Comparative Biochemistry and Physiology, Part B 188 (2015) 37–45

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Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases Naoki Kabeya a, Yoji Yamamoto a, Scott F. Cummins b, Abigail Elizur b, Ryosuke Yazawa a, Yutaka Takeuchi c, Yutaka Haga a, Shuichi Satoh a, Goro Yoshizaki a,⁎ a b c

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs, QLD 4556, Australia Research Center for Advanced Science and Technology, Tokyo University of Marine Science and Technology, Banda 670, Tateyama-shi, Chiba 294-0308, Japan

a r t i c l e

i n f o

Article history: Received 2 February 2015 Received in revised form 17 June 2015 Accepted 17 June 2015 Available online 23 June 2015 Keywords: Desaturase Elongase Marine fish Docosahexaenoic acid Eicosapentaenoic acid

a b s t r a c t To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via β-oxidation. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are defined as PUFA with ≥ 20 carbon chain length and ≥ 3 double bonds (Bell and Tocher, 2009). In general, these fatty acids are synthesized from their precursor, α-linolenic acid (18:3n-3) based on sequential reactions catalyzed by fatty acyl desaturase (Fads) and elongation of very-long-chain fatty acids (Elovl) enzymes (Fig. 1) (Miyazaki and Ntambi, 2008). The Fads introduce a double bond into fatty acyl chain and the Elovl are responsible for a condensation reaction that is the first step in the addition of two carbons to the carboxyl end of a fatty acyl chain (Miyazaki and Ntambi, 2008). The functions of these enzymes from several teleost species have been extensively characterized using yeast heterologous expression system to determine their capacity of LC-PUFA biosynthesis.

⁎ Corresponding author. Tel./fax: +81 3 5463 0558. E-mail address: [email protected] (G. Yoshizaki).

http://dx.doi.org/10.1016/j.cbpb.2015.06.005 1096-4959/© 2015 Elsevier Inc. All rights reserved.

The amino acid sequence of Fads from teleost species is more similar to the mammalian Δ6 desaturase (Fads2), which mainly catalyzes the conversion of 18:2n-6 to 18:3n-6 and 18:3n-3 to 18:4n-3 in the LCPUFA biosynthetic pathway (Fig. 1) (Zheng et al., 2004, 2009; Tocher et al., 2006; Gonzalez-Rovira et al., 2009; Gregory et al., 2010; Kim et al., 2011; Morais et al., 2011; Wang et al., 2014; Xie et al., 2014). On the other hand, orthologues of the mammalian Δ5 desaturase (Fads1) have not been found in any teleost species (Castro et al., 2012). Particularly, the Fads2 enzyme from most marine fish species showed Δ6/Δ8 bifunctional desaturase activity (Fig. 1) (Monroig et al., 2011a, 2013; Tu et al., 2012). Unlike these species, the herbivorous marine fish species, rabbitfish (Siganus canaliculatus) has two Fads2 enzymes: a bifunctional Δ6/Δ5 Fads and a monofunctional Δ4 Fads (Li et al., 2010) (Fig. 1). The monofunctional Δ4 Fads has also been found in marine flatfish species, Senegalese sole (Solea senegalensis), a carnivorous species occupying higher trophic level compared to rabbitfish (Morais et al., 2012) (Fig. 1). Thus, marine fish species vary in their capacity to biosynthesize LC-PUFA depending on their complement of these key enzymes. However, besides these studies, other Δ6 Fads activity toward 24:5n-3, which is an essential activity for DHA synthesis from 24:5n-3 in species

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Fig. 1. Polyunsaturated fatty acid biosynthetic pathway found in teleost species. 1Fads2 isolated from most marine fish species lacks this activity. Fads2 with relatively high Δ5 desaturase activity was found in zebrafish Danio rerio (Hastings et al., 2001), Atlantic salmon Salmo salar (Hastings et al., 2005), rabbitfish Siganus canaliculatus (Li et al., 2010), and pike silverside Chirostoma estor (Fonseca-Madrigal et al., 2014). 2elovl2 gene has been isolated only from freshwater species: Atlantic salmon (Morais et al., 2009) and zebrafish (Monroig et al., 2009). 3 Fads2 with Δ4 desaturase activity has been isolated from rabbitfish (Li et al., 2010), Senegalese sole Solea senegalensis (Morais et al., 2012), pike silverside (Fonseca-Madrigal et al., 2014), and striped snakehead Channa striata (Kuah et al., 2015). 4Δ6 desaturase activity toward 24:6n-3 has been identified only in Atlantic salmon (Hastings et al., 2005; Bell and Tocher, 2009) and zebrafish (Tocher et al., 2003).

