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Enrichment of long-chain polyunsaturated fatty acids by coordinated expression of multiple metabolic nodes in the oleaginous microalga Phaeodactylum tricornutum Xiang Wang, Yu-Hong Liu, Wei Wei, Xia Zhou, Wasiqi Yuan, Srinivasan Balamurugan, Ting-Bin Hao, Wei-Dong Yang, Jie-Sheng Liu, and Hong-Ye Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02397 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017
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Journal of Agricultural and Food Chemistry
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Enrichment of long-chain polyunsaturated fatty acids by coordinated expression
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of multiple metabolic nodes in the oleaginous microalga Phaeodactylum
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tricornutum
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Xiang Wang+, Yu-Hong Liu+, Wei Wei, Xia Zhou, Wasiqi Yuan, Srinivasan
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Balamurugan, Ting-Bin Hao, Wei-Dong Yang, Jie-Sheng Liu, Hong-Ye Li*
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Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher
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Education Institute, College of Life Science and Technology, Jinan University,
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Guangzhou 510632, China.
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Correspondence to H. Li: email:
[email protected]; tel: +86-20-85228470.
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+These authors contributed equally to this work.
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Abstract
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Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as
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promising alternatives to depleting fish oils. However, the overproduction of
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LC-PUFAs in microalgae has remained challenging. Here, we report a sequential
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metabolic engineering strategy that systematically overcomes the metabolic
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bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein
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transacylase, catalyzing the first committed step in type II fatty acid synthesis, and
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desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in
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Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs,
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with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to
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18.98 µg/mg and 9.15 µg/mg (dry weight), respectively. Importantly, eicosapentaenoic
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acid (EPA) was accumulated up to a highest record of 85.35 µg/mg by metabolic
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engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas
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DHA was found exclusively in phospholipids. Combinatorial expression of these
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critical enzymes led to the optimal increment of LC-PUFAs without unbalanced
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metabolic flux and demonstrated the practical feasibility of generating sustainable
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LC-PUFA production.
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Key words: metabolic engineering, coordinate expression, PUFA, MCAT, desaturase,
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microalga.
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Introduction
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In microalgae, fatty acids are initially synthesized in plastids and are desaturated to
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produce a wide array of unsaturated fatty acids, including the two essential fatty acids,
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linoleic acid (C18:2) and linolenic acid (C18:3), and their long chain (≥20 carbon)
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derivatives, long-chain polyunsaturated fatty acids (LC-PUFAs). Among them,
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omega-3 and -6 fatty acids, such as arachidonic acid (ARA; C20:4 ∆5,8,11,14),
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eicosapentaenoic acid (EPA; C20:5 ∆5,8,11,14,17) and docosahexaenoic acid (DHA;
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C22:6 ∆4,7,10,13,16,19), are of particular interest due to their established human
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health benefits, such as neonatal retinal and neurological development, cardiovascular
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protection, and blood pressure regulation
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predominantly synthesized by certain marine bacteria and microalgae and are
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aggregated in the lipids of marine fish by an aquatic food chain. Therefore, fish
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oils/lipids have been considered as the primary dietary source of these PUFAs.
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However, declining marine fish stocks and contamination of fish oils with toxic
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pollutants have provoked the development of sustainable and cost-effective
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alternative sources
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factories for the production of biofuels, pharmaceuticals, nutraceuticals, value-added
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food supplements and a wide array of commodity components through metabolic
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engineering
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possess the unique biosynthetic machinery to synthesize LC-PUFAs directly from
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malonyl-CoA by fatty acid synthases and the elongase-desaturase pathway 9. However,
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only a few microalgal species possess the capability to hyper-accumulate LC-PUFAs.
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. These LC-PUFAs are almost
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. Photosynthetic microalgae have emerged as the potential cell
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. Additionally, unlike terrestrial plants, numerous microalgal systems
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A strategy to overcome this econo-technical obstacle is needed, and this has prompted
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extensive research efforts, such as heterotrophic cultivation
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application of nutrient stress
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previous efforts, microalgal metabolic rewiring strategies have attracted attention as a
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potential tool to overproduce high levels of LC-PUFAs, which could provide an
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economically feasible source of LC-PUFAs. However, this requires the coordinated
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regulation of various key genes and the provision of sufficient precursors and
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cofactors. In this study, we report a potential strategy to overproduce LC-PUFAs,
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generating engineered microalgae that coordinately expressed MCAT (Malonyl
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CoA-acyl carrier protein transacylase
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malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis, and
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PtD5b (Desaturase 5b 19) simultaneously. Our findings successfully demonstrate that
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LC-PUFAs can be increased by 2.69-fold in the model oleaginous microalga P.
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tricornutum and provide insight into the generation of a novel metabolic circuit for
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the large-scale overproduction of high-value components.
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Materials and methods
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Microalga and cultivation conditions
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Phaeodactylum tricornutum (No: CCMP-2561) was acquired from Provasoli-Guillard
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National Center for Marine Algae and Microbiota. Algae were cultured in f/2 medium
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without Si and cultivated as reported previously 8. Microalgal density was measured
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by the direct-count method as reported previously 8.
