Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

Abstract

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Keywords: acyl specificity; lysophosphatidic acid acyltransferase; microalgae; oil content; plastid transformation; triacylglycerols.

Figure 1

CrLPAAT1 is a homolog of the Arabidopsis plastidial protein LPAAT1 (ATS2). (a) Amino acid sequence alignment of CrLPAAT1 and AtATS2/AtLPAAT1. Black and grey boxes represent conserved and similar residues, respectively. Red boxes in the first row of the alignment indicate the plastid targeting transit peptides predicted using PredAlgo (Tardif et al., 2012). Motifs and amino acids identified as being important (Yamashita et al., 2007) are highlighted: NHX4D (motif I, purple line), GVIFIDR (motif II, blue line), EGTR (motif III, orange line), IVPIVM (motif IV, green line) and amino acid residues (red dots). Yellow boxes indicate hypothetical membrane‐spanning domains predicted by TMHMM (Krogh et al., 2001). (b) A phylogenetic tree of lysophosphatidic acid acyltransferase based mostly on sequences from C. reinhardtii and Arabidopsis thaliana. The protein sequences of C. reinhardtii were obtained from Phytozome v10.3: CrLPAAT1 (Cre09.g398289.t1.1); CrPGA2 (Cre05.g248150.t1.2); CrPGA3 (Cre17.g707300.t1.2); CrPGA4 (Cre10.g460350.t1.1); and CrPGA5 (Cre17.g738350.t1.2). The protein sequences of A. thaliana were obtained from TAIR: AtATS2/AtLPAAT1 (At4 g30580.1); AtLPAAT2 (At3 g57650.1); AtLPAAT3 (At1 g51260.1); AtLPAAT4 (At1 g75020.1); AtLPEAT1 (At1 g80950.1); and AtLPEAT2 (At2 g45670.1). LPAATs of Escherichia coli (EcPlsC, M63491) and Saccharomyces cerevisiae (ScSLC1, L13282) are included. The phylogenetic tree was inferred using MEGA6 and the maximum‐likelihood method with 1000 bootstrap replications. LPAAT: Lysophosphatidic acid acyltransferase, PGA: Phospholipid/Glycerol Acyltransferase. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Scale bar represents substitutions per unit distance.

Figure 2

CrLPAAT1 functions as an LPAAT. (a) CrLPAAT1 expression rescued the high temperature sensitivity of the E. coli plsC mutant, which is deficient in LPAAT. (b) Membrane fractions (60 μg protein) isolated from the transgenic E. coli cells catalysed phosphatidic acid formation in the presence of 200 μm LysoPA‐18:1, 25 μm 18:1‐CoA (white bars) and 25 μm 16:0‐CoA (black bars). Data are means of three replicates with standard deviations shown. Note that the buffer control has been subtracted from individual results.

Figure 3

Green fluorescent protein (GFP) was targeted to the plastid when fused to the transit peptide (TP) of CrLPAAT1 protein in tobacco epidermal cells. (a) A schematic diagram of the construct used to test the putative transit peptide of CrLPAAT1. (b) GFP fluorescence when tobacco epidermal cells were transformed with the TP_{C r LPAAT 1}‐sGFP fusion protein. (c) Chlorophyll autofluorescence. (d) Overlay of TP_{C r LPAAT 1}‐sGFP and chlorophyll fluorescence. (e) Overlay of TP_{C r LPAAT 1}‐sGFP, chlorophyll fluorescence and bright field. The construct was agro‐infiltrated into tobacco leaves and visualized by confocal microscopy. Bars = 10 μm.

Figure 4

CrLPAAT1 was localized to C. reinhardtii plastids. Chlamydomonas reinhardtii (strain CC‐125) was transformed with pOpt‐CrLPAAT1‐Clover. From left to right: Clover fluorescence, chlorophyll fluorescence, overlay of Clover and chlorophyll fluorescence, and bright field. The numbers to the left of each figure indicate the name of each individual transformant expressing CrLPAAT1–Clover. Bars = 10 μm.

Figure 5

Quantitative RT‐PCR analysis of the transcript level of CrLPAAT1 under nitrogen starvation. Expression levels of CrLPAAT1 were normalized to that of the housekeeping gene RACK1. Error bars represent standard errors based on three biological replicates.

Figure 6

Immunodetection of CrLPAAT1 expression in C. reinhardtii cells expressing HA‐tagged CrLPAAT1 using anti‐HA antibodies. (a) Immunoblot analysis of C. reinhardtii cells expressing HA‐tagged CrLPAAT1 using anti‐HA antibodies. Two bands were detected; the upper band corresponds to the full protein (36.5 kDa), and the lower band corresponds to the mature protein (31.5 kDa) lacking the transit peptide. (b) The SDS‐PAGE gel was stained with blue dye (ProSieve EX Safe Stain—LONZA) as a loading control for the immunoblot shown in (a). Twenty micrograms of total proteins were loaded onto an SDS‐PAGE gel, and expression of CrLPAAT1 was detected using anti‐HA antibodies. A duplicate of the gel was also visualized after staining with blue dye for 1 h. OE: pLM21‐CrLPAAT1‐HA overexpressors; three transformants (OE1, OE2 and OE3) are shown.

Figure 7

Oil content is increased by up to 20% in plastidial transgenic lines overexpressing CrLPAAT1 under nitrogen starvation. Cells were harvested from nitrogen‐starved cells (TAP‐N for 3 days). Data are means of three biological replicates (OE1, OE2 and OE3 shown in Figure 6) together with three technical replicates for each biological replicate; error bars denote 95% confidence intervals. *: denotes significant increases. Statistical analysis was carried out using the Student's t‐test (P < 0.05).

Figure 8

The involvement of CrLPAAT1 in oil synthesis in C. reinhardtii. HG, head group; G3P, glycerol‐3‐phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol; R, an acyl group. Arrows indicate catalytic steps, and enzymes are indicated above the arrows (CrLPAAT1 and C16 are in red).