lacking Δ4 Fads, has not been characterized in any marine fish species examined to date. Functional characterization of Elovl enzymes isolated from various marine fish species has also been performed using the yeast expression system. These studies suggested that marine fish species lack Elovl2, which is preferentially responsible for the elongation step from C22 to C24 LC-PUFA; this enzyme has been found in freshwater species (Monroig et al., 2009; Morais et al., 2009). In particular, the elongation step from 22:5n-3 to 24:5n-3 is essential for enzymes in DHA biosynthesis from 22:5n-3 for species lacking Δ4 Fads (Fig. 1). However, Elovl5 and Elovl4 have been isolated from several marine fish species. Elovl5 has the ability to preferentially elongate C18 (18:3n-6 and 18:4n-3) and C20 (20:4n-6 and EPA) fatty acids, with only low activity toward C22 fatty acids (Agaba et al., 2005; Zheng et al., 2009; Gregory et al., 2010; Mohd-Yusof et al., 2010; Morais et al., 2011, 2012; Monroig et al., 2013; Wang et al., 2014). In contrast to Elovl5, Elovl4, which has been isolated from cobia, Rachycentron canadum (Monroig et al., 2011b) and rabbitfish (Monroig et al., 2012), could elongate C20 fatty acids to longer-chain fatty acids (up to C36). Accordingly, it is expected that this enzyme could supply Elovl2 activity in other marine fish species (Monroig et al., 2011b, 2012). Although most marine fish species are rich in n-3 LC-PUFA, particularly DHA, it is considered that these fish species just retain the fatty acid content in their diets because their capacity of LC-PUFA biosynthesis is limited. Because insufficient supply of DHA (and EPA) to their feeds is a cause of high mortality and poor development in several marine fish species (Tocher, 2010, 2015), fish oil is used as an EPA and DHA source in aquatic feed. However, the limited and unstable supply of fish oil hinders the development of marine fish aquaculture (Naylor et al., 2009). Accordingly, to reduce fish oil use in aquaculture, we have been developing a transgenic method for modifying the endogenous fatty acid synthetic pathway in the freshwater zebrafish (Alimuddin et al., 2005, 2007, 2008). We also recently established a transgenic marine fish, a Nibe croaker (Nibea mitsukurii) strain carrying the elovl2 gene isolated from Masu salmon (Oncorhynchus masou) (Kabeya et al., 2014). Fatty

acid profile analysis of the transgenic Nibe croaker juveniles and adults showed that EPA, which is a substrate of Elovl2, was decreased and 22:5n-3, which is an elongation product of Elovl2, was increased in the liver, compared to non-transgenic fish. Furthermore, 24:5n-3, which is another elongation product of Elovl2, was consistently detected in the livers of transgenic fish but was not detected in any nontransgenic fish. However, DHA content showed no difference between non-transgenic and transgenic fish. Larval-stage fish of this strain cannot be reared with DHA-deficient feed (Kabeya et al., 2015). Based on these results, we hypothesize that the Nibe croaker endogenous Fads had no activity toward 24:5n-3, given that although 24:5n-3 level was elevated, DHA level was not elevated in transgenic Nibe

Table 1 List of primers used for cloning, RT-PCR and construction of yeast expression vector. Restriction sites are underlined. Step

Primer name

cDNA synthesis

Oligo(dT) primer

Sequence

5′-GTAATACGACTCACTATAGGGCACGCGT GGTCGACGGCCCGGGCTGGTTTTTTTTTTT TTTTTT-3′ Cloning Marinefish_Elovl4_Fw1 5′-TTGCCTTGTCYAGTTTCTTG-3′ Marinefish_Elovl4_Rv1 5′-CAGACACAYCCACRAAYACACAC-3′ Marinefish_Elovl4_Fw2 5′-ATCAAATTGCACCGGATGTC-3′ Marinefish_Elovl4_Rv2 5′-ACACAYCCACRAAYACACACUGC-3′ Construction Fads2-F 5′-GGTAAGCTTAATAATGGGAGGTGGAGG CCAGCTGAC-3′ Fads2-R 5′-AGTTCTAGATCATTTGTGGAGATATGC ATCGAGC-3′ Elovl5-F 5′-ATTAAGCTTCAAATGGAGACCTTCAAT CATAAAC-3′ Elovl5-R 5′-GCATCTAGATCAATCCACCCTCAGTTTCT TGTG-3′ Elovl4-F 5′-ATTAAGCTTGCCATGGAGGCTGTAACA CATT-3′ Elovl4-R 5′-AAACTCGAGTTACTCCCTCTTCGCTCT TC-3′

