The wild-type M. alpina ATCC 32222 strain was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and conserved in our lab; the M. alpina uracil-auxotrophic strain (CCFM501, Culture Collection of Food Microorganisms, Jiangnan University, Jiangsu Province, PR China) was modified from wild-type M. alpina in our lab (Hao et al., 2014). Agrobacterium tumefaciens C58C1 was provided by Yasuyuki Kubo (Kyoto Prefectural University, Kyoto, Japan) and used as a transfer DNA (T-DNA) donor for fungal transformation. The Escherichia coli TOP10 strain was used for plasmid storage. The pYES2/NT C plasmid containing the galactose-inducible GAL1 promoter was purchased from Invitrogen (Carlsbad, CA, USA); the pBIG2-ura5s-ITs plasmid was modified from pBIG2RHPH2 (provided by Yasuyuki Kubo).

Saccharomyces cerevisiae cultures were grown in yeast extract/peptone/dextrose medium at room temperature for 2 days with constant agitation at 200 rpm. The S. cerevisiae strain INVSc 1 (his3Δ1/his3Δ1 leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52) was grown on YEP medium (10 g L⁻¹ tryptone, 10 g L⁻¹ yeast extract, and 5 g L⁻¹ NaCl) at 28°C. Wild-type M. alpina ATCC 32222 was grown on GY solid medium (30 g L⁻¹ glucose, 5 g L⁻¹ yeast extract, 2 g L⁻¹ KNO₃, 1 g L⁻¹ NaH₂PO₄, and 0.3 g L⁻¹ MgSO₄⋅7H₂O). The auxotrophic CCFM 501 strain was maintained on GYU solid medium (30 g L⁻¹ glucose, 5 g L⁻¹ yeast extract, 2 g L⁻¹ KNO₃, 1 g L⁻¹ NaH₂PO₄, and 0.3 g L⁻¹ MgSO₄⋅7H₂O supplemented with 0.05 g L⁻¹ uracil) at 25°C. Transformation media included synthetic complete (SC) medium, minimal medium (MM), and induction medium (IM) (Michielse et al., 2008). For fatty acid accumulation, each transformant and wild-type M. alpina were grown for 7 days at 28°C in broth medium (50 g L⁻¹ glucose, 5 g L⁻¹ yeast extract, 10 g L⁻¹ KNO₃, 1 g L⁻¹ KH₂PO₄, and 0.25 g L⁻¹ MgSO₄⋅7H₂O, pH 6.0).

The PaD17 sequence was optimized based on the codon preferences of S. cerevisiae; the resulting codon-optimized gene, oPAD17 (1,077 bp in length), was synthesized and sub-cloned into the pUC57-simple vector. The oPAD17 F/oPAD17 R primer pair (Table 1) was used to amplify oPAD17 under PCR cycling conditions of 94°C for 3 min, 30 cycles of amplification at 94°C for 30 s, 58°C for 30 s, and 68°C for 1.5 min, and a final extension at 68°C for 7 min. The oPAD17 coding sequence fanked with restriction endonuclease sites EcoR I and Xho I was ligated into pYES2/NT C (with T7 promoter and T7 terminator) to yield the plasmid pYES2/NT C-oPAD17. The insert was confirmed by sequencing, after which the plasmid containing oPAD17 was electro-transformed into E. coli TOP10 for storage. The haploid strain INVSc1 was transformed with pYES2/NT C-oPAD17 and selected on minimal uracil-free agar plate to yield the strain INVSc 1 (pYES2/NT C-oPAD17).

Total RNA was isolated from pelleted transformants using TRIzol reagent (Invitrogen) and reverse-transcribed using the Prime Script RT reagent kit (Takara, Otsu, Shiga, Japan) according to the manufacturers’ instructions. The q-oPAD17 F/q-oPAD17 R primer pair used for RT-qPCR is summarized in Table 1. An ABI-Prism 7900 sequence detection system and Power SYBR Green PCR Master Mix (both Applied Biosystems, Foster City, CA, USA) were used for the RT-qPCR analysis according to the manufacturer’s instructions. The reaction mixtures comprised 10 µL of SYBR Green PCR Master Mix, 0.5 µL of each primer pair, 8 µL of distilled water, and 1 µL of DNA template or distilled water for a no-template control. The PCR cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of amplification at 95°C for 15 s and 60°C for 30 s. The housekeeping gene 18S rRNA was used as the normalization standard for gene expression. The primer pair 18S F/18S R was used as indicated in Table 1.

Western blot analysis was performed as described previously (Chen et al., 2013). One hundred micrograms of cellular protein extract per lane (in duplicate) were subjected to SDS-PAGE (12% gel), followed by western blotting analysis or coomassie blue staining. PVDF membranes (Millipore, Billerica, MA, USA) were used for western blot analysis. To detect recombinant proteins, membranes were incubated with an anti-His primary antibody (Nanjing GenScript Biotechnology Corporation, Nanjing, PR China), followed by a horseradish peroxidase-conjugated goat anti-mouse IgG (Nanjing GenScript Biotechnology Corporation, Nanjing, PR China).

