Identification of a Δ11 desaturase from the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal 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 homologs of Lepidopteran fatty acyl-CoenzymeA Δ11 desaturases from Rhizophagus irregularis. Both DES1 and DES2 are expressed in intraradicle 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.


Introduction
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 synthesise 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 characterised, but it's 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 2monoacylglycerols 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 Δ11cis ), 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 Δ11cis is unusual in that it contains a double bond at the ω5 (or Δ11) position and it has been used as a biomarker for arbuscular mycorrhization because it is not found 3 in plants and it is rarely present in other soil microorganisms [10]. 16:1 Δ11cis 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 Δ11cis is made in the intraradicle 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 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 Δ11 desaturase capable of synthesising 16:1 Δ11cis .

Expression of DES1 and DES2 in S. cerevisiae
The open reading frames of DES1 and DES2 were codon optimised for expression in S. cerevisiae capillary column (Agilent) as described previously [26].

Identification of putative ∆11 desaturases
To identify candidate ∆11 desaturases from AM fungi we performed a blastp search of the R.
Expression of DES1 and DES2 in R. irregularis To investigate whether DES1 and DES2 are expressed in R. irregularis we analysed a RNAsequencing 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, 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 Δ11cis 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 wild type S. cerevisiae and the desaturation-deficient ole1Δ knock-out 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 translational elongation factor EF-1α (TEF1) promoter [22]. The ole1Δ strain is completely deficient in fatty acid desaturation and can only grow on media that is supplemented with exogenous long-chain unsaturated fatty acids [24,29]. A plate test of ole1Δ 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].
Analysis of the fatty acyl composition of lipids from wild type S. cerevisiae cells 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 (C18:1 Δ9cis ), as compared to the empty vector control (EVC) (Fig. 3; Table S1). By contrast, DES2 expression in wild type 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  Table S1; Fig. S1). Further analysis of the fatty acyl composition of ole1Δ cells expressing DES1 or DES2 confirmed that, with the substrates that are available, DES1 preferentially produces C18:1 Δ9cis over C16:1 Δ9cis , whereas DES2 produces 16:1 Δ11cis and to a lesser extent 18:1 Δ11cis (Fig. 3; Table S1).
Desaturases are classified based on their ability to recognise either the ω (methyl) or Δ (carboxyl) end of the fatty acyl moiety for insertion of the double bond [36]. The ability of DES1 and DES2 to produce Δ9 and Δ11 fatty acids using substrates with different chain lengths (C16 and C18) suggests that both are front-end desaturases that count carbon atoms from the carboxylterminus 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 Tyr 133, which is located on the second transmembrane helix and blocks access of acyl chains longer than C18 [31,37]. DES1 also possess Tyr in the corresponding position ( Fig. 1). One helical twist above Tyr 133 in M. musculus SCD3, and therefore facing the binding pocket, is Ile 137 [31]. Mutant analysis suggests that when Ile 137 is substituted for Ala (as is found in SCD1) SCD3 substrate preference 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 DES1 prefers a C18 substrate and DES2 prefers C16.  [5][6][7][8][9] and so these substrates might be more abundant. R.
irregularis also contains a comparatively low level of 18:1 Δ9cis [4,12] that is likely to be produced by DES1, given its activity in S. cerevisiae. In addition to R. irregularis, 16:1 Δ11cis 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 [38], but not in those of non-mycorrhizal fungi. Interestingly, G. rosea is one of the species from the family Gigasporaceae that does not contain 16:1 Δ11cis [11,12]. It is therefore possible that G. rosea DES2 either has a different activity (i.e. is not a Δ11 desaturase) or is not expressed. At present it is not known why many