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Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae
© The Author(s) 2018
Received: 7 April 2017
Accepted: 2 January 2018
Published: 13 January 2018
Dunaliella salina is a unicellular green alga with a high α-linolenic acid (ALA) level, but a low eicosapentaenoic acid (EPA) level. In a previous analysis of the catalytic activity of delta 6 fatty acid desaturase (FADS6) from various species, FADS6 from Thalassiosira pseudonana (TpFADS6), a marine diatom, showed the highest catalytic activity for ALA. In this study, to enhance EPA production in D. salina, FADS6 from D. salina (DsFADS6) was identified, and substrate specificities for DsFADS6 and TpFADS6 were characterized. Furthermore, a plasmid harboring the TpFADS6 gene was constructed and overexpressed in D. salina. Our results revealed that EPA production reached 21.3 ± 1.5 mg/L in D. salina transformants. To further increase EPA production, myoinositol (MI) was used as a growth-promoting agent; it increased the dry cell weight of D. salina transformants, and EPA production reached 91.3 ± 11.6 mg/L. The combination of 12% CO2 aeration with glucose/KNO3 in the medium improved EPA production to 192.9 ± 25.7 mg/L in the Ds-TpFADS6 transformant. We confirmed that the increase in ALA was optimal at 8 °C; the EPA percentage reached 41.12 ± 4.78%. The EPA yield was further increased to 554.3 ± 95.6 mg/L by supplementation with 4 g/L perilla seed meal (PeSM), 500 mg/L MI, and 12% CO2 aeration with glucose/KNO3 at varying temperatures. EPA production and the percentage of EPA in D. salina were 343.8-fold and 25-fold higher than those in wild-type D. salina, respectively.
FADS6 from Thalassiosira pseudonana, which demonstrates high catalytic activity toward α-linolenic acid, was used to enhance EPA production by Dunaliella salina. Transformation of FADS6 from Thalassiosira pseudonana into Dunaliella salina with myoinositol, CO2, low temperatures, and perilla seed meal supplementation substantially increased EPA production in Dunaliella salina to 554.3 ± 95.6 mg/L. Accordingly, D. salina could be a potential alternative source of EPA and is suitable for its large-scale production.
Recent clinical and epidemiological studies have indicated that polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5Δ5, 8, 11, 14, 17), are essential nutrients and play crucial roles in the treatment of various human diseases [1–3], such as neuropsychiatric disorders , rheumatoid arthritis , inflammatory diseases [6, 7], hypertension ,and cardiovascular diseases . Currently, marine fish oil is the richest source of EPA; however, the depletion of global fisheries, high cost of EPA purification, and pollution of the marine environment  have prompted a search for alternative sources. Microorganisms, including microalgae, fungi, and bacteria, are the primary producers of EPA; microalgae are the most abundant source of EPA. Many recent studies have developed technologies to accumulate EPA directly from various microalgae .
Furthermore, we investigated various factors influencing EPA production in D. salina. Inositols are potentially valuable supplements for microalgal growth, especially myoinositol (MI) [14, 15]. Carbon sources provide energy necessary for algal growth. D. salina can use CO2 as a carbon source under photoautotrophic conditions . D. salina is also capable of utilizing organic carbon sources, such as perilla seed meal (PeSM), which may promote its growth and/or EPA production . PeSM solubility reaches 80% at pH 8.0, and it contains comprehensive essential amino acids and has a high ALA content; accordingly, it is a potential substrate for EPA production in D. salina.
In this study, we identified the DsFADS6 gene from D. salina and characterized the substrate specificities for DsFADS6 and TpFADS6. Additionally, each FADS6 was overexpressed separately in D. salina. Furthermore, transgenic algae expressing each transgene were grown in Ben-Amotz medium to examine EPA production. Finally, we investigated the factors that influence EPA production by D. salina, including MI, CO2, low temperature, and PeSM supplementation.