INVSc 1 (pYES2/NT C-oPAD17) strains were grown on SC medium. Overnight cultures were diluted to an optical density at 600 nm (OD₆₀₀) of 0.4 and subsequently aliquoted into three 20-ml cultures containing 0.2 mM each of LA, GLA, DGLA, and ARA, respectively. Next, different concentrations of AA (0.05, 0.1, and 0.2 mM) were added to the culture media to investigate the AA to EPA conversion activity. After incubation for another 48 h, the cultures were harvested by centrifugation and subjected to further analysis.

The PaD17 gene was codon-optimized based on the codon preferences of M. alpina; the resulting gene, oPaFADS17 (1,077 bp in length, GenBank accession No. KT372000) was synthesized and sub-cloned into the pUC57-simple vector. The oPaFADS17 F/oPaFADS17 R primer pair (Table 1) was used to amplify oPaFADS17 under PCR conditions of 94°C for 3 min, 30 amplification cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 1.5 min, and a final extension step at 68°C for 7 min. The T-DNA binary vector pBIG2-ura5s-ITs, which contains a ura5s cassette as the selection marker, contains the His550 promoter and trpCt transcription terminator, was used as the backbone for expression vector construction, The oPaFAD17 coding sequence fanked with restriction endonuclease sites Hind III and Xho I was cloned into this vector to generate pBIG2-ura5s-oPaFADS17. Validated binary vectors carrying the oPaFADS17 gene were electrotransformed into E. coli TOP10 and A. tumefaciens C58C1 for storage or A. tumefaciens-mediated transformation (see below).

Agrobacterium tumefaciens-mediated transformation was performed using an appropriately modified version of a previously described protocol (Ando et al., 2009). Uracil-auxotrophic M. alpina CCFM501 spores were harvested from 30-day cultures grown on GY agar supplemented with 0.05 g L⁻¹ uracil and suspended. Positive A. tumefaciens transformants carrying pBIG2-ura5s-oPaFADS17 were confirmed by PCR with the HisproF/TrpCR primer pair (Table 1). A. tumefaciens transformants were grown at 28°C for 48 h to an OD₆₀₀ of 1.2 in MM medium supplemented with 100 µg mL⁻¹ kanamycin and 100 µg mL⁻¹ rifampicin.

Bacterial cells were harvested by centrifugation, washed with fresh IM medium, and diluted to an OD₆₀₀ of 0.3 in 20 ml of fresh IM. After preincubation for 12 h at 25°C with shaking (200 rpm), the culture was diluted to an OD₆₀₀ of 0.4–1.2, after which 100 µL of bacterial cell suspension was mixed with an equal volume of a spore suspension (10⁷/100 μL) and spread on cellophane membranes. The membranes were placed on cocultivation medium (similar to IM, except with a glucose concentration of 0.9 g L⁻¹) and incubated at 23°C for approximately 36 h in a dark incubator. After cocultivation, the membranes were transferred to uracil-free SC agar plates containing 100 µg mL⁻¹ cefotaxime and 100 µg mL⁻¹ spectinomycin and cultured at 18°C for 12 h to inhibit the A. tumefaciens growth, and then at 25°C until colonies appeared. Hyphae from visible fungal colonies were then transferred to fresh uracil-free SC agar plates. This transfer process was repeated three times to obtain stable transformants. All experiments were performed in triplicate.

Transformants were cultivated in liquid broth medium at 28°C with shaking at 200 rpm for 7 days. M. alpina mycelia were harvested by filtration and washed twice with sterile water. The integration of T-DNA into M. alpina genomic DNA was verified by PCR. One pair of primers (HisproF1/TrpCR1; Table 1) was designed to target promoter and terminator-specific regions and confirm successful gene integration. The PCR conditions were 94°C for 3 min, 30 amplification cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, and a final extension step of 72°C for 10 min. Positive transformants were expected to yield 818 and 1,244 bp fragments.

Mortierella alpina strains were cultivated in broth medium at 28°C with shaking at 200 rpm for 7 days. M. alpina mycelia were harvested by filtration and immediately frozen in liquid nitrogen for storage. TRIzol-mediated total RNA extraction and RT-qPCR were performed as described above. The primer pairs used for RT-qPCR were shown in Table 1. The internal control gene 18S rRNA was used as the normalization standard for gene expression.