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croaker. However, there is no clear information concerning the functional roles of Nibe croaker Fads and Elovl enzymes. An understanding of the endogenous LC-PUFA biosynthetic pathway in Nibe croaker is important for the establishment of a desirable transgenic strain with the complete biosynthetic pathway of DHA. We have isolated cDNA clones encoding fads2- and elovl5-like genes from Nibe croaker in a previous study (Yamamoto et al., 2010). In the present study, with the aim of elucidating the LC-PUFA biosynthetic pathway in Nibe croaker, we newly cloned the full-length cDNA of an elovl4-like gene and functionally characterized Fads2, Elovl5 and Elovl4 using heterologous expression of these genes in yeast.

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2. Materials and methods 2.1. Cloning and sequence analysis of a putative elovl4 cDNA from Nibe croaker Because a putative elovl4 cDNA from Nibe croaker showed high expression in the eye but not in the liver in our previous study (Kabeya et al., 2014), total RNA was extracted from the whole eye of Nibe croaker using Isogen reagent (Nippon Gene Co., Ltd., Tokyo, Japan) following the manufacturer's instructions. First strand cDNA was synthesized from 1 μg of the total RNA using SuperScript® Reverse Transcriptase

Fig. 2. Analysis of the deduced amino acid sequence of the elovl4 gene sequence from Nibe croaker Nibea mitsukurii. (A) Comparison of the deduced amino acid sequence of the Nibe croaker elovl4 gene with those of human Homo sapiens, zebrafish Danio rerio (isoforms Elovl4_a and Elovl4_b), Atlantic salmon Salmo salar, rabbitfish Siganus canaliculatus and cobia Rachycentron canadum. Identical sequences are shaded black. The amino acid sequence of Nibe croaker Elovl4 contained two motifs that are typically conserved in members of the microsomal Elovl family, including histidine box motif (HXXHH) and the endoplasmic reticulum retrieval signal (ER). In addition, hydropathy analysis revealed that Nibe croaker Elovl4 protein contained six (I–VI) putative membrane-spanning domains. (B) Hydrophobicity plot of the amino acid sequence of Nibe croaker Elovl4. Shaded areas (I–VI) indicate putative membranespanning domains.

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(Invitrogen, Carlsbad, CA, USA) with the Oligo(dT) primer (Table 1). The first polymerase chain reaction (PCR) amplification of the putative elovl4 cDNA was performed using Marinefish_Elovl4_F1 primer and Marinefish_Elovl4_R1 primer (Table 1) and nested PCR was performed using Marinefish_Elovl4_F2 primer and Marinefish_Elovl4_R2 primer (Table 1) with PrimeSTAR® Max DNA polymerase (TaKaRa Bio, Shiga, Japan). The primers were designed to anneal to the sequences of untranslated regions of the cobia and rabbitfish elovl4 gene (GenBank accessions HM026361 and JF320823, respectively). Both 1st and nested PCR amplifications were performed with 35 cycles comprising denaturation for 10 s at 98 °C, annealing for 5 s at 55 °C, and extension for 80 s at 72 °C. The nested PCR products were cloned into the pGEM®-T Easy vector (Promega, Madison, WI, USA) after adding 3′ adenosine overhangs to PCR products using Taq DNA polymerase (TaKaRa Taq™, TaKaRa Bio). The cloned PCR fragments were sequenced by cycle sequencing method (Mardis and McCombie, 2012) (DNA sequencing service, TaKaRa Bio). Hydropathy analysis of the deduced amino acid sequence was performed using TopPred II software for membrane protein structure predictions (Claros and von Heijne, 1994) with the Goldman, Engelman, and Steitz (GES) scale (Engelman et al., 1986). The phylogenetic tree of amino acid sequences was constructed by the neighbor-joining method (Saitou and Nei, 1987) with bootstrapping analysis (1000 replicates).

