Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Soft cheese with white rind lacks essential fatty acids (EFAs), and as a result its long‐term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid (OA) and linoleic acid (LA) to produce LA and α‐linolenic acid (ALA), respectively, was isolated from G. candidum, and then cloned and heterologously expressed in Saccharomyces cerevisiae. This gene, named GcFADS12, had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that GcFADS12 had the ability to convert OA to LA and LA to ALA, and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of GcFADS12 catalyzes the conversion of the intermediate product (LA) to ALA by addition of OA as the sole substrate. The catalytic activity of GcFADS12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that GcFADS12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFAs by G. candidum in cheese.

The industry of making mold cheese, a kind of cheese fermented by mold, is in its initial stage in China [1]. Mold cheese contains rich nutrients such as protein, calcium, fatty acids and vitamins, among which fatty acids account for 24-28%. Fatty acids in mold cheese play an important role in the formation of cheese flavors and are especially indispensable for the human diet [2]. However, cheese lacks polyunsaturated fatty acids (PUFAs), especially essential fatty acids (EFAs) including linoleic acid (LA, 18:2 D9,12 ) and a-linolenic acid (ALA, 18:3 D9,12,15 ), and this problem has not been solved so far. This has been the main reason why the proportion of saturated and unsaturated fatty acids in cheese is seriously unbalanced. Therefore, the longterm consumption of cheese containing a large amount of saturated fatty acids and lacking EFAs may lead to various kinds of cardiovascular and cerebrovascular diseases such as hyperlipidemia, hypertension and atherosclerosis.
[3-6]. G. candidum is safe for food use; its safety has been studied since the 1960s and its fermented products have a high level of safety for consumers [7]. G. candidum has been used widely to produce lipase [8-10], but it has not been applied in EFA biosynthesis in the mold cheese-making industry thus far.
In EFA biosynthesis, D12 fatty acid desaturase (FADS12), D15 fatty acid desaturase (FADS15) and D6 fatty acid desaturase (FADS6) play a key role in regulating the level of EFAs. In a previous study, the molecular mechanism, substrate specificity and catalytic activity for FADS6 were analyzed [11,12] and applied to PUFA synthesis [13,14]. FADS12 is a key bifunctional membrane-bound desaturase that converts oleic acid (OA, 18:1 D9 ) to linoleic acid (LA, 18:2 D9,12 ) and LA to a-linolenic acid (ALA, 18:3 D9,12,15 ) by introducing a double bond between the carbons 12 and 13 from the carboxyl end of the substrate in the biosynthesis of EFAs [15][16][17]. The genome of G. candidum has been sequenced and submitted to the Gen-Bank database (LOCUS: CCBN010000001) by Casaregola, and we found from its genome sequence that there was a sequence encoding D12 fatty acid desaturase of G. candidum (GcFADS12). However, its function has been not analyzed. The level of OA, as a substrate of FADS12, accounts for about one-quarter of the total fatty acid in mold cheese. Many studies have reported that the FADS12 gene from various species is overexpressed for LA production, and the relative LA level was increased up to fivefold under flask culture conditions in Rhodosporidium toruloides [16,18,19]. It was inferred that the catalytic properties of FADS12 directly determine the EFA level in mold cheese. Thus, it is necessary to clone the GcFADS12 gene and characterize the function of GcFADS12.
In the present study, the relative transcript level of the GcFADS12 gene of G. candidum was determined. Furthermore, the GcFADS12 gene was cloned, and the function of the gene product characterized following expression in Saccharomyces cerevisiae. In addition, multiple sequence alignment of FADS12s and a phylogenetic analysis were performed. Finally, the effect of substrate concentration on the conversion rate and kinetics of GcFADS12 was analyzed to assess its affinity for both substrates.

Strains and plasmids
Geotrichum candidum was isolated from mold cheese and preserved in our Animal-derived Foods Processing and Utilization lab. Plasmid pYES2/NT C (Invitrogen, Shanghai, China) with 6 9 His tag was used for expression of the GcFADS12 gene and detection of GcFADS12.

Media and cultural conditions
LB agar plates for plasmid construction in Escherichia coli Top 10 and SC-U synthetic minimal medium for gene expression in S. cerevisiae were used as described previously [12]. G. candidum was grown on Yeast Peptone Dextrose medium (containing 10 gÁL À1 yeast extract, 10 gÁL À1 peptone, 10 gÁL À1 glucose and 20 gÁL À1 agar) at 28°C.