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Gene cloning, vector design and co-transformation of recombinant vectors
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, light treatment
and metabolic engineering approaches
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11–13
,
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. Among
), catalyzing the formation of the
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The amino acid and nucleotide sequences of MCAT and PtD5b from P. tricornutum
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were retrieved from the National Center for Biotechnology Information (NCBI). The
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conserved domains of MCAT and PtD5b were predicted by SMART. The
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phylogenetic tree was constructed by MEGA7 using the neighbor-joining algorithm
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based on the amino acids of MCAT or PtD5b from several organisms retrieved from
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NCBI. The subcellular localization of MCAT and PtD5b was predicted by WoLF
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PSORT and LocTree3.
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Total RNA was extracted using the Plant RNA Kit (Omega, Norcross, GA, USA)
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according to the manufacturer’s instructions. The cDNA was transcribed from total
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RNA using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).
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The full-length coding regions of MCAT and PtD5b were PCR amplified, respectively,
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using primers (Table 1). The gene fragments were purified using the Gel Extraction
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Kit (Omega, Norcross, GA, USA) and were cloned separately into the expression
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vectors pHY-18 and pHY-21
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II one-step kit (Vazyme, Nanjing, China) according to the manufacturer’s protocol
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(Figure 1C and 1D). Both recombinant plasmids (pHY18-MCAT and pHY21-D5b)
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were electroporated into microalgae together using the GenePulser Xcell apparatus
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(Bio-Rad, Hercules, CA, USA) as described previously
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microalgae were first incubated in f/2 liquid medium for 48 h and then were screened
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on the f/2 solid medium plate with chloramphenicol (250 mg/L) and zeocin (100
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mg/L). The surviving colonies that appeared after one month were picked and
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transferred into fresh f/2 medium containing chloramphenicol and zeocin. The
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by homologous recombination using the ClonExpress
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. The transformed
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transformed lines were sub-cultured once a week in fresh f/2 medium supplemented
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with respective antibiotics for further purification and propagation. Microalgae
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cultivated in the f/2 medium without antibiotics were verified by genomic PCR and
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were subjected to further analyses.
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Molecular verification of engineered lines
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The relative transcript level of MCAT and PtD5b was determined by quantitative
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real-time PCR (qPCR) as previously described
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blotting was performed to detect the expression level of recombinant proteins in
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transformants according to the method described previously
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Cambridge, UK) and anti-flag (Sigma, St. Louis, MO, USA) were used as the primary
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antibodies. Endogenous β-actin protein was used as an internal reference, while the
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anti-β-actin (Sangon, Shanghai, China) was used as the primary antibody. The
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enzymatic activity of MCAT was also measured using the Plant MCAT Elisa Kit
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(Bangyi, Shanghai, China) following the manufacturer’s instructions.
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Photosynthetic efficiency
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The chlorophyll fluorescence parameter Fv/Fm (the maximum quantum efficiency of
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photosystem II) was measured using the Phyto-PAM phytoplankton analyzer (WALZ,
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Effeltrich, Germany) according to the manufacturer’s instructions.
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Lipid analysis
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The relative neutral lipid content of microalgae was first determined by Nile red
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(Sigma, St. Louis, MO, USA) staining as described previously 21. Briefly, 30 µL Nile
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red solution was added into 3 mL microalgae (v/v = 1:100), the mixture was incubated
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using primers (Table 1). Western
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. Anti-c-Myc (Abcam,
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for 20 min at 37°C in the darkness. All the stained samples, unstained samples and
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stained medium were added to 96-well plate and determined by a microplate reader
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(Bio-Tek, Winooski, VT, USA) at excitation wavelength of 530 nm and emission
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wavelength of 592 nm. Total lipids (TLs) of P. tricornutum were isolated according to
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the method described from Bligh and Dyer
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after N2 flow. TLs were fractionated by solid-phase extraction (SPE) into neutral lipid
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(NL), phospholipid (PL) and glycolipid (GL) using pre-packed silica cartridges
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(Waters, Milford, MA, USA) as described previously
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solvents were evaporated under a N2 stream, and the fractionated lipids were
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determined gravimetrically.
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and were determined gravimetrically
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. Upon fractionation, the
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Triacylglycerides (TAGs) were isolated by thin-layer chromatography using a
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solvent system of hexane/diethyl ether/acetic acid (v/v/v = 85:15:1). Isolated TAGs
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were visualized under iodine vapor. Silica containing TAG and fractionated lipids was
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gathered and trans-methylated
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GC-MS using methyl nonadecylate (Aladdin, Shanghai, China) as the FAME (fatty
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acid methyl ester) standard. For continuous illumination treatment, microalgae were
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incubated at 20 ± 0.5°C in an artificial climate incubator provided with cool-white
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fluorescence light of 200 µmol photons m-2 s-1 for 96 h, and the cells were harvested
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for fatty acid analysis according to the protocol mentioned above.
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Statistical analysis
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All of the experiments in this study were performed at least in triplicate, and the data
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are represented as the means ± standard deviation (SD). The statistical significance of
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. Fatty acid composition analysis was performed by
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the difference was tested by Student’s t-test and was indicated as p < 0.05 (*) or p
