T. aureum ATCC 34304 was purchased from the American Type Culture Collection. A. limacinum mh0186 was identified based on the sequence of the 18S ribosomal DNA (rDNA) (DDBJ accession number AB362211). The restriction enzymes and T4 DNA ligase were purchased from Nippon Gene (Tokyo, Japan). Synthetic oligonucleotides were obtained from Hokkaido System Science (Hokkaido, Japan) and Genenet (Fukuoka, Japan). The antibiotic neomycin (G418) was purchased from Nacalai Tesque (Kyoto, Japan). Eicosatetraenoic acid (ETA) (C20:4n − 3), dihomo-γ-linoleic acid (DGLA) (C20:3n − 6), docosapentaenoic acid (DPA) (C22:5n − 3) and docosatetraenoic acid (DTA) (C22:4n − 6) were purchased from Cayman Chemical Co. (Michigan). Eicosadienoic acid (EDA) (C20:2n − 6), linoleic acid (LA) (C18:2n − 6), and α-linolenic acid (ALA) (C18:3n − 3) were obtained from Sigma. Eicosatrienoic acid (ESA) (C20:3n − 3) was purchased from Biomol. Sealife was obtained from Marinetech (Tokyo, Japan). All other reagents were of the highest purity available.
Cloning of cDNA encoding TauΔ5des.
To obtain a DNA fragment encoding the fatty acid Δ5 desaturase (TauΔ5des) from T. aureum ATCC 34304, a set of primers was designed based on the sequence of the fatty acid Δ5 desaturase from Thraustochytrium sp. ATCC 26185 (DDBJ accession number FJ821482): 3F (5′-TAC TGG AAG AAC CAG CAC AGC AAG CAC CAC-3′)/1RNES (5′-CGC CGT GGG GAA GAG GTG GTG CTC GAT CTG-3′). PCR was then performed with the T. aureum ATCC 34304 cDNA library as a template using Advantage 2 polymerase mix (Clontech, California). The cycling parameters for PCR were 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and the number of cycles was 30. A 550-bp PCR product was subcloned into the pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, California) and a DNA sequencer (ABI model 3130; Applied Biosystems). The sequence of an insert, showing high identity with the Δ5 desaturase gene of Thraustochytrium sp. ATCC 26185, was used to design the primers for 5′ and 3′ rapid amplification of cDNA ends (RACE). A 3′-RACE product was obtained using RACEd5F (5′-TGT CCT GCT TCC TGG TTG GTC TC-3′) and RACEd5FNES (5′-TCT GGA CCC TGT TTC TGC ACC CGC-3′). The cycling parameters for PCR were as described above. A 5′-RACE product, however, was not amplified by this method, and thus, the LA PCR in vitro cloning kit (Takara Bio, Japan) was used along with GSP1 (5′-ACCGCAAAGTTGGTGAAGATG-3′) and GSP2 (5′-CAAAGCCAAAGGTGGCCATGTAGAGAC-3′). PCR was then carried out with the T. aureum ATCC 34304 genome (BglII digest) as a template using the Advantage 2 polymerase mix. The cycling parameters for PCR were 94°C for 30 s, 60°C for 30 s, and 72°C for 3 min, and the number of cycles was 30. As a result, we obtained a 2.5-kbp PCR product corresponding to the 5′ region of TauΔ5des. Finally, the open reading frame (ORF) of the TauΔ5des gene was determined by comparing the ORF of the fatty acid Δ5 desaturase gene of Thraustochytrium sp. ATCC 26185.
Amino acid sequences of various fatty acid desaturases, including TauΔ5des, were aligned using the ClustalW 1.81 software program (24
), and a phylogenetic tree was constructed with the neighbor-joining methods using the MEGA4 program (www.megasoftware.net
). The percentage of replicate trees was calculated by the bootstrap test (1,000 replicates).
Generation of S. cerevisiae harboring the TauΔ5des gene.
The ORF of TauΔ5des was amplified by PCR using d5fulllengthF, containing an EcoRI site (5′-CGA ATT C
AT GGG ACG CGG CGG CGA AGG TCA G-3′), and d5fulllengthR, containing a XhoI site (5′-GCT CGA G
TT GGG TCG GGA TAA AAT AAA TGG C-3′). PrimeSTAR HS DNA polymerase (Takara Bio) was used for amplification. The cycling parameters for PCR were the same as described above. The PCR products were digested with EcoRI and XhoI and cloned into the same sites of pYES2/CT (Invitrogen, California). The TauΔ5des expression vector, designated pYpΔ5Des, was introduced into Saccharomyces cerevisiae
INVSc1 (Invitrogen) using the lithium acetate method (25
). The transformants were selected on agar plates lacking uracil (0.67% Difco yeast nitrogen base without amino acid, 2% glucose, 0.059% CSM [complete supplement mixture minus adenine, histidine, leucine, tryptophan, and uracil], 0.002% adenine, 0.002% l
-histidine, 0.01% l
-leucine, 0.002% l
-tryptophan). The transformants harboring the TauΔ5des gene were designated scΔ5ura, and the mock transfectants were designated scura.
Generation of A. limacinum mh 0186 harboring the TauΔ5des gene.
