DES2 is a fatty acid Δ11 desaturase capable of synthesizing palmitvaccenic acid in the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi are oleaginous organisms, and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1Δ11cis). However, it is not known how this uncommon fatty acid species is made. Here, we have cloned two homologues of lepidopteran fatty acyl‐coenzyme A Δ11 desaturases from the AM fungus Rhizophagus irregularis. Both enzymes, DES1 and DES2, are expressed in intraradical mycelium and can complement the unsaturated fatty acid‐requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1Δ9cis) production, whereas DES2 expression results in the production of 16:1Δ11cis and vaccenic acid (18:1Δ11cis). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1Δ11cis in R. irregularis.

Arbuscular mycorrhiza (AM) is the most common plant-microbe symbiotic association [1]. AM fungi are obligate biotrophs and receive organic carbon from their host plants in return for mineral nutrients [1]. Lipids are the major carbon currency in the AM fungal mycelium, and they are transported to vesicles and spores where they are stored [2]. It was thought that AM fungi most likely synthesize their lipids de novo from sugars, which they receive from their host plant [3]. However, genomic analysis has suggested that AM fungi are fatty acid auxotrophs [4] and subsequent studies have shown that they rely on their host plant to supply them with long-chain fatty acyl moieties so that they can make fungal lipids [5][6][7][8]. The plant metabolic pathway that supplies fatty acyl moieties to AM fungi has been partially characterized, but it is not yet clear precisely where this pathway ends and those of the fungus begin [5][6][7][8]. However, it is currently proposed that long-chain saturated fatty acyl moieties are most likely being transferred as 2-monoacylglycerols or free fatty acids [5][6][7][8][9].
The lipids in many (but not all) AM fungi are dominated by a single molecular species of monounsaturated fatty acid called 11-cis-palmitvaccenic acid (16:1 D11cis ), which can account for over 70 mol% of the fatty acyl moieties in their spores and is present mainly in the form of triacylglycerols [4,[10][11][12]. 16:1 D11cis is unusual in that it contains a double bond at the x5 (or D11) position, and it has been used as a biomarker for arbuscular mycorrhization because it is not found in plants and it is rarely present in other soil microorganisms [10]. 16:1 D11cis has also been used in chemotaxonomy, because it is abundant in many AM fungi (Glomeromycota) but is lacking in certain species of the families Glomeraceae and Gigasporaceae [11].
It is thought that 16:1 D11cis is made in the intraradical mycelium of AM fungi, but it is not known how [4,12]. The discovery that AM fungi receive fatty acyl moieties from their host plant [5][6][7][8] also raises the possibility that 16:1 D11cis might be a product of plant metabolism. Understanding how and where 16:1 D11cis is made is therefore important to define how lipid metabolic pathways function within arbuscular mycorrhiza. D11 desaturases have previously been cloned from insects [13,14] and marine diatoms [15], but we are not aware of any that have been characterized in fungi. The genomes of several AM fungi have now been sequenced, including Rhizophagus irregularis [16], which contains 16:1 D11cis [4]. A blastp search (https:// www.ncbi.nlm.nih.gov/) of the R. irregularis genome using known lepidopteran fatty acyl-coenzyme A (CoA) D11 desaturases [13,14] revealed two potential homologues (DES1 and DES2). It is problematic to test the function of these genes in AM fungi because they are not amenable to genetic modification. We therefore characterized DES1 and DES2 by heterologous expression in Saccharomyces cerevisiae [13] and showed that DES2 encodes a fungal D11 desaturase capable of synthesizing 16:1 D11cis .