at 98 °C, annealing for 5 s at 55 °C, and extension for 90 s (fads2) or 60 s (elovl5 and elovl4) at 72 °C. The amplified fragments were ligated into the pYES2 yeast expression vector (Invitrogen) using the LigaFast™ Rapid DNA Ligation System (Promega) and then the ligation products were used to transform ECOS™ Competent Escherichia coli DH5α cells (Nippon Gene Co., Ltd.) following the manufacturer's instructions. After plasmid purification by alkaline lysis method using FastGene™ Plasmid Mini Kit (Nippon Genetics Co., Ltd.) following the manufacturer's instructions, yeast transformation and yeast culture were performed following previously described methods (Li et al., 2010). The following fatty acids (Larodan Fine Chemicals, Malmo, Sweden) were used as substrates for yeast transformed with fads2: 18:3n-3, 18:2n-6, 20:3n-3, 20:2n-6, 20:4n-3, 20:3n-6, 22:5n-3, 22:4n6 and 24:5n-3, elovl5: 16:3n-3, 18:3n-3, 18:2n-6, 18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3 and 22:4n-6, elovl4: 18:3n-3, 18:4n-3, 20:5n-3 and 22:5n-3. Each fatty acid substrate except 24:5n-3 was added to a final concentration of 0.5 mM and 24:5n-3 was added to 1.0 mM. After 48 h of culture at 30 °C in the presence of each substrate fatty acid, the yeast cells were collected, washed twice with ice-cold Hank's balanced salt solution and stored at − 80 °C until fatty acid analysis.

2.2. Functional characterization of the Nibe croaker Fads2, Elovl5 and Elovl4 cDNAs by heterologous expression in yeast

Fatty acid methyl esters (FAME) were prepared using the Fatty Acid Methylation Kit (Nacalai Tesque, Inc., Kyoto, Japan) and purified using the Methylated Fatty Acid Purification Kit (Nacalai Tesque, Inc.) following the manufacturer's instructions. The FAME were analyzed using a gas chromatograph (GC-2025; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a silica capillary column (L × I.D. 30 m × 0.32 mm, df 0.25 μm, SUPELCOWAX® 10, Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas. The temperature of GC oven was initially set at 170 °C and then increased to 260 °C at a rate of 2 °C/min. The temperatures of the injection port and detector were

The fads2, elovl5, and elovl4 cDNAs were PCR amplified from the liver (fads2 and elovl5) and eye (elovl4) of Nibe croaker using primers listed in Table 1 (fads2: Fads2-F and Fads2-R, elovl5: Elovl5-F and Elovl5-R, elovl4: Elovl4-F and Elovl4-R) with PrimeSTAR®Max DNA polymerase (TaKaRa Bio). These primers contained Hind III and Xba I (fads2 and elovl5) and Hind III and Xho I (elovl4) restriction enzyme sites. Amplifications were performed with 30 cycles comprising denaturation for 10 s

2.3. Fatty acid analysis of the yeast

Fig. 3. Phylogenetic tree comparing the deduced amino acid sequence of the Nibe croaker elovl4 gene with other known Elovl amino acid sequences. The GenBank accession numbers of reference cDNA sequences are shown in parentheses. The horizontal branch length is proportional to amino acid substitution rate per site. The numbers represent the frequencies (%) with which the tree topology presented was replicated after 1000 iterations. The scale bar indicates amino acid substitution rate per site along the horizontal branch.

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both set to 260 °C. FAME peaks were identified by comparison of the retention time of each with that of the appropriate FAME standard (Qualmix Fish S, 18:3n-6, 20:2n-6, 20:3n-6, 20:3n-3, 20:4n-3, 22:2n-6, 22:3n-3, 22:5n-6; Larodan Fine Chemicals). Other fatty acids (16:3n-3, 22:3n-6, 24:5n-3 and 24:6n-3; Larodan Fine Chemicals) that are not commercially available as FAME standard were used after methylation using the above-mentioned kit. Desaturase and elongase activities were calculated as the proportion of fatty acid substrate converted to desaturated product as [product area / (product area + substrate area)] × 100 (%).

3. Results 3.1. Sequence analysis of putative elovl4 cDNA from Nibe croaker The putative elovl4 from Nibe croaker consisted of an open reading frame (ORF) of 918 base pairs encoding 305 amino acids (GenBank accession KM606993) (Fig. 2). The amino acid sequence of the putative

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Elovl4 contained a histidine box (HXXHH) and putative endoplasmic reticulum retention signal at its carboxyl end, which are typical for members of the microsomal Elovl family (Fig. 2). Hydropathy analysis revealed that the predicted protein was composed of six transmembrane regions (Fig. 2). Phylogenetic analysis of teleost species showed that Nibe croaker Elovl4 clustered in the Elovl4 group (Fig. 3).