To express the TauΔ5des gene in thraustochytrids, a linear DNA cassette containing a TauΔ5neor gene driven by the ubiquitin promoter/terminator and a neomycin resistance (Neor) gene driven by the EF1α promoter/terminator was constructed. For mock transformants, the TauΔ5des gene with the ubiquitin promoter/terminator was omitted from the cassette. The EF1-α promoter/terminator and ubiquitin promoter/terminator were cloned from T. aureum ATCC 34304. The DNA linear cassette was introduced into A. limacinum mh0186 cells by electroporation. Cells (5 × 106) and 5 μg DNA in 80 μl of Nucleofector solution L (Amaxa Biosystems, Maryland) were transferred to a 0.1-cm-gap cuvette and electroporated (pulsed conditions: 50 μF, 50 Ω, 7.5 kV/cm, 2 times) by using a Gene Pulser system (Bio-Rad, CA). The cells were then immediately resuspended in 1 ml of GY medium (3% glucose, 1% yeast extract, and 1.75% Sealife), incubated at 25°C for 1 day, and spread on PD agar plates (0.48% potato dextrose broth, 1.75% Sealife, and 1.5% agar) containing neomycin at 0.5 mg/ml. After incubation at 25°C for 2 to 5 days, colonies appearing on the plates were regarded as transformants. The transformants harboring the TauΔ5des gene were designated mhΔ5neor, and the mock transfectants were designated mhneor.
Total RNA was prepared from mhΔ5neor and mhneor with Sepazol RNA I Super (Nacalai Tesque), the RNeasy Mini kit (Qiagen, Tokyo, Japan), and DNase I (TaKaRa Bio) and reverse transcribed to cDNA using PrimeScript reverse transcriptase (RT) (TaKaRa Bio). Then, PCR amplification was performed using, for the TauΔ5des gene, D5EVF (5′-TTG ATA TCA TGG GAC GCG GCG GCG AAG GTC AGG T-3′) and D5EVR (5′-TTG ATA TCC TAA GCG GCC TTG GCC GCC GCC TG-3′), and for the Neor gene, proGF (5′-GCG ACC TAA GCA ACA CTA GCC AAC ATG ATT GAA CAG GAC GGC CTT CAC-3′) and GterR (5′-AGT ATA GCA CAT ACT ACA GAT AGC TCA AAA GAA CTC GTC CAG GAG GC-3′). The cycling parameters for PCR were 98°C for 10 s, 60°C for 30 s, and 72°C for 1.5 min, and the number of cycles was 30. For semiquantitative RT-PCR, the template concentration and cycle number of PCR were adjusted by using respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as a standard. PCR amplifications of GAPDH genes of thraustochytrids and yeasts were performed using mhg3pdhF2 (5′-CAC CGG CTC TGA CTA CGT TGT GG-3′) plus mhg3pdhR2 (5′-CTT CAT GGC GGC GCA GAT CTC CTC-3′) and scg3pdhF1 (5′-TAT GCT GCT TAC ATG GTC AAG TAC G-3′) plus scg3pdhR1(5′-ACA ACG GCA TCT TCG GTG TAA CCC-3′), respectively. The cycling parameters for PCR were 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and the number of cycles was 30 for the GAPDH gene and 25 for the TauΔ5des gene.
After digestion of the genomic DNAs of mhΔ5neor and mhneor with the restriction enzyme BglII, the digestion products were subjected to electrophoresis using a 0.7% agarose gel and then transferred to the Biodyne membrane (PALL Gelman Laboratory). Part (600 bp) of the TauΔ5des gene was amplified by PCR and used as a probe after labeling with digoxigenin (DIG). The DIG probe was prepared by using a PCR DIG probe synthesis kit (Roche, Germany). The PCR was carried out using D5ORFcomF (5′-GAC GCG GCG GCG AAG GTC AGG-3′) and D5ORFcomR (5′-CTT GCT GTG CTG AAC GCG CCA C-3′) as primers. The cycling parameters for PCR were 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and the number of cycles was 30.
Fatty acid analysis.
Fatty acid methyl esters (FAMEs) were prepared as described previously (20
). FAMEs were analyzed by gas-liquid chromatography (GC) and GC-mass spectrometry (GC-MS) using a Shimadzu GC-MS QP-5000 instrument (Shimadzu Co., Kyoto, Japan) as reported previously (1
). The rate of conversion of the substrate into the product was calculated as follows: conversion rate (percent) = [GC area for the product/(GC area for the product plus GC area for the substrate)] × 100.
Conversion of fatty acids in transgenic S. cerevisiae and A. limacinum mh 0186.
The scΔ5ura and scura cells were cultured for 2 days at 25°C in 5 ml of Ura− medium containing 2% glucose, harvested (1,500 × g for 3 min), and washed with distilled water. To induce the expression of the TauΔ5des gene, the cells were suspended in 10 ml of Ura− medium containing 2% galactose. The cells were harvested and then recultured at 25°C for 1 day in Ura− medium containing various fatty acids at the concentrations indicated. The mhΔ5neor and mhneor cells were cultured for 2 days at 25°C in 5 ml of GY medium, harvested (3,500 × g for 5 min), and washed with 1.75% Sealife. The cells were cultured at 25°C for 1 day in PD medium containing various fatty acids at the concentrations indicated. Total fatty acids were extracted, and FAMEs were analyzed by GC and GC-MS.