Identification of DsFADS6 from D. salina
To identify genes encoding delta 6 desaturases involved in the biosynthesis of PUFAs in D. salina, a pair of degenerate primers was designed to target sequences corresponding to the heme binding motif of the cyt b5-like domain (HPGG) and the third His-rich motif (QIEHH) in DsFADS6. A 975-bp cDNA fragment from D. salina encoded a partial amino acid sequence containing a cyt b5-like domain in the N terminus and a His-rich motif in the C terminus; this region had high homology with delta 6 desaturases in other species.
To isolate the full-length cDNA clone, the insert was used as a probe to screen a cDNA library of D. salina. A full-length cDNA (DsFADS6) was identified by alignment and sequence analyses. The open reading frame of DsFADS6 was 1329 bp and it encoded 442 amino acids.
Characterization of the substrate specificity for DsFADS6 and TpFADS6
Fatty acid compositions (% w/w) of the total lipid contents of yeast transformants harboring the control plasmid (pYES2) and the recombinant plasmids (pYES2-DsFADS6 and pYES2-TpFADS6)
+LA and +ALA
+LA and +ALA
+LA and +ALA
22.9 ± 0.2
25.6 ± 0.2
20.5 ± 0.1
25.5 ± 0.4
21.9 ± 0.1
22.7 ± 0.3
23.4 ± 0.3
22.0 ± 0.6
24.0 ± 0.4
12.7 ± 0.3
14.6 ± 0.2
12.9 ± 0.3
14.4 ± 0.2
16.3 ± 0.3
11.8 ± 0.2
14.6 ± 0.2
14.4 ± 0.4
13.1 ± 0.6
11.4 ± 0.4
10.2 ± 0.6
11.8 ± 0.7
10.9 ± 0.4
12.5 ± 0.3
10.2 ± 0.4
12.0 ± 0.7
14.3 ± 0.5
9.3 ± 0.2
12.7 ± 0.8
10.3 ± 0.7
11.6 ± 0.4
12.5 ± 0.2
11.8 ± 0.6
12.6 ± 0.4
11.3 ± 0.4
11.2 ± 0.3
10.3 ± 0.4
18:2 (LA, ω-6)
38.2 ± 0.3
23.6 ± 0.1
29.6 ± 0.6
16.1 ± 0.2
11.5 ± 0.5
5.9 ± 0.6
18:3 (ALA, ω-3)
39.6 ± 0.5
21.2 ± 0.6
37.5 ± 0.2
21.6 ± 0.3
4.3 ± 0.6
2.7 ± 0.2
18:3 (GLA, ω-6)
7.2 ± 0.1
5.0 ± 0.1
27.3 ± 0.3
13.3 ± 0.3
18:4 (SDA, ω-3)
34.9 ± 0.4
16.7 ± 0.2
LA conversion ratea
24.3 ± 1.3
23.8 ± 1.8
70.4 ± 0.2
69.3 ± 0.3
ALA conversion ratea
89.0 ± 2.2
86.1 ± 1.3
When a single substrate was added, both substrates were catalyzed by TpFADS6 and their conversion rates were 70.4 ± 0.2 and 89.0 ± 2.2%, respectively. When both substrates were added simultaneously, the conversion rates of TpFADS6 for both substrates were similar to those observed when they were added individually, consistent with the results of Thierry et al. . These results show that TpFADS6 is highly capable of catalyzing the conversion of both substrates to GLA and SDA. However, TpFADS6 did not exhibit substrate specificity.
Substrate concentration has no effect on catalytic activity in yeast expressing DsFADS6; similarly, we found that there was no substrate specificity for TpFADS6, irrespective of fatty acid concentration (data not shown).
Overexpression of TpFADS6 in D. salina
To construct expression plasmids, a 1329-bp fragment was amplified using pGEM-FDs/pGEM-RDs primers, and a 1455-bp fragment was amplified using pGEM-FTp/pGEM-RTp primers. Both fragments were ligated into the pGEM-CAT vector to obtain the pGEM-CAT-DsFADS6 and pGEM-CAT-TpFADS6 vectors (Additional file 2: Fig. S1a, b). The resulting pGEM-CAT-DsFADS6 and pGEM-CAT-TpFADS6 vectors were electrotransformed separately into D. salina. After transformation, small green colonies appeared at a frequency of 10–20 colonies/plate after 15 days. Ten pGEM-CAT-DsFADS6 transformants and 10 pGEM-CAT-TpFADS6 transformants were picked and cultured in liquid medium.