Lipid extraction and analysis
Cultures were normalized for cell volume based on OD600 measurements and the cells were pelleted by centrifugation at 2400 g, the supernatant discarded, and the pellets frozen in liquid nitrogen and stored at À80°C. Heptadecanoic acid (17:0) was added to the cell pellets to provide an internal standard (IS). Fatty acid methyl esters (FAMEs) were then prepared from the cell pellets by transmethylation in 1 mL of methanol/toluene/dimethoxypropane/H 2 SO 4 (66 : 28 : 2 : 1 by volume) at 80°C for 40 min, before 0.5 mL hexane and 1 mL KCl (0.88% w/v) were added and the contents were vortexed and centrifuged, and the upper hexane phase was transferred to a fresh vial. Extraction with hexane was repeated twice to ensure extraction of all FAMEs and the three extracts pooled. The FAMEs were dried down under N 2 and reconstituted in 0.5 mL heptane, and 75 µL was taken for analysis by gas chromatography (GC) coupled to mass spectrometry (MS) or flame ionization detector (FID). The position of double bonds in monounsaturated FAMEs was determined by preparing dimethyl disulfide (DMDS) adducts [25]. FAMEs (0.1-1 µg) in 50 µL hexane were combined with 5 µL 50 mgÁmL À1 iodine in diethyl ether and 50 µL DMDS and were vortexed and heated at 40°C for 15 h. Then, 5 µL 5% (w/v) sodium thiosulfate and 200 µL hexane were added, vortexed and centrifuged to separate the phases. The hexane layer was removed, dried under N 2 and  Separation of FAMEs and DMDS adducts was performed by 6890N Network GC System (Agilent Technologies, Santa Clara, CA, USA) fitted with a 30 m 9 0.25 mm, 0.25 µm film thickness, HP1-MS-UI capillary column (Agilent Technologies). FAME/DMDS adducts (1 µL) were injected (splitless) at 280°C and He used as the carrier gas (0.6 mLÁmin À1 ) at a constant flow. The oven program was as follows: 70°C (1 min), 40°CÁmin À1 ramp to 150°C, 4°CÁmin À1 ramp to 300°C (2 min), 325°C (18 min). For FAME/DMDS adduct identification, GC was coupled to a 5975B mass selective detector (Agilent Technologies) with a 3.5-min solvent delay, on constant scan mode 42-500 m/z. The detection and quantification of FAMEs by GC-FID was also performed, using a DB-23 capillary column (Agilent Technologies) as described previously [26].
Expression of DES1 and DES2 in R. irregularis To investigate whether DES1 and DES2 are expressed in R. irregularis, we analysed a RNA-sequencing data set that includes structures from both asymbiotic and symbiotic stages of the AM fungal life cycle such as germ tubes, runner hyphae, intraradical mycelium, arbuscules, branched absorbing structures and immature and mature spores [33]. A search for the corresponding transcripts of DES1 and DES2 within this data set revealed that both genes are expressed in all seven AM fungal structures, but DES2 appears to be the more strongly expressed of the two genes, particularly in intraradical mycelium, arbuscules and spores ( Table 1). A desaturase responsible for producing 16:1 D11cis in R. irregularis should be expressed in these structures since this fatty acyl moiety is most abundant in triacylglycerol that accumulates first in lipid droplets that form in the intraradical mycelium proximal to arbuscules [2,34].

Functional analysis of DES1 and DES2 by expression in S. cerevisiae
To test the enzymatic function of DES1 and DES2, we transformed WT S. cerevisiae and the desaturationdeficient ole1D knockout strain [24,29] with the high-copy-number plasmids pHEY-DES1 and pHEY-DES2, designed to express the two genes under the control of the strong constitutive TEF1 promoter [22]. The ole1D strain is completely deficient in fatty acid desaturation and can only grow on media that are supplemented with exogenous long-chain unsaturated fatty acids [24,29]. A plate test of ole1D harbouring either pHEY-DES1 or pHEY-DES2 showed that cell growth could be rescued by expression of DES1 or DES2 (Fig. 2), suggesting that both proteins can function as desaturases [24,29]. Fatty acid methyl ester analysis of lipids from WT S. cerevisiae cells [24,29] expressing DES1 revealed that there was no change in the molecular species that were produced (Fig. 3). However, there was a significant (P > 0.05) increase in the relative abundance of oleic acid (18:1 D9cis ), as compared to the EVC (Fig. 3; Table S1). By contrast, DES2 expression in WT cells led to the appearance of two major new molecular species of fatty acyl moiety (Fig. 3), which GC-MS analysis indicated were isomers of 16:1 (m/z 268) and 18:1 (m/z 296). Further analysis of the double bond positions by extraction of the molecular ions of DMDS adducts [25] revealed the characteristic fragment ions of 16:1 D11cis (m/z 117, 245) and 11-cis-vaccenic acid (18:1 D11cis ) (m/z 145, 245) (Fig. S2). Small amounts of 13-cis-octadecenoic (18:1 D13cis ) (m/z 117, 273) were also detected ( Fig. 3; Table S1; Fig. S2). Further analysis of the fatty acyl composition of ole1D cells expressing DES1 or DES2 confirmed that with the substrates that are available, DES1 preferentially produces 18:1 D9cis over 16:1 D9cis , whereas DES2 produces 16:1 D11cis and to a lesser extent 18:1 D11cis (Fig. 3; Table S1).