Single-factor experiments and a designed orthogonal experiment were performed to increase the EPA yield. Part 1 involved an investigation of the effects of different carbon sources, including glucose, corn starch, soluble starch, potato starch, and glycerol, on dry biomass, lipid, and EPA production. Part 2 compared an inexpensive defatted soybean meal (DSMO) with a costly yeast extract as organic nitrogen sources. Part 3 evaluated various concentration of inorganic nitrogen KNO₃. Finally, the effects of EPA production were investigated through an orthogonal experimental design in which the following three factors were analyzed: carbon source (factor A), DSOM concentration (factor B), and KNO₃ concentration (factor C). An OA₉ (3³), or orthogonal array of three factors and three levels, was employed to assign the factors for consideration, as shown in Table 2. Nine trials were performed to complete the optimization process according to the OA₉. A range analysis was used to determine the optimal EPA production conditions.

A range analysis includes two parameters, K_ji and R_j. K_ji is defined as the sum of the indexes of all levels in each factor j and k_ji is the average value of each experimental factor at the same lever i; R_j is defined as the range between the maximum and minimum values of k_ji. As k_ji is used to evaluate the importance of factors, a large R_j indicates greater importance of a particular factor.

Fatty acids were extracted and methyl-esterified from approximately 50 mg of freeze-dried cells, as described previously (Chen et al., 2013). FAME profiles were analyzed using gas chromatography/mass spectrometry (GC/MS; GC-2010 Plus; GCMS-QP2010 Ultra, Shimadzu Co., Kyoto, Japan) with a 30 m × 0.25 mm Rtx-Waxetr column (film thickness: 0.25 µm) and helium as the carrier gas. The following temperature programme was set: 40°C for 5 min, ramp to 120°C at 20°C per min, ramp to 190°C at 5°C per min, hold for 5 min, ramp to 220°C at 5°C per min, and hold for 17 min. Fatty acid quantification was performed using peak-height area integrals. Pentadecanoic acid was used as the internal standard to quantify FAMEs with aliphatic chains ≤18, and nonadecanoic acid was used as the internal standard to quantify FAMEs with aliphatic chains >18.

All experiments were performed independently at least three times, and the mean values ± SDs were presented. Statistical analysis was performed with one-way analysis of variance with Tukey’s test with SPSS 20, and P < 0.05 was considered to indicate statistical significance.

The optimized oPAD17 gene was used to construct pYES2/NT C-oPAD17, which was successfully transformed into S. cerevisiae (Figure 2A). RT-qPCR (Figure 2B) and western blotting (Figure 2C) revealed normal levels of PaD17 transcription and translation, indicating successful target gene transcription and expression in the recombinant strain.

Figure 3 shows the successful functional expression of INVSc 1 (pYES2/NT C-oPAD17), along with representative GC-MS analyses of FAMEs in yeast cultures. Gas chromatograms (Figure 3C) detected a peak corresponding to the C20:5 (5,8,11,14,17) standard (Figure 3A) that was not present in the INVSc1-pYES2/NT C control strain (Figure 3B). To gain qualitative insight into the substrate requirements of the oPAD17-encoded protein, various possible fatty acid substrates were supplied to yeast cultures expressing this enzyme.

INVSc 1 (pYES2/NT C-oPAD17) strains were grown at room temperature (28°C) on YEP medium supplemented with linoleic acid (LA, C18:2, ω-6), γ-linolenic acid (GLA, C18:3, ω-6), dihomo-γ-linolenic acid (DGLA, C20:3, ω-6), and AA (C20:4, ω-6) (Figure 4A). LA, GLA, DGLA, and AA were converted into ALA (C18:3, ω-3), stearidonic acid (SDA, C18:4, ω-3), eicosateteraenoic acid (ETA, C20:4, ω-3) and eicosapentaenoic acid (EPA, C20:5, ω-3), respectively. Although the oPAD17 product exhibited poor 18 C-PUFA conversion efficiency (<5%), it exhibited much better efficiency for 20 C-PUFAs (>10%), especially AA for which the conversion rate was almost 50%. We also determined desaturase activities at a low temperature (12°C), and observed lower conversation rate (Figure 4A) relative to those obtained at room temperature. Furthermore, we evaluated the effects of different AA concentration on desaturase activity (Figure 4B). As shown in Figure 4B, the AA to EPA conversion rate decreased with increasing AA concentration. When exposed to 0.05 mM AA, INVSc 1 (pYES2/NT C-oPAD17) strains exhibited conversion rate as high as 68%, consistent with the reported rate for Y. lipolytica.

The optimized oPaFADS17 gene was heterologously expressed in the M. alpina auxotrophic CCFM 501 strain, and putative transformants were selected randomly by subculture on uracil-free SC medium. Stable transformants were then obtained by successive sub-culturing on uracil-free SC agar plates for three generations, and were transferred to GY agar plates for further analysis. We eventually obtained six mitotically stable oPaFADS17 transformants.