3.2. Functional characterization of Nibe croaker Fads2, Elovl5, and Elovl4 cDNAs in yeast The substrate specificities of Nibe croaker Fads2, Elovl5, and Elovl4 enzymes were determined by heterologous expression in yeast Saccharomyces cerevisiae grown in the presence of potential fatty acid substrates. The predominant fatty acids in control yeast cells carrying the empty pYES2 vector were 16:0, 16:1 isomers, 18:0 and 18:1n-9, as well as any exogenously added fatty acid substrates (data not shown). This result indicated that yeast carrying the empty pYES2 vector expresses no fatty acid-metabolizing enzymes with activities toward

Fig. 4. Chromatograms of fatty acid methyl esters (FAMEs) extracted from yeast transformed with the coding sequence of Nibe croaker fads2. The yeast was grown with no substrate (A) or in the presence of one of the exogenously added substrates 18:3n-3 (B), 18:2n-6 (C), 20:3n-3 (D), 20:2n-6 (E), 20:4n-3 (F), 20:3n-6 (G), 22:5n-3 (H), 22:4n-6 (I) and 24:5n-3 (J) written in bold letters. Asterisks indicate additional peaks. Peaks representing 16:0, 16:1 isomers, 18:0 and 18:1n-9 are the major endogenous fatty acids in the yeast. Vertical and horizontal axes indicate FID response and retention time, respectively.

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PUFA substrates, in agreement with earlier observations (Hastings et al., 2001; Agaba et al., 2004). Fatty acid analyses of the yeast expressing each enzyme cultured in medium lacking any fatty acid supplement were also performed (Figs. 4A, 5A, 6A). The results showed that the Nibe croaker Elovl5 has some capability to elongate yeast endogenous monounsaturated fatty acids (Fig. 5A, 18:1n-7, 20:1n-9, and 20:1n-7), as previously reported in several fish species (Hastings et al., 2005; Morais et al., 2009; Mohd-Yusof et al., 2010; Monroig et al., 2013). The yeast cells expressing the Nibe croaker fads2 ORF cultured in the presence of 18:3n-3 or 18:2n-6 showed peaks corresponding to 18:4n-3 and 18:3n-6 (Fig. 4B, C). The conversion rates of 18:3n-3 to 18:4n-3 and 18:2n-6 to 18:3n-6 were 24.2% and 16.3%, respectively (Table 2). Moreover, Fads2 showed some activity toward 20:3n-3 and 20:2n-6, which were desaturated to 20:4n-3 and 20:3n-6, respectively (Fig. 4D, E and Table 2). However, these conversion rates were very low when

compared with those for 18:3n-3 and 18:2n-6 (Table 2). In addition, comparing activities toward n-3 and n-6 PUFA substrates, the Nibe croaker Fads2 desaturated n-3 more efficiently than n-6 PUFA substrates (Table 2). No evidence of desaturation of 20:3n-6, 20:4n-3, 22:4n-6, 22:5n-3, or 24:5n-3 was observed (Fig. 4F–J). These results indicated that Fads2 from Nibe croaker had Δ6 desaturase activity toward 18:3n-3 and 18:2n-6 but not 24:5n-3. When the Nibe croaker Elovl5 cDNA was expressed in the yeast cells, evidence of elongation of almost all fatty acids except for 22:4n-6 was observed (Fig. 5, Table 3). These results indicated that Nibe croaker Elovl5 had activity toward substrates with 16 to 22 carbons, with apparent preference for C16, C18 and C20 over C22 FA substrates (Table 3). Transgenic yeast cells expressing the Nibe croaker elovl4 ORF cultured in the presence of C18 (18:3n-3 and 18:4n-3), C20 (20:5n-3), or C22 (22:5n-3) substrates showed peaks corresponding to chain-elongated

Fig. 5. Chromatograms of fatty acid methyl esters (FAMEs) extracted from yeast transformed with the coding sequence of Nibe croaker elovl5. The yeast was grown with no substrate (A) or in the presence of one of the exogenously added substrates 16:3n-3 (B), 18:2n-6 (C), 18:3n-3 (D), 18:3n-6 (E), 18:4n-3 (F), 20:4n-6 (G), 20:5n-3 (H), 22:4n-6 (I) and 22:5n-3 (J) written in bold letters. Asterisks indicate additional peaks. Peaks representing 16:0, 16:1 isomers, 18:0 and 18:1n-9 are the major endogenous fatty acids in yeast. Peaks representing 18:1n-7, 20:1n9 and 20:1n-7 are presumed elongation products of the yeast endogenous monounsaturated fatty acids. Vertical and horizontal axes indicate FID response and retention time, respectively.

