesamol on the Activities of Key Enzymes Related to Fatty Acid Biosynthesis. It is well-known that the supply of reducing power in the form of NADPH plays an important role in fatty acid biosynthesis in oil-rich microorganisms.15 NADPH is mainly used for acyl chain growth and desaturation of fatty acids, and its supply is thought to be positively linked to fatty acid biosynthesis and accumulation.35 Efforts remain to be made to overcome the provision limitation of NADPH for enhancing lipid synthesis in oleaginous microorganisms.13 There are several intracellular enzymes involved in the generation of NADPH, including malic enzyme (ME), isocitrate dehydrogenase ICDH, and glucose-6-phosphate dehydrogenase (G-6-PDH). In C. cohnii, regardless of sesamol supplementation, the activity of ME increased as the culture time went and reached its maximum at 72 h, followed by a slight decline (Figure 5A). However, sesamol inhibited ME activity during the whole culture period, for example, 174.9 nmol min−1 (mg protein)−1 at 72 h, which is 37% less as compared to the control cultures. In contrast, there was no significant difference in ICDH activity between the cultures with and without sesamol, except at 72 h, when sesamol attenuated the activity of ICDH (Figure 5B). Interestingly, unlike Thraustochytrium sp., in which a high level of G-6-PDH activity was observed, we detected no G-6-PDH activity in all samples of C. cohnii. G-6-PDH is a key enzyme involved in the pentose phosphate pathway (PPP) and serves as the prime alternative for producing NADPH.13 Our results suggested that C. cohnii might utilize ME and ICDH rather than
Figure 4. Effect of sesamol on DHA accumulation in C. cohnii: (A) DHA productivity under different concentrations of sesamol; (B) time course of DHA percentage per TFA (circles) and volumetric DHA yield (squares) in C. cohnii with (solid) or without (open) 1 mM sesamol supplementation. D
DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
finding that sesamol has the potential to enhance DHA production by C. cohnii is scientifically interesting and provides valuable future directions for lowering production costs in industrial applications.
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ASSOCIATED CONTENT
S Supporting Information *
Response factors of the representative fatty acids related to the internal standard of C17:0 (Table S1). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01441.
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AUTHOR INFORMATION
Corresponding Author
*(F.C.) Phone: +86-10-62745356. Fax: +86-10-62757427. Email:
[email protected]. Funding
This study was supported by a 985 Project of Peking University, the 863 Plan of Ministry of Science and Technology of China (Grant 2012AA023107), Public Science and Technology Research Funds Projects of Ocean (Grant 201505032), and the National Natural Science Foundation of China (Grant 31471717). Notes
Figure 5. Effect of sesamol on the activities of ME (A) and ICDHC (B) of C. cohnii growing with 1 mM (solid) or without sesamol (open).
The authors declare no competing financial interest.
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REFERENCES
(1) Mayurasakorn, K.; Williams, J. J.; Ten, V. S.; Deckelbaum, R. J. Docosahexaenoic acid: brain accretion and roles in neuroprotection after brain hypoxia and ischemia. Curr. Opin. Clin. Nutr. Metab. Care 2011, 14, 158−167. (2) Karr, J. E.; Alexander, J. E.; Winningham, R. G. Omega-3 polyunsaturated fatty acids and cognition throughout the lifespan: a review. Nutr. Neurosci. 2011, 14, 216−225. (3) Gogus, U.; Smith, C. n-3 Omega fatty acids: a review of current knowledge. Int. J. Food Sci. Technol. 2010, 45, 417−436. (4) Chitranjali, T.; Anoop, P. A.; Kurup, G. M. Omega-3 fatty acid concentrate from Dunaliella salina possesses anti-inflammatory properties including blockade of NF-κB nuclear translocation. Immunopharmacol. Immunotoxicol. 2015, 37, 81−89. (5) Liu, J.; Sommerfeld, M.; Hu, Q. Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production. Appl. Microbiol. Biotechnol. 2013, 97, 4785−4798. (6) Ganuza, E.; Anderson, A. J.; Ratledge, C. High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fedbatch system. Biotechnol. Lett. 2008, 30, 1559−1564. (7) Lian, M.; Huang, H.; Ren, L. J.; Ji, X. J.; Zhu, J. Y.; Jin, L. J. Increase of docosahexaenoic acid production by Schizochytrium sp. through mutagenesis and enzyme assay. Appl. Biochem. Biotechnol. 2010, 162, 935−941. (8) Pleissner, D.; Eriksen, N. T. Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii. Biotechnol. Bioeng. 2012, 109, 2005−2016. (9) Kuratko, C. N.; Salem, N. Docosahexaenoic acid from algal oil. Eur. J. Lipid Sci. Technol. 2013, 115, 965−976. (10) Mendes, A.; Reis, A.; Vasconcelos, R.; Guerra, P.; da Silva, T. L. Crypthecodinium cohnii with emphasis on DHA production: a review. J. Appl. Phycol. 2009, 21, 199−214. (11) de Swaaf, M. E.; de Rijk, T. C.; van der Meer, P.; Eggink, G.; Sijtsma, L. Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by C-13 labelling and desaturase inhibitor experiments. J. Biotechnol. 2003, 103, 21−29. (12) Song, X. J.; Tan, Y. Z.; Liu, Y. J.; Zhang, J. T.; Liu, G. L.; Feng, Y. G.; Cui, Q. Different impacts of short-chain fatty acids on saturated
G-6-PDH to produce NADPH and that sesamol attenuated the intracellular NADPH level, to a large degree, by inhibiting ME activity instead of ICDH or G-6-PDH. So far, the DHA biosynthetic pathway is not completely defined in C. cohnii.11 DHA can be synthesized via the conventional route, which involves a series of aerobic NADPHdependent desaturation reactions (Figure 1). Alternatively, DHA biosynthesis can utilize the PKS route, which is thought to be anaerobic and desaturase-free and present in Schizochytrium sp. and certain related thraustochytrid marine protists.15 In our work, accompanied by the sesamol-mediated decrease in NADPH level, C. cohnii showed a drop in TFA content but an increase in DHA proportion per TFA (Figure 3A and Table 1). These results may suggest that C. cohnii is likely to employ, at least partially, the PKS pathway for DHA biosynthesis. In the presence of sesamol, NADPH production is attenuated due to the inhibition of malic enzyme, leading to a decrease in the accumulation of these de novo synthesized saturated and monounsaturated fatty acids and thus the buildup of fatty acid precursors in C. cohnii. The precursors, on the other hand, may be redirected to the biosynthesis of DHA via the desaturase-free PKS pathway, which required much less NADPH, resulting in the possible increase in the relative DHA content. Sesamol is well-known as a potent antioxidant and inhibitor of malic enzyme. In the present study, sesamol was used to improve DHA production by the marine microalga C. cohnii for the first time. Sesamol attenuated the intracelluar ROS level and promoted cell density, DHA proportion per TFA, and DHA productivity of C. cohnii, with concomitant decreases in NADPH level and TFA content. Our results suggest that C. cohnii, to a large degree, utilizes ME rather than ICDH or G-6PDH to produce NADPH for the de novo fatty acid biosynthesis. DHA, on the other hand, is likely synthesized, at least partially, via the alternative PKS pathway, which involves no oxygen or NADPH-dependent desaturases. The E
DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.5b01441 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