RNA isolation and RT-qPCR analysis
Approximately 0.5 g (dry weight) of G. candidum cells was ground to a fine powder with a precooled mortar and pestle using liquid nitrogen. Total RNA was isolated using the RNAprep Pure Plant Kit, and reverse-transcribed with QuantScript RT Kit as described previously [13]. RT-qPCR was performed as described previously [14], and the transcript levels were calculated using the 2 ÀDDCt method.

Primer design, PCR amplification and sequence analysis for the GcFADS12 gene
To identify genes encoding GcFADS12, a PCR-based cloning strategy was adopted. According to the available sequence information of FADS12 from Galactomyces candidum WGS (protein id: CDO51572.1; GenBank accession no. for its genome: CCBN010000001), two highly degenerate primers (shown in Table S1) were designed to target sequences corresponding to the first and the third His-rich motif in GcFADS12. Two primers were designed to clone the upstream sequence from 'HGKHHK' and the downstream sequence from 'HDIIETHVLHH' (shown in Table S1). After the full-length cDNA of GcFADS12 was amplified, it was ligated into pMD19-T simple vector and sequenced.
Yeast transformation, heterologous expression of the GcFADS12 gene in S. cerevisiae and western blot analysis of GcFADS12 Plasmid pYES2/NT C-GcFADS12 was transformed into S. cerevisiae and induced. After induction, cultures were supplemented with 0.25 mM cis-OA or 0.25 mM cis-LA and 1% Tergitol Nonidet P-40 for the solubilization of fatty acids. For substrate concentration experiments, 0.125, 0.25 or 0.5 mM cis-OA or cis-LA was added in the induced medium. For western blotting analysis, THE ™ His tag antibody (Invitrogen) and horseradish peroxidase-conjugated goat anti-mouse IgG (H&L) (Bio-Rad, Shanghai, China) were used for GcFADS12 detection. The protocol of induction, transformation and western blotting used was the same as described previously [11].

Phylogenetic analysis and topology prediction
Multiple sequence alignment of FADS12s from various species was performed and a phylogenetic tree was constructed using DNAMAN software (Lynnon, Suite D, San Ramon, CA, USA) and visualized with TREEVIEW. A predicted topology model for GcFADS12 was performed with TMHMM.

Lipid extraction and fatty acid analysis
Lipids from an equivalent weight of cells were extracted and methyl esterified, and fatty acid content was analyzed by gas chromatography as described previously [14].

Results
Relative transcript level of the GcFADS12 gene in Geotrichum candidum To analyze whether the relative transcript level of the GcFADS12 gene is stable during various fermentations  of G. candidum, RT-qPCR was performed. The results revealed that the transcript level of the GcFADS12 gene in G. candidum had no significant difference among various fermentation periods with a slight increase that ranged from 1.2 to 1.5 (Fig. 1), suggesting that GcFADS12 relative transcript level was stable in culture of G. candidum.

Cloning of the GcFADS12 gene
To clone the GcFADS12 gene, a 600-bp DNA fragment was amplified from our previously screened G. candidum, using degenerate primers derived from highly conserved histidine sequences of D12 fatty acid desaturase. Cloning of the full-length

Multiple sequence alignment and phylogenetic analysis of FADS12s
The amino acid sequence of GcFADS12 showed a significant similarity (66.44%) to D12 fatty acid desaturase of several species in a multiple sequence alignment (Fig. 2). In addition, the GcFADS12 sequence had 94.30% identity with FADS12 from G. candidum WGS.
A phylogenetic tree was constructed for GcFADS12 and other FADS12s from various species. Phylogenetic analysis of the putative GcFADS12 gene showed that it had a close relationship with the FADS12 gene sequence in G. candidum WGS. SlFADS12 and both GcFADS12s clustered in a major clade, FADS12s from Kluyveromyces marxianus, Clavispora lusitaniae and Ascoidea rubescens formed a separate cluster at the bottom of the tree, and other FADS12s were in another group (Fig. 3).

Characterization of GcFADS12 function
The expression level of the GcFADS12 gene in S. cerevisiae was determined by SDS/PAGE and western blot. The GcFADS12 band was generally not visible in SDS/PAGE as the expression of the membrane enzyme was low in S. cerevisiae. But western blot analysis showed that there was a specific protein band with a molecular mass of 50 kDa, which was in accordance with the theoretical molecular mass; and there was no significant difference among various samples (Fig. 4A). The function of GcFADS12 was characterized by adding various fatty acid substrates. The results of gas chromatography showed that it had the function of FADS12, but its ability to transform OA and LA substrates was weak (Fig. 4A,B). These findings revealed that GcFADS12 encoded a fatty acid desaturase possessing FADS12 activity, which could successfully convert exogenous OA to LA and LA to ALA. The conversion rate from OA to LA and LA to ALA was 20.40 AE 0.66% and 6.40 AE 0.57%, respectively (Fig. 4A). In addition, GcFADS12 also continued to transform LA (the product of OA) to ALA by addition of OA as the substrate, and the conversion rate from LA to ALA was 9.80 AE 0.25% (Table 1).