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Table 2 Substrate conversions in Nibe croaker fads2-transformed yeast. Conversion (%) indicates the percentage of substrate fatty acid converted to the product fatty acid. Substrate

Product

Conversion (%)

Activity

n-3 18:3n-3 20:3n-3 20:4n-3 22:5n-3 24:5n-3

18:4n-3 20:4n-3 20:5n-3 22:6n-3 24:6n-3

24.2 ± 3.29 4.2 ± 0.37 0.0 0.0 0.0

Δ6 Δ8 Δ5 Δ4 Δ6*

n-6 18:2n-6 20:2n-6 20:3n-6 22:4n-6

18:3n-6 20:3n-6 20:4n-6 22:5n-6

16.3 ± 0.65 1.6 ± 0.06 0.0 0.0

Δ6 Δ8 Δ5 Δ4

Δ6* indicates Δ6 desaturase activity toward C24 fatty acid substrates.

Fig. 6. Chromatograms of fatty acid methyl esters (FAMEs) extracted from yeast transformed with the coding sequence of Nibe croaker elovl4. The yeast was grown with no substrate (A) or in the presence of one of the exogenously added substrates 18:3n-3 (B), 18:4n-3 (C), 20:5n-3 (D) and 22:5n-3 (E) written in bold letters. Asterisks indicate additional peaks. Peaks representing 16:0, 16:1 isomers, 18:0 and 18:1n-9 are the major endogenous fatty acids in yeast. Vertical and horizontal axes indicate FID response and retention time, respectively.

products of each fatty acid (Fig. 6, Table 4). In particular, Nibe croaker Elovl4 was able to convert both 20:5n-3 and 22:5n-3 to 24:5n-3, the C24 substrate for Δ6 Fads in DHA (22:6n-3) synthesis. 4. Discussion In the present study, we confirmed that the Nibe croaker Fads2 desaturase has Δ6 desaturase activity toward 18:3n-3 and 18:2n-6 but not toward 24:5n-3 and slight Δ8 desaturase activity toward 20:3n-3 and 20:2n-6. The Elovl5 showed preferential elongase activity toward C16, C18 and C20 PUFA. The deduced amino acid sequence of the newly cloned elovl4-like cDNA showed all the features found in previously cloned elovl4 cDNAs from several species. Although Elovl4 may be able to elongate longer-chain fatty acids, it showed most preferential elongase activity toward C20. These results indicate that the Nibe croaker is not capable of synthesizing EPA from 18:3n-3 and DHA from 22:5n-3.

In our previous study, we observed an EPA concentration decrease, a 22:5n-3 concentration increase, a 24:5n-3 accumulation but no DHA level alteration in the livers of transgenic Nibe croaker expressing the Masu salmon Elovl2 compared with those in their non-transgenic counterparts (Kabeya et al., 2014). Based on these results, we expected that the establishment of new transgenic strain(s) carrying other genes such as Δ5 desaturase, Δ4 desaturase and crosses of the new strain(s) with the existing strain would be needed. However, before starting our next project, the determination of the precise endogenous LC-PUFA biosynthetic pathway in Nibe croaker was required, because no DHA concentration alteration between transgenic and non-transgenic fish conflicted with our expectation that Nibe croaker was able to synthesize DHA from EPA as long as they carry foreign elovl2 gene. Indeed, in this study, the Nibe croaker endogenous Fads2 showed no Δ6 desaturase activity toward 24:5n-3, which is an essential activity for DHA synthesis from EPA in species lacking Δ4 Fads in yeast. In our previous in vivo studies, the DHA concentration was not altered between transgenic and non-transgenic fish during DHA deficiency (Kabeya et al., 2015), although the expression level of the fads2 in Nibe croaker was elevated as per the decrease in DHA concentration in their feeds (Yamamoto et al., 2010). In addition, although the brain and eye of the transgenic fish contains 24:5n-3, 24:6n-3, a desaturation product of 24:5n-3 was not detected despite Fads2 expression in these tissues (Kabeya et al., 2014). These results suggest that the desaturation pathway from 24:5n-3 to 24:6n-3 is probably absent in the Nibe croaker. Given that Nibe croaker may harbor other Fads with activities other than Δ6, it is now desirable to clone other Fads from Nibe croaker, although this task may be difficult owing to the lack of current genomic or transcriptomic information for this species. However, currently available genomic data for large yellow croaker, Larimichthys crocea (Wu et al., 2014), Pacific bluefin tuna Thunnus orientalis (Nakamura et al., 2013) and Atlantic cod Gadus morhua (Star et al., 2011) show that these species most likely carry a single copy of the fads2 gene (data not shown). Although Δ6 activity toward 24:5n-3 in zebrafish (Tocher et al., 2003) and Atlantic salmon (Hastings et al., 2005; Bell and Tocher, 2009) has been reported, there have been no such studies in marine species. Accordingly, our study provides the first evidence of a Fads, lacking activity toward 24:5n-3, in a marine fish species. In addition, as in most marine fish species, Nibe croaker Fads showed no Δ4 desaturase activity toward 22:5n-3. The functional characterization of Nibe croaker Elovl5 revealed that the most preferential substrate of Elovl5 is C20 fatty acid (20:4n-6 and EPA). These characteristics were similar to those of other Elovl5 isolated from several species to date (Zheng et al., 2009; Morais et al., 2011, 2012). Nibe croaker Elovl5 also showed high activity toward 16:3n-3, like Elovl5 isolated from meager (Argyrosomus regius) which is other sciaenid species in the Mediterranean (Monroig et al., 2013). This result suggests that Nibe croaker is able to endogenously convert 16:3n-3 to 18:3n-3. However, Elovl5 showed relatively low activity toward 22:5n-3. This result is consistent with the observation that 24:5n-3,