The T-DNA region of the binary vector pBIG2-ura5s-oPaFADS17 (Figure 5) should have integrated into the genomic DNA of M. alpina during the A. tumefaciens transformation process. Accordingly, we verified the presence of the ura5 and oPaFADS17 expression cassettes in the transformant genomes using PCR. The presence of two fragments (818 and 1,244 bp) confirmed that the oPaFADS17 expression cassettes were randomly inserted into the M. alpina genome, whereas the control strain (wild-type M. alpina) did not yield corresponding DNA fragments (Figure 5).

RT-qPCR analysis of oPaFADS17 transcript levels revealed the successful transcription of this construct in all heterologous expression strains, whereas no transcription was detected in wild-type M. alpina (Figure 6A). The stable transformant and wild-type M. alpina strains were cultivated in broth medium for 7 days at 28°C prior to the evaluation of fatty acid productivity and composition. These strains had similar fatty acid components but significantly different AA and EPA contents (Figures 6B,C), and the latter differed among transformants according to differences in integration sites. High EPA-producing transformants exhibited sharp decreases in AA and dramatic increases in EPA level. Among all transformants, MA-oPaFADS17-3 (CCFM 695) produced yields of 18.7% EPA and 18.8% AA among TFA (Table 3), indicating the conversion of almost half of the AA present in M. alpina and an EPA production of 617 mg L⁻¹.

Broth medium (pH 6.0) was used in studies of the effects of different carbon sources on biomass, lipid, and EPA production by M. alpina CCFM 695. Of the tested sources, soluble starch yielded the highest level of mycelial growth (14.50 g L⁻¹; Table 3), whereas maximum EPA production was achieved in medium containing corn starch (1.2 g L⁻¹). Starch, an energy storage polysaccharide produced widely by plants such as wheat, maize, rice, and potato, is one of the most abundant carbohydrates in nature (Ledesma-Amaro and Nicaud, 2016). As reported previously, glucose was the optimal carbon source for fatty acid accumulation by M. alpina, whereas starch was a poor growth supporter and only allowed the generation of 1.6 g L⁻¹ dry biomass (Nisha and Venkateswaran, 2011). In contrast, our results showed that a medium containing soluble starch or corn starch generated much higher biomass levels relative to glucose (10.9 g L⁻¹; Table 3), which we attribute to potential differences among strains. However, soluble starch was not conducive to TFA production (2.8 g L⁻¹; Table 3). Potato starch yielded the lowest amount of dry cell biomass, and glycerol yielded the poorest EPA production by M. alpina CCFM 695. In summary, various carbon sources had specific effects on EPA production.

Broth medium was also used to study the effects of nitrogen sources on biomass, lipid, and EPA production by M. alpina CCFM 695. In these experiments, yeast extract was replaced by different concentrations of DSOM (Table 3), an abundant and inexpensive agricultural product. However, as DSOM contains insoluble materials and the lipids are intracellular products of fungal cells, the direct addition of soy flour to the medium would increase the difficulty of biomass separation from soybean solids. We therefore supplemented the culture media with a filtrate of DSOM that had been boiled for 20 min. The addition of 50 g L⁻¹ of DSOM promoted M. alpina CCFM 695 growth and led to an increase in biomass to 14.2 g L⁻¹, a much greater elevation than that observed with yeast extract. Furthermore, DSOM increased EPA production by more than twice over yeast extract (1.5 g L⁻¹ vs. 617 mg L⁻¹). In other words, DSOM might not only decrease the cost of fermentation, but it could also serve as a good nitrogen source for M. alpina CCFM 695 in the industrial production of EPA. Furthermore, a study of the inorganic nitrogen KNO₃ concentration revealed that a low concentration of this component could promote EPA accumulation.

Nine experiments were conducted per the OA₉ matrix, and the resulting of dry biomass, TFA and EPA production were presented in Table 4. EPA production varied from 376 to 1,734 mg L⁻¹, dry biomass and TFA content increased to 16.55 and 6.46 g L⁻¹, respectively. Table 5 listed the mean values of K (k_ji) for different factors at different levels in the range analysis. A higher mean k_ji value indicated that the corresponding level had a greater effect on EPA production. Therefore, we determined the following optimal parameters: the optimal carbon source was glucose, the optimal DSOM concentration was 50 g L⁻¹, and the optimal KNO₃ concentration was 5 g L⁻¹ (A₁B₂C₂). This combination yielded EPA production level as high as 1.73 g L⁻¹. Meanwhile, the R_j value demonstrates the significance of a factor’s influence, and a larger value indicates that the factor had a greater effect on EPA production. In Table 5, the factor significance level, in decreasing order, was: Carbon source (R_A) > DSOM concentration (R_B) > KNO₃ concentration (R_C). The range value R_A was the largest, indicating that the carbon source had the most significant effect on EPA production.