Cloning of Acyl-ACP Thioesterase FatA from Arachis hypogaea L. and Its Expression in Escherichia coli

In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli.


Introduction
In higher plants, fatty acid biosynthesis is catalyzed by the action of a type II fatty acid synthase, located in plastids [1][2][3][4]. The reaction includes the condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP derivatives resulting in the acyl-ACP chain successively elongated with two carbon units [5,6]. The final acyl chain elongation product is terminated by acyl-ACP thioesterases (Fats) that hydrolyze the thioester bond of the acyl-ACP and release free fatty acids, which are quickly exported to the cytosol via acyl-CoA synthetase [3,7,8].
Plant acyl-ACP thioesterases are plastid-targeted and nuclear-encoded proteins. Based on their sequence identity and substrate specificity, there are two gene families: FatA and FatB [9][10][11]. The FatA gene is one of the key genes involved in the plastidial fatty acid biosynthesis pathway and encodes thioesterase, with a higher specificity for 18:1-ACP and a lower activity for 18:0-ACP and 16:0-ACP [5,[12][13][14][15]. The FatA thioesterase determines which fatty acids are available for the biosynthesis of membrane lipids and allows the transport of fatty acids out of the plastids to incorporate into glycerolipids. On the other hand, the FatB gene encodes thioesterases with a preference for saturated fatty acids with 8-18 carbons [4,5,7,16].
Recently, several FatA and FatB cDNAs have been cloned and characterized following recombinant expression in E. coli and in plants [4,5,13,[17][18][19][20]. However, there has not yet been a similar report regarding peanut Fat genes. In the present study, we report the isolation of the AhFatA gene and the characterization of the mechanisms and expression levels of FatA in A. hypogaea L. We believe this is the first such work reported for AhFatA and that it will provide information for the genetic manipulation of A. hypogaea L. fatty acid. We also demonstrate significant changes in fatty acid profiles as a result of heterologous expression in E. coli.

Plant Materials.
Peanut cultivar "Luhua 14" was used in this study. Roots, stems, leaves, flowers, and seeds of

Expression Analysis of AhFatA in Different Tissues and Seed Developmental Stages by Quantitative Real-Time PCR.
Quantitative real-time PCR (qRT-PCR) examination of AhFatA expression was carried out with a Bio-Rad iQ5. Peanut β-actin (primers of Actin-F and Actin-R, Table 1) was used as an internal control for normalization of the cDNA. We also designed the AhFatA qRT-PCR primers, AhFatA-F and AhFatA-R (Table 1). Reactions were prepared following the manufacturer's instructions, and qRT-PCR was performed using the Bio-Rad iQ5. Each PCR was repeated four times in a total volume of 20 μL containing 2×SYBR Green I PCR Master Mix (TaKaRa), 100 nM of each primer, and 1 μL diluted (1 : 20) template cDNA.
Reactions were carried out in 96-well optical-grade PCR plates and the matched optical-grade membrane (TaKaRa). The amplification program was as follows: an initial denature step consisting of 1 min at 95 • C, followed by 42 cycles of 10 s at 95 • C, 30 s at 60 • C and 30 s at 72 • C, and an additional cycle of 10 s at 95 • C, 30 s at 58 • C and 5 min at 72 • C, and 10 s at 95 • C for melting curve analysis. The data obtained were analyzed with Bio-Rad iQ5 software. The relative expression of AhFatA in different tissues and at various developmental stages of the immature seeds was calculated using the relative 2 −ΔΔCt method [21]; the error bars indicate SD (n = 4).

Lipid Extraction and Fatty Acid Methyl Ester (FAME)
Analysis of E. coli Samples. Bacterial membrane lipid extraction was carried out as described by Bligh and Dyer [22], with modifications. Wet cell samples were heated at 40 • C to obtain 300 mg dry cell paste. The dry cell paste was diluted with 4 mL chloroform/methanol (1 : 10, v/v), and a suspension of 1 mL hexane containing C19:0 internal standard (1 mg/mL) was added. The mixture was heated at 80 • C for 2 hours in a water bath, and then after cooling, 5 mL of 7% potash was added and mixed. After 10 min, the mixture was centrifuged at 10,000 ×g for 10 min. The supernatants (bacterial sample FAME eluate) were subjected to gas chromatography (GC) using the Elite-wax column in a Perkin-Elmer instrument (ASXL). The flame-ionization detection (FID) temperature was 250 • C, and the operating temperature was maintained at 220 • C. The data presented in this paper are the average of three experiments for each sample.