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Table 3 Substrate conversions in Nibe croaker elovl5 transformed yeast. Conversion (%) indicates the percentage of substrate fatty acid converted to the product fatty acid. Accumulated conversions were calculated by summing the individual conversion for a particular product and those for longer products. Substrate n-3 16:3n-3 18:3n-3 18:4n-3 20:5n-3 22:5n-3 n-6 18:2n-6 18:3n-6 20:4n-6 22:4n-6

Product

Individual conversion (%)

Accumulated conversion (%)

Activity

18:3n-3 20:3n-3 20:3n-3 22:3n-3 20:4n-3 22:4n-3 22:5n-3 24:5n-3 24:5n-3

49.8 ± 0.33 41.5 ± 1.59 45.2 ± 3.09 2.6 ± 0.37 41.5 ± 3.62 47.7 ± 5.01 74.4 ± 2.32 12.0 ± 0.98 3.6 ± 0.86

91.3 ± 1.30 41.5 ± 1.59 47.8 ± 3.43 2.6 ± 0.37 89.2 ± 1.39 47.7 ± 5.01 86.4 ± 1.89 12.0 ± 0.98 3.6 ± 0.86

C16 → 18 C18 → 20 C18 → 20 C20 → 22 C18 → 20 C20 → 22 C20 → 22 C22 → 24 C22 → 24

20:2n-6 22:2n-6 20:3n-6 22:3n-6 22:4n-6 24:4n-6 24:5n-6

33.3 ± 0.59 2.5 ± 0.17 59.9 ± 1.84 10.9 ± 2.05 74.4 ± 2.66 1.5 ± 0.77 0.0

35.7 ± 0.73 2.5 ± 0.17 70.8 ± 1.18 10.9 ± 2.05 75.9 ± 1.97 1.5 ± 0.77 0.0

C18 → 20 C20 → 22 C18 → 20 C20 → 22 C20 → 22 C22 → 24 C22 → 24

which is an elongation product of 22:5n-3, was not detected in various tissues of non-transgenic Nibe croaker, although the elovl5 gene was expressed in several tissues (Yamamoto et al., 2010). In the present study, we also isolated and functionally characterized an Elovl4 cDNA, with the aim of determining how this enzyme participates in the DHA biosynthetic pathways. Based on the deduced amino acid sequence, Nibe croaker Elovl4 exhibits characteristic features of microsomal membrane-bound enzymes by containing a single histidine box, an endoplasmic reticulum retention signal and multiple transmembrane regions (Leonard et al., 2004; Jakobsson et al., 2006). Phylogenetic analysis suggested that the protein encoded by the elovl4 cDNA is more similar to Elovl4 proteins isolated from various fish species than to Elovl5 and Elovl2. Functional characterization showed that the Elovl4 enzyme was able to elongate all fatty acids tested including 18:3n-3, 18:4n-3, 20:5n-3 and 22:5n-3. Although conversion rates of fatty acids were relatively low compared to those obtained with Elovl5, these results support the participation of Elovl4 in the biosynthesis of n-3 PUFA. In addition, there is a possibility that the Elovl4 can convert longer-chain (C24 to C36) fatty acids equally as well as the previously characterized Elovl4 isolated from cobia (Monroig et al., 2011b) and rabbitfish (Monroig et al., 2012). However, although the elovl4 gene was dominantly expressed in the eye (Kabeya et al., 2014) and the endogenous Fads could not convert 24:5n-3 to 24:6n-3, 24:5n-3 was not detected in the eye of non-transgenic Nibe croaker. These results suggest that the Elovl4 activity toward 22:5n-3 is insufficient to produce a detectable level of 24:5n-3 or that the converted 24:5n-3 is immediately elongated by Elovl4 to longer-chain fatty acids including 26:5n-3 to 36:5n-3. In any case, combining the results of both the transgenic study and current Elovl4 functional characterization, we conclude that the elongase activities of the Elovl4 protein did not majorly affect the biosynthesis of 24:5n-3 from 22:5n-3 in the whole body. It is considered that the inability of marine fish lacking Δ4 Fads to biosynthesize DHA is due not only to a lack of Elovl2 and Δ5 Fads but also to a lack of Δ6 Fads, which acts by converting 24:5n-3 to 24:6n-3. Table 4 Substrate conversions in Nibe croaker elovl4-transformed yeast. Conversion (%) indicates the percentage of substrate fatty acid converted to the product fatty acid. Substrate