The expression levels of the DsFADS6 and TpFADS6 genes in all samples (including wild-type [WT] D. salina, pGEM-CAT-DsFADS6 transformants, and pGEM-CAT-TpFADS6 transformants) were analyzed after a 15-day cultivation period. Based on RT-qPCR, the transcript level of the DsFADS6 gene in pGEM-CAT-DsFADS6 transformants was approximately two to ninefold higher than that in WT D. salina (Fig. 3c). The No. 9 pGEM-CAT-DsFADS6 transformant (Ds-DsFADS6-9) was used as a negative control; it had the highest transcript level of DsFADS6 (9.4-fold higher than that in WT D. salina). There were significant differences in the transcript levels of TpFADS6 among pGEM-CAT-TpFADS6 transformants. The No. 4 pGEM-CAT-TpFADS6 transformant (Ds-TpFADS6-4 transformant) had the highest TpFADS6 transcript level (8.5-fold higher than that in WT D. salina), followed by the Nos. 8 and 10 pGEM-CAT-TpFADS6 transformants (7.1- and 7.6-fold higher than that in WT D. salina, respectively), and the transcript level was lower for the other pGEM-CAT-TpFADS6 transformants (Fig. 3d). The fatty acid profiles of Ds-TpFADS6-4, Ds-TpFADS6-8, and Ds-TpFADS6-10 were examined.
Fatty acid profile of transformants
Principal fatty acid profiles of each D. salina transformant
C16 series (mg/L)a
The percentage of EPA (TFA %)
WT D. salina
266.0 ± 30.7
78.6 ± 1.6
41.2 ± 1.5
5.3 ± 0.2
26.6 ± 4.7
1.6 ± 0.2
1.91 ± 0.21
273.4 ± 22.0
73.2 ± 1.6
38.9 ± 2.6
5.4 ± 0.3
27.3 ± 2.5
1.5 ± 0.1
2.04 ± 0.14
261.8 ± 18.2
74.1 ± 4.9
37.6 ± 1.9
2.0 ± 0.4
2.6 ± 0.6
20.7 ± 3
28.12 ± 5.75
239.0 ± 33.5
79.9 ± 5.5
35.5 ± 2.6
2.1 ± 0.2
2.8 ± 0.2
21.3 ± 1.5
26.72 ± 2.26
274.5 ± 19.4
83.8 ± 3.3
38.9 ± 2.1
2.2 ± 0.2
3.3 ± 0
19.1 ± 2.3
22.9 ± 3.65
Biomass-enhancing effect of MI supplementation
Although TpFADS6 can promote the conversion of ALA to EPA in Ds-TpFADS6 transformants, the biomass of transformants was still low, limiting large-scale production. We attempted to resolve this problem by supplementing cultures with MI to stimulate the growth of transformants. Kichul et al. showed that supplementation with 500 mg/L MI had the greatest growth-promoting effect on D. salina (1.34 times higher growth than that of the control) .
Lipid enhancement by supplementation with glucose/KNO3 and CO2
Glucose and CO2 in the culture medium can be used as carbon sources for algal growth, and their levels have significant effects on lipid accumulation in D. salina [16, 18]. We combined CO2 with glucose/KNO3 and examined DCW and lipid accumulation in D. salina transformants.