In WT S. cerevisiae cells, trace amounts of 16:1 D11cis and 18:1 D11cis were also detected (Table S1). 16:1 D11cis is known to be a product of 9-cis-myristoleic acid (14:1 D9cis ) elongation by Elo1p [35], and 18:1 D11cis is most likely an elongation product of 16:1 D9cis . 16:1 D11cis elongation is also likely to explain the small amounts of 18:1 D13cis detected in both WT and ole1D cells expressing DES2. To test this hypothesis, ole1D cells expressing DES2 were supplemented with 16:0 or 18:0 free fatty acids to increase the respective amounts of substrate available for desaturation. The addition of 16:0 resulted in a significant increase in 16:1 D11cis and 18:1 D13cis (P > 0.05), which is consistent with a precursor-product relationship (Table S1). Addition of 18:0 resulted in a significant increase in 18:1 D11cis (P > 0.05), but not in 18:1 D13cis (Table S1), suggesting that these MUFAs are not products of the same substrate. Taken together, these data suggest that the 18:1 D13cis is not a direct product of 18:0 desaturation, but of 16:1 D11cis elongation.
Desaturases are classified based on their ability to recognize either the x (methyl) or D (carboxyl) end of the fatty acyl moiety for insertion of the double bond [37]. The ability of DES1 and DES2 to produce D9 and D11 fatty acids using substrates with different chain lengths (C16 and C18) suggests that both are front-end desaturases that count carbon atoms from the carboxyl terminus for insertion of the double bond. The structural basis of chain length specificity has been studied previously in fatty acyl-CoA desaturases [31]. The substrate binding channel of Mus musculus SCD1 is capped by Tyr104, which is located on the second transmembrane helix and blocks access of acyl chains longer than C18 [31,38]. DES1 also possess Tyr in the corresponding position, while DES2 possesses a less bulky Cys residue (Fig. 1). One helical twist above Tyr104 in MmSCD1, and therefore facing the binding pocket, is Ala108 [31]. Mutant analysis suggests that when the Ile residue present at this position in MmSCD3 is substituted for Ala, the substrate preference of MmSCD3 changes from C16 to C18 [31]. Ile has a bulkier side chain than Ala and may therefore shorten the substrate channel [31]. DES1 has Gly in this position (Fig. 1), which has a small side chain. DES2 has Met in this position (Fig. 1), which has a slightly larger side chain. The residues occupying these positions might therefore explain why both DES1 and DES2 accept a C18 substrate. Although 16:1 D11cis is highly abundant in R. irregularis, the levels of 18:1 D11cis are much lower [4,12]. Given that DES2 can synthesize both MUFAs in S. cerevisiae, it is possible that the predominance of 16:1 D11cis in R. irregularis is the result of substrate availability rather than acyl chain length specificity [39]. It is thought that Fig. 2. Plate test illustrating the ability of DES1 and DES2 to rescue the unsaturated fatty acid auxotrophic phenotype of Saccharomyces cerevisiae ole1D. A 0.1-OD600 culture was successively diluted 10-fold to 10 À4 , and 2-µL drops were added to plates with or without a MUFA supplement, using 1 mM 15:1 D10cis . Image was taken after 72 h of growth at 30°C.  R. irregularis receives fatty acyl moieties from its host plant that are mainly C16 [5][6][7][8][9] and so this substrate is likely to be most abundant. However, it is also conceivable that 16:1 D11cis might be preferentially incorporated into triacylglycerol, owing to the activities of lipid assembly and remodelling enzymes that are present in R. irregularis but have yet to be characterized [4]. R. irregularis also contains a comparatively low level of 18:1 D9cis [4,12] that is likely to be produced by DES1, given its activity in S. cerevisiae. In addition to R. irregularis, 16:1 D11cis is present in many Glomeromycota and putative orthologues of DES2 can also be found in the R. diaphanous, R. clarus, R. cerebriforme and Gigaspora rosea genomes [40], but not in those of nonmycorrhizal fungi. Interestingly, G. rosea is one of the species from the family Gigasporaceae that does not contain 16:1 D11cis [11,12]. It is therefore possible that G. rosea DES2 either has a different activity (i.e. is not a D11 desaturase) or is not expressed. At present, it is not known why many Glomeromycota make 16:1 D11cis and some do not. The identification of DES1 and DES2 may help in future studies to better understand the physiological role of the different molecular species of MUFAs found in AM fungi.