Overexpression of Peanut Diacylglycerol Acyltransferase 2 in <i>Escherichia coli</i>

<div><p>Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, <i>Arachis hypogaea</i> type 2 <i>DGAT</i> (<i>AhDGAT2</i>) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in <i>Escherichia coli</i> Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in <i>E. coli</i>. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in <i>E. coli.</i></p> </div

Fatty-acid content of wild-type <i>E. coli</i> strains and strains transformed with empty vector, AhDGAT2a–GST, and AhDAGT2b–GST.

<p>Open columns represent the values in un-induced strains and filled columns the values in IPTG-induced cultures after 6 h. Vertical bars represent the standard deviations of three replicates. a,c,e, significant at the 0.05 level; b,d,f, significant at the 0.01 level. a and b, compared with WT; c and d, compared with the empty-vector strain; e and f, compared with AhDGAT2a-GST vector strain. <i>P</i> values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061363#pone.0061363.s002" target="_blank">Table S1</a>.</p

SDS–PAGE analysis of the time course for expression of AhDGAT2a and AhDGAT2b recombinant fusion proteins in <i>E. coli</i> cell extracts.

<p>Recombinant proteins transformed into <i>E. coli</i> were induced with IPTG and their expression levels evaluated after 0, 2, 4, and 6 h. Molecular weight standards are shown on the left. The relative mobilities of GST (26.97 kDa), AhDGAT2a–GST (64.5 kDa), and AhDGAT2b–GST fusion proteins (64.5 kDa) are indicated on the right.</p

Growth curves of the wild type and transformed <i>E. coli</i> strains in liquid culture.

<p>The optical density (OD₆₀₀) of the bacterial cultures is shown on the y axis. Bacteria were incubated aerobically at 37°C with shaking at 170 rpm. Vertical bars represent the standard deviation of three replicates.</p

Comparison of the amino acid sequences of AhDGAT2a and AhDGAT2b.

<p>The three amino acid differences are shaded in gray. Two black underlined regions (amino acids 40–62 and 67–82) highlight the two predicted transmembrane domains. The red underline shows the conserved LPLAT_MGAT-like domain (amino acids 104–321).</p

Cell sizes (mean±SE) of the recombinant <i>Escherichia coli</i> strains.

<p>a: Significantly different from WT at the 0.05 level.</p><p>b: Significantly different from WT at the 0.01 level.</p

Expression of AhDGAT2–GST fusion proteins after induction with 1 mM IPTG at 37°C for 6 h.

<p>M: Protein molecular weight marker. (A) Lanes 1, 3: AhDGAT2a–GST and AhDGAT2b–GST extracted from the cytoplasmic fraction; Lanes 2, 4: AhDGAT2a–GST and AhDGAT2b–GST extracted from inclusion bodies. (B) Western blot analysis of the AhDGAT2–GST fusion proteins using anti-GST tag monoclonal antibody. Lane 1: Wild-type <i>E. coli</i> Rosetta (DE3) strain; Lane 2, GST expression from the empty-vector transformed strain; Lanes 3, 5: AhDGAT2a–GST and AhDGAT2b–GST from the cytoplasmic fraction; Lanes 4, 6: AhDGAT2a–GST and AhDGAT2b–GST from inclusion bodies.</p

Phylogenetic tree showing relationships among the DGAT2 protein sequences from various plant species.

<p>The tree was generated using DNAMAN software. Branch lengths indicate evolutionary distance. The GenBank protein ID numbers for the DGAT2s are as follows: MtDGAT2, <i>Medicago truncatula</i>, ACJ84867.1; AtDGAT2, <i>Arabidopsis thaliana</i>, NP_566952.1; EaDGAT2, <i>Euonymus alatus</i>, ADF57328.1; HaDGAT2, <i>Helianthus annuus</i>, ABU50328.1; OeDGAT2, <i>Olea europaea</i>, ADG22608.1; RcDGAT2, <i>Ricinus communis</i>, XP_002528531.1; VfDGAT2, <i>Vernicia fordii</i>, ABC94473.1; ZmDGAT2, <i>Zea mays</i>, NP_001150174.1; OsDGAT2, <i>Oryza sativa</i> Japonica Group, NP_001057530.1. The two peanut DGAT2s are underlined.</p

Morphology of wild type and transformed <i>E. coli</i> before and 6 h after the addition of IPTG into the growth medium.

<p>Cells were sampled and Gram stained before induction (A, C, E, G) and 6 h after induction (B, D, F, H). (A, B) wild-type <i>E. coli</i> cells; (C, D) empty-vector transformed cells; (E, F) AhDGAT2a–GST vector transformed cells; (G, H) AhDGAT2b–GST vector transformed cells.</p

Additional file 7: of Transcriptome analysis of alternative splicing in peanut (Arachis hypogaea L.)

Table S7. Fatty acid related genes. (XLSX 250 kb