The effect of substrate concentration on the conversion rate for GcFADS12
To characterize the effect of substrate concentration on the conversion rate of GcFADS12, S. cerevisiae transformants harboring pYES2/NT C-GcFADS12 plasmid and the control (harboring pYES2/NT C) were incubated with 0.125/0.250/0.500 mM OA or 0.125/0.250/0.500 mM LA as fatty acid substrates for 12 h at 28°C, and the resulting fatty acid composition is listed in Table 1. The results revealed that substrate concentration had little effect on the conversion rate in yeast expressing GcFADS12, whereas there was significantly augmented OA conversion from 25.04 AE 0.45% to 17.72 AE 1.02% and LA conversion from 10.54 AE 0.08% to 9.71 AE 0.54% by OA addition. LA conversion ranged from 7.12 AE 0.36% to 5.80 AE 0.84% by LA addition (Table 1).

Kinetics of GcFADS12
The affinity of GcFADS12 was next assessed by evaluating its reaction rate in the presence of two fatty acid concentrations of OA or LA, where the values of maximal reaction velocity (V m ) and K m were calculated using the Michaelis-Menten equation (Table 2). The kinetics results showed that the K m value for OA as substrate was lower than that for LA as substrate for the pYES2-GcFADS12 transformant, whereas V m for OA as substrate was more than double that for LA as substrate (Table 2). In addition, the K m value for LA as substrate, which was the intermediate product of OA of GcFADS12, was markedly decreased, compared to that for LA as the substrate; but it corresponded to that for OA as the substrate. The V m for LA as substrate (the intermediate product of OA) was much lower than that for OA as the substrate, as there was a low quantity of the intermediate product of LA (Table 2). Together, these results suggest that GcFADS12 had stronger affinity for OA than for LA as substrate.

Discussion
In this study, FADS12 cDNA from G. candidum (GcFADS12) was cloned and its functional characterization was reported. Based on the multiple sequence alignment of the other FADS12 enzymes, the functional domains of GcFADS12 were highly conserved among various species (Fig. 2). GcFADS12 possessed typical characteristics of other FADS12 family members, including three histidine-rich conserved motifs (HXXHHK, HWXVXXTXLQH and HLVHH) and six transmembrane domains (Fig. 5), and these observations were in agreement with our previous reports on D6 fatty acid desaturase [12,16]. The amino acid sequence of GcFADS12 was highly similar to the one that S. Casaregola submitted to the GenBank (protein id: CDO51572.1), which was in agreement with the observation from our phylogenetic analysis of FADS12 (Fig. 3).
The bifunctional FADS12 enzyme has been widely characterized as being responsible for converting the OA and LA fatty acid substrates to LA and ALA, respectively, in many species [17,20,21]: functional analysis results showed that FADS12 from Rhodosporidium kratochvilovae converted 11.4% of OA to LA, and 19.1% of LA to ALA; Wei et al. [20] reported that FADS12 from Rhizopus arrhizus had a high conversion rate of 16.0% for OA when it was expressed in yeast; and the conversion rate of OA by FADS12 from Mortierella alpina 1S-4 was approximately 24.1%, and that of LA was 36.8%. FADS12 isolated from G. candidum in cheese in this study was also bifunctional for both OA and LA, and we found from our functional analysis that GcFADS12 should continually catalyze the product (LA) of OA to ALA. This plays an important role in enhancing and especially balancing the level of essential fatty acids (LA and ALA) from G. candidum in cheese.
We found that the conversion rates of OA and LA for GcFADS12 showed a downward trend as the substrate concentration (as shown in Table 1). However, as the extent of the decline became smaller, it conformed to the curve of the Michaelis-Menten equation. The kinetics of GcFADS12 showed that it had a strong affinity and conversion rate for OA, which was consistent with the results of GcFADS12's functional characterization.
In this study, we newly isolated and identified a gene coding for D12 fatty acid desaturase from G. candidum, characterized its function with OA and LA, and assessed its affinity for both substrates. Our study presents a step forward to producing EFAs with G. candidum in cheese.