Expression Patterns of the AhFatA Gene.
To investigate the expression patterns of the AhFatA gene in a range of organs and at different growth stages of the peanut, the relevant samples were analyzed by qRT-PCR. Results showed that in wild-type A. hypogaea L., transcripts were detected in every tissue, but most strongly in seeds and most weakly in roots ( Figure 1). The expression of the AhFatA gene in seeds at different developmental stages (10, 20, 30, 40, 50, 60, and 70 DAP) was also examined by qRT-PCR, and results showed that the AhFatA transcript levels were higher at 60 DAP than at other stages ( Figure 2). These results showed that AhFatA allows fatty acid accumulation in the seeds of A. hypogaea L., indicating that the seeds may have thioesterase activity that is different from that of plants with a shortage of fatty acids.  that AhFatA had the highest expression levels, and the fusion protein GST-AhFatA was soluble after being induced by IPTG for 4 h at 37 • C. The E. coli cells with the pGEX-4T-1 plasmid also had the same expression levels of AhFatA (Figure 3).

Overexpression of AhFatA in E. coli Affects Bacterial
Growth. The AhFatA gene was expressed from the lac promoter in E. coli BL21 (DE3) and induced by IPTG. Our results showed no significant change in bacterial growth rate of either control cells or transformants before IPTG induction in 37 • C. In contrast, after IPTG induction, the growth rate of E. coli BL21 (DE3) with pGEX-4T-1-AhFatA decreased over time compared to the cells with pGEX-4T-1, and cells with pGEX-4T-1 plasmid maintained the higher growth rate at 37 • C (Supplementary Figure 3A). Approximately 5 hours after-induction, growth of the E. coli cells harboring pGEX-4T-1-AhFatA, or pGEX-4T-1 slowed and eventually stopped. The growth rate of the E. coli cells (control and transformants) showed the same trends at 25 • C (Supplementary Figure 3B) as at 37 • C, although growth was slower at 25 • C.

Discussion
In this study, we cloned a 1119-bp gene from A. hypogaea L. and showed that it had high similarity with the FatA genes from other plants. The gene, called AhFatA, represents the first FatA from A. hypogaea L. to be studied. Realtime quantitative PCR analysis of the AhFatA expression pattern revealed that AhFatA was expressed in all tissues and was quite similar to AtFatA from A. thaliana and many other plants [1,24]. Of all plant thioesterases, FatA is essential for plant viability and plays an important role in transferring acyl chains to the extraplastidial glycerolipid and determining the metabolic flux into triacylglycerols. AhFatA transcript levels were higher in seeds and higher at 60 DAP than at other stages, showing that this gene functions largely in fatty acid accumulation, which is in accordance with the oil accumulation in seeds.
It has been reported that thioesterase types A have a higher specificity for 18:1-ACP. We expressed AhFatA in E. coli BL21 (DE3) using the pGEX-4T-1 vector with the lac promoter and demonstrated high levels of expression of AhFatA. Our results showed that the fatty acid composition of the recombinants changed greatly towards C18:1 and C16:1. There is also a relative increase in the accumulation of C18:2 in direct of the proportion of 18:1. Therefore, it could be hypothesized that the bacteria compensate for the available fatty acids by increasing the saturated fatty acids. The results are similar to those in other plants such as Garcinia mangostana [13], Arabidopsis thaliana, Coriandrum sativum [2], Brassica campestris [23], and Ricinus communis L. [4]. Therefore, AhFatA may function mainly to provide palmitoleic acid and oleic acid.
The effect of AhFatA gene overexpression on cell growth at different temperature was examined. The specific growth rates of E. coli BL21 (DE3) harboring pGEX-4T-1-AhFatA during the exponential phase were much lower than the host with pGEX-4T-1 plasmid. This phenomenon may due to the following reasons. The accumulation of AhFatA protein affected the lipid metabolism in the E. coli BL21 (DE3) harboring pGEX-4T-1-AhFatA, and then the release of free fatty acids limited the cells growth. The presented results demonstrate that the transformed cells may use the plasmidencoded FatA gene to produce fatty acids.

Conclusions
In conclusion, we cloned the AhFatA gene of A. hypogaea L., a member of the acyl-ACP thioesterases and described the functional characterization of AhFatA in E. coli BL21 (DE3). Our results showed that the expression of AhFatA, which was higher in seeds than other tissues, has a high specificity for 18:1-ACP and 16:1-ACP. Overexpression of AhFatA in E. coli BL21 (DE3) leads to the accumulation of palmitoleic acid and oleic acid. This research provides the basis not only for the cloning and expression of the AhFatA gene, but also for modifying fatty acid composition through genetic engineering of the acyl-ACP thioesterases in plants and microorganisms. We are currently developing transgenic A. hypogaea L. and cyanobacteria to enhance downstream fatty acid production by termination of fatty acyl chainelongation with AhFatA. We believe that the AhFatA gene will be helpful in transgenic lines and that it will be a suitable tool for genetic modification of oil crops to generate improved crops in the future.