Promoting the conversion of LA to ALA
To maximize the ALA level, we maintained the culture temperature at the optimal temperature (8 °C) from 20 to 25 days. The DCW and TFA levels at varying temperatures decreased slightly relative to those in a constant temperature (Fig. 7a, b). LA levels decreased sharply between 20 and 25 days, from 16.1 ± 1.7 to 7.3 ± 0.8 mg/L in WT D. salina (from 7.0 ± 0.8 to 2.1 ± 0.3 mg/L in the Ds-TpFADS6-8 transformant) (Fig. 7c). By contrast, ALA levels increased rapidly during this time period, from 115.6 ± 11.8 to 233.1 ± 24.1 mg/L in WT D. salina (from 6.4 ± 0.6 to 24.7 ± 2.1 mg/L in the Ds-TpFADS6-8 transformant) (Fig. 7d). We confirmed that the DsFADS15 enzyme presented high catalytic activity at 8 °C. The EPA level in the Ds-TpFADS6-8 transformant reached 216.8 ± 22.6 mg/L, which was only 24 mg/L higher than the yield obtained at a constant temperature (Fig. 7e). However, the percentage of EPA (TFA %) increased to 41.12 ± 4.78% at the end of cultivation, which was 1.5-fold higher than that observed at a constant temperature (27.64 ± 1.30%) (Fig. 7f).
EPA enhancement by PeSM supplementation
Most algae synthesize VLC-PUFA via the FADS6 pathway (Fig. 1) . Although D. salina is rich in ALA, the substrate specificity of DsFADS6 was unclear. We confirmed that it only catalyzed LA, and its conversion rate was low (24.3 ± 1.3%). In addition, TpFADS6 had no substrate specificity for the two substrates LA and ALA, but had a much higher ability to catalyze ALA and LA relative to that of FADS6 in Micromonas pusilla . Therefore, we reasoned that transformation of the TpFADS6 gene into D. salina may promote ω3-PUFA accumulation.
Cho et al. reported that exogenous genes can be easily introduced into D. salina . We achieved the successful transformation of D. salina with the DsFADS6 and TpFADS6 genes using the electroporation method. However, the expression levels of the genes varied among transformants. This variation has two potential explanations: the DsFADS6 or TpFADS6 gene was randomly inserted into the D. salina genome at one or more sites, or the integration sites were random.
Both LA and ALA levels decreased in TpFADS6 transformants, and ALA levels decreased more substantially. In contrast, EPA levels and the percentage of EPA (TFA %) increased. The conversion rate of ALA to EPA in TpFADS6 transformants was greater than that in S. cerevisiae. This may be explained by the optimization of codon usage for TpFADS6 in D. salina, but not in S. cerevisiae. It may be also explained by differences in the levels of LA and ALA in D. salina. TpFADS6 had a higher conversion rate for ALA in D. salina (which had higher levels of ALA) than in S. cerevisiae (in which similar levels of both substrates were added).
Many previous studies have shown that auxins play key roles in algal growth [15, 21]. In this study, the DCWs of WT D. salina and the transformants were significantly increased by MI supplementation, which was expected, as MI is involved in many physiological functions [15, 22]. MI is safe for use in humans and its cost is relatively low, supporting its use as a growth-promoting agent for large-scale PUFA production in D. salina. Many studies have reported that CO2 fixation rates in microalgae are much greater than those in terrestrial plants . Many microalgae tolerate CO2 levels of up to 12.0% , but algal growth can be affected by high CO2 levels . Our results were consistent with these previous results. At a high CO2 level, the DCW of D. salina may be decreased, owing to the reduced levels of dissolved oxygen in the medium, and lipid biosynthesis in D. salina may increase. Aeration with CO2 in D. salina culture medium not only increases lipid levels but also promotes the conversion of CO2 (inorganic substance) to lipids (organic substance).
Surprisingly, after Ds-TpFADS6 transformants were maintained at 4 °C for 5 days, their ALA levels increased and LA levels decreased. We reasoned that the activity of the DsFADS15 enzyme was enhanced at the low temperature, promoting the conversion of LA to ALA and increasing the ALA level. These findings are similar to those of Okuda , who observed accumulation of EPA in Mortierella alpina at a low temperature (below 15 °C). However, FADS15 in M. alpina had high activity for C20 substrates, whereas DsFADS15 had high activity for C18 substrates. Ds-TpFADS6 transformants exhibited greater EPA accumulation under varying temperatures than under constant temperatures, suggesting that the LA-ALA-EPA pathway was more active under varying temperatures, and there was less LA flux through the ω6 pathway. Additional studies are needed to clarify the molecular mechanism of DsFADS15 activity under low temperatures.