Product

Conversion (%)

Activity

18:3n-3 18:4n-3 20:5n-3

20:3n-3 20:4n-3 22:5n-3 24:5n-3 24:5n-3

7.0 ± 0.02 5.5 ± 0.01 12.0 ± 0.85 6.7 ± 1.66 3.6 ± 0.38

C18 → 20 C18 → 20 C20 → 22 C22 → 24 C22 → 24

22:5n-3

Thus, to achieve our final goal of generating transgenic marine fish strains that are capable of endogenously synthesizing DHA, we need to produce at least two more strains carrying Δ5 and Δ6 Fads, which can convert 24:5n-3 to 24:6n-3. However, although a Fads with Δ5 desaturase activity has been isolated from several teleost species (Hastings et al., 2001, 2005; Li et al., 2010), a Fads enzyme with Δ6 desaturase activity mainly toward 24:5n-3 has not been reported to date. Accordingly, we hypothesize that the Δ4 pathway is a more promising candidate for producing a transgenic strain capable of synthesizing DHA from 22:5n-3, given that the Δ4 desaturation pathway does not the Δ6 desaturation step from 24:5n-3 to 24:6n-3 (see Fig. 1). Given that the Δ4 Fads has been isolated from rabbitfish (Li et al., 2010), Senegalese sole (Morais et al., 2012), pike silverside (Chirostoma estor) (Fonseca-Madrigal et al., 2014), and striped snakehead (Channa striata) (Kuah et al., 2015), genes encoding these Fads would be ideal for use in future transgenesis experiments. Acknowledgments The authors are grateful to members of the aquaculture group of the GeneCology Research Centre of the University of the Sunshine Coast for help with the yeast experiment. References Agaba, M.K., Tocher, D.R., Dickson, C.A., Dick, J.R., Teale, A.J., 2004. Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Mar. Biotechnol. 6, 251–261. Agaba, M.K., Tocher, D.R., Zheng, X., Dickson, C.A., Dick, J.R., Teale, A.J., 2005. Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish. Comp. Biochem. Physiol. B 142, 342–352. Alimuddin, Yoshizaki, G., Kiron, V., Satoh, S., Takeuchi, T., 2005. Enhancement of EPA and DHA biosynthesis by over-expression of masu salmon Δ6-desaturase-like gene in zebrafish. Transgenic Res. 14, 159–165. Alimuddin, Yoshizaki, G., Kiron, V., Satoh, S., Takeuchi, T., 2007. Expression of masu salmon Δ5-desaturase-like gene elevated EPA and DHA biosynthesis in zebrafish. Mar. Biotechnol. 9, 92–100. Alimuddin, Kiron, V., Satoh, S., Takeuchi, T., Yoshizaki, G., 2008. Cloning and overexpression of a masu salmon (Oncorhynchus masou) fatty acid elongase-like gene in zebrafish. Aquaculture 282, 13–18. Bell, M.V., Tocher, D.R., 2009. Biosynthesis of polyunsaturated fatty acids in aquatic ecosystems: general pathways and new directions. In: Arts, M.T., Brett, M.T., Kainz, M.J. (Eds.), Lipids in Aquatic Ecosystems, 1st ed. Springer, New York, pp. 211–236. Castro, L.F.C., Monroig, Ó., Leaver, M.J., Wilson, J., Cunha, I., Tocher, D.R., 2012. Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates. PLoS One 7, e31950. Claros, M.G., von Heijne, G., 1994. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10, 685–686.

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