Many plant seeds are rich in ALA, such as linseeds, tree peony seeds, sesame seeds, and perilla seeds [27–30]; among them, perilla seeds have the highest ALA content . Accordingly, perilla seed oil has applications for both food and medicine. The byproduct of perilla seed oil processing (PeSM) is not widely used. Most PeSM is used as a protein source for animal feed . In addition to ALA, PeSM also contains a high protein content and many other bioactive compounds, making it an inexpensive and undervalued algal material. D. salina is capable of using organic nitrogen for its growth [32, 33], and PeSM is a suitable nitrogen source. The solubility of PeSM is much greater than that of peony seed meal (PSM), which is used for M. alpina growth . However, a high concentration of PeSM can still affect D. salina cell growth owing to increased levels of insoluble substances from PeSM in cultures under high concentrations.
EPA is found in a wide variety of marine microalgae. Recently, some remarkable findings in the generation of transgenic microalgae for enhanced EPA production have been reported [35–37]. D. salina is widely used for β-carotene production [38, 39]. In addition, it accumulates high levels of lipids and triacylglycerides. However, its EPA levels are low, and EPA production by D. salina has not been evaluated. In this study, several strategies were used to improve EPA accumulation by D. salina. However, investigations of EPA production by microalgae are still in the early stages, and an in-depth understanding of the factors that affect EPA production is still needed. Genetic engineering may be the most efficient means to improve EPA production in microalgae.
In this study, we identified FADS6 from D. salina. DsFADS6 had a preference for LA as a substrate and TpFADS6 had no substrate preference when expressed in S. cerevisiae. We successfully overexpressed the TpFADS6 gene in D. salina; to our knowledge, this represents the first report of EPA bioproduction via the ω3 pathway in D. salina. EPA production in Ds-TpFADS6 transformants was enhanced to various degrees by MI, CO2, low temperature, and PeSM supplementation.
Strains and plasmids
Dunaliella salina (D. salina) was newly isolated from the reef on the beach in the northeast region of China. TpFADS6 gene (GenBank accession AY817155) was synthesized by Shanghai Sunny Biotechnology Co. Ltd; INVSc1 yeast strain (Invitrogen) was used for heterogeneous expression and substrate preference determination. Plasmid pGEM-CAT was used for FADS6 expression in D. salina.
Media and cultural conditions
D. salina was grown at 26 °C on Ben-Amotz medium  in 12 h light (4500 lx)/12 h dark cycle. SC-U was synthetic minimal defined medium for Saccharomyces cerevisiae . Biomass-enhancing medium was the Ben-Amotz modified medium by supplementation with 500 mg/L myo-inositol (MI) . Lipid-producing medium was the Ben-Amotz modified medium by aerating with 12% CO2 level mixed with ambient air , and supplementation with 50 mM glucose (9 g/L) and 10 mM KNO3 (1 g/L) which was the result of our preliminary experiment for lipid production in D. salina. PeSM (1, 2 and 4 g/L) was added in lipid-producing medium as exogenous substrates for more lipid production, and was prepared by previous procedure for peony seed meal .
RNA isolation and gene synthesis
1 μg RNA of D. salina cells was reverse-transcribed with QuantScript RT Kit according to the manufacturer’s instruction. The cDNA transcribed was used as a template for DsFADS6 amplification with primers. Codon-optimized TpFADS6 gene was synthesized by Biotechnology Co. Ltd and subcloned into the vector pUC57 and transformed into DH5α.
Primer design, PCR amplification, and sequence analysis for DsFADS6 and TpFADS6
To identify genes encoding DsFADS6, a PCR-based cloning strategy was adopted. According to the available sequences information of FADS6 from Parietochloris incisa, Thalassiosira pseudonana Glossomastix chrysoplasta and Phaeodactylum tricornutum (GenBank accession nos. GU390532, AY817155, AAU11445 and AY082393), two highly degenerate primers (Additional file 1: Table S1) were designed to target sequences corresponding to the heme-binding motif of the cyt b5-like domain and the third His-rich motif in DsFADS6. The amplified product of expected length (DsFADS6 partial sequence, about 700 bp) was ligated into pMD19-T simple vector and then sequenced. DsFADS6 partial sequence obtained was used to do BLAST search on GenBank (NCBI). Two degenerate primers (Additional file 1: Table S1) were designed to clone the upstream sequence from HPGG and the downstream sequence from QIEHH. Both amplified products were sequenced and located the start codon and the stop codon. After the full length cDNA of DsFADS6 was amplified, it was then ligated into pMD19-T simple vector and sequenced.
For DsFADS6 and TpFADS6 gene amplification, primers were synthesized based on DsFADS6 and TpFADS6 gene sequences. The forward primers were FDs & FTp and the reverse primers were RDs and RTp (all primers are listed in Additional file 1: Table S1). PCR amplification, expression vector construction and sequencing were based on previous study , and recombinant plasmids were designated pYES2/NT C-DsFADS6 and pYES2/NT C-TpFADS6.
Yeast transformation, heterologous expression in S. cerevisiae, and determination of substrate preference for DsFADS6 and TpFADS6
pYES2/NT C-DsFADS6 and pYES2/NT C-TpFADS6 were transformed into S. cerevisiae using the lithium acetate transformation method . The selection procedure, SDS-PAGE gel and Western blotting for expression of DsFADS6 and TpFADS6 were analyzed as described previously . After induction, cultures were supplemented with substrates (as described previously ). Substrate concentration experiment, lipids extraction and determination were also analyzed as described previously .
Plasmid construction in D. salina
DsFADS6 and TpFADS6 genes were amplified with pGEM-FDs/pGEM-RDs and pGEM-FTp/pGEM-RTp primers, respectively, as shown in Additional file 1: Table S1. To construct an expression vector pGEM-CAT, the Ubiquitin-Ω (Ubil-Ω) promoter and nos terminator was amplified and cloned into the pGEM control vector to generate the expression vector pGEMΩ-CAT containing the Ubil-Ω promoter, nos terminator and CAT gene. DsFADS6 was digested and subcloned into Hind III and Xho I sites to generate a plasmid designated pGEMΩ-CAT-DsFADS6 while TpFADS6 was subcloned into EcoR I and Xho I sites to generate a plasmid designated pGEMΩ-CAT-TpFADS6. The expression plasmids (pGEMΩ-CAT-DsFADS6 and pGEMΩ-CAT-TpFADS6) were transferred into D. salina cells according to the method described by Wang et al. .
CAT assays and RT-qPCR analysis
The pre-procedure of CAT assays for stably D. salina transformants was followed by Wang’s description . RT-qPCR analysis procedure were followed as previously described . The transcript levels were calculated using the 2−ΔΔ Ct method .
PUFAs production for D. salina transformants
D. salina transformant cells in the liquid medium were grown for 30 days. The D. salina lysates obtained above were also used to determine dry cell weight (DCW) and fatty acid profiles in triplicates. FA profiles were investigated by using Gas Chromatography (GC) analysis as described previously . D. salina transformant with the highest EPA production and the control were grown in biomass-enhancing medium for biomass enhancement, and grown in the lipid-producing medium for lipid enhancement. Finally, they were separately grown in EPA-enhancing medium for more EPA production.
HS designed and carried out this work, and drafted the manuscript. XL analyzed the data and helped to draft the manuscript. RW supervised the research and helped to draft the manuscript. XY conceived the study and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
There was no new software, database or application/tool used in our manuscript.
Consent for publication
All authors have read and approved this version of the article, and consented for publication.
Ethics approval and consent to participate
This study was supported by the National Natural Science Foundation of China (No. 31471713) and Program for Liaoning Excellent Talents in University (LR2015059).
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