A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of Cuphea Seed Oil

Abstract

Seed oils of many Cuphea sp. contain >90% of medium-chain fatty acids, such as decanoic acid (10:0). These seed oils, which are among the most compositionally variant in the plant kingdom, arise from specialized fatty acid biosynthetic enzymes and specialized acyltransferases. These include lysophosphatidic acid acyltransferases (LPAT) and diacylglycerol acyltransferases (DGAT) that are required for successive acylation of medium-chain fatty acids in the sn-2 and sn-3 positions of seed triacylglycerols (TAGs). Here we report the identification of a cDNA for a DGAT1-type enzyme, designated CpuDGAT1, from the transcriptome of C. avigera var pulcherrima developing seeds. Microsomes of camelina (Camelina sativa) seeds engineered for CpuDGAT1 expression displayed DGAT activity with 10:0-CoA and the diacylglycerol didecanoyl, that was approximately 4-fold higher than that in camelina seed microsomes lacking CpuDGAT1. In addition, coexpression in camelina seeds of CpuDGAT1 with a C. viscosissima FatB thioesterase (CvFatB1) that generates 10:0 resulted in TAGs with nearly 15 mol % of 10:0. More strikingly, expression of CpuDGAT1 and CvFatB1 with the previously described CvLPAT2, a 10:0-CoA-specific Cuphea LPAT, increased 10:0 amounts to 25 mol % in camelina seed TAG. These TAGs contained up to 40 mol % 10:0 in the sn-2 position, nearly double the amounts obtained from coexpression of CvFatB1 and CvLPAT2 alone. Although enriched in diacylglycerol, 10:0 was not detected in phosphatidylcholine in these seeds. These findings are consistent with channeling of 10:0 into TAG through the combined activities of specialized LPAT and DGAT activities and demonstrate the biotechnological use of these enzymes to generate 10:0-rich seed oils.

Figure 1.

Expression of CpuDGAT1, CpuDGAT2_A, CpuDGAT2_B, and CpuDGAT2_C transcripts in different organs of C. avigera var pulcherrima. RT-PCR analysis of DGAT1 and DGAT2 transcripts in different organs of C. avigera var pulcherrima. Eukaryotic initiation factor 4A (eIF4) and actin genes were used as internal controls. PCR products were obtained with gene-specific primers for CpuDGAT1, CpuDGAT2_A, CpuDGAT2_B, or CpuDGAT2_C (Supplemental Table S1).

Figure 2.

Alignment of deduced amino acid sequence of CpuDGAT1 with selected plant and mammalian homologs. Amino acid alignment of CpuDGAT1 with plant and mammalian DGAT1 homologs. Amino acid sequences were obtained from the NCBI. DGAT sequences are shown and NCBI accession numbers are as follows: CpuDGAT1, C. avigera var pulcherrima, CpuDGAT1 (KU055625); AtDGAT1, Arabidopsis, AtDGAT1 (CAB44774); BnDGAT1, B. napus, BnDGAT1 (AF164434); RcDGAT1, R. communis RcDGAT1 (XP002514132); VfDGAT1, Vernicia fordii, VfDGAT1 (ABC94471); OeDGAT1, Olea europaea, OeDGAT1(AAS01606); EgDGAT1, E. guineensis DGAT1 (XP010924968); RnDGAT1, Rattus norvegicus, RnDGAT1 (NP445889); HsDGAT1, Homo sapiens, HsDGAT1 (NP036211). The residues blocked on red background are 100% conserved bases in seven known motifs (in dotted boxes) of DGAT1s. Black arrows show the CpuDGAT1 residues differing from those of other plant DGAT1s within the seven motifs. White triangles show the catalytic active site residues (Asn-381) and (His-417) found in all MBOAT family enzymes (Chang et al., 2011). The ER retrieval motif is indicated with an orange box (within motif VII).

Figure 3.

Analysis of neutral lipids from S. cerevisiae H1246 strain expressing Cuphea and Arabidopsis DGATs. Thin layer chromatographic analysis of neutral lipids of S. cerevisiae H1246 strain expressing Cuphea DGAT2 cDNAs (CpuDGAT1_A, CpuDGAT2_B, and CpuDGAT2_C), full-length CpuDGAT1, CpuDGAT1 lacking the coding sequence for the first 70 amino acids (CpuDGAT1trunc), and the Arabidopsis DGAT1 (AtDGAT1) in the vector pYes2. CpuDGAT1trunc, CpuDGAT2_C, and AtDGAT1 expression functionally complement TAG biosynthesis in S. cerevisiae H1246 mutant. A, Neutral lipids from yeast cells expressing CpuDGAT1 and CpuDGAT1trunc. B, Neutral lipids from control yeast cells (pYes2) and yeast cells expressing CpuDGAT2_A and CpuDGAT2_B. C, Neutral lipids from yeast cells expressing CpuDGAT2_C and AtDGAT1.

Figure 4.

DGAT activity in extracts of developing seeds from transgenic camelina plants expressing CpuDGAT1. Measurement of DGAT activity in crude extracts from developing wild-type camelina seeds (22 DAF) and developing camelina seeds expressing CvFatB1 alone, coexpressing CvFatB1 and CpuDGAT1 (CvFatB1+CpuDGAT1), or coexpressing CvFatB1, CvLPAT2, and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Results are for assays using [1-¹⁴C] 10:0-CoA and DAG species: 10:0/10:0 and 18:1/18:1, respectively. Values are mean ± sd (n = 3). Asterisks denote statistically significant differences (*P < 0.0001) as compared to wild type, as determined by two-tailed Student’s t test. Wt, wild type.

Figure 5.

Fatty acid composition of TAG and sn-2 position of TAG in mature seeds of wild-type camelina and camelina lines engineered for expression of CvFatB1 alone or with combinations of CpuDGAT1 and CvLPAT2. A, Fatty acid composition of TAG in wild-type camelina seeds and camelina seeds engineered for expression of CvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown are the means of mol % of each fatty acid and ± sd of the mean for three biological replicates. Asterisks denote statistically significant difference from wild type (or CvFatB1 in the case of 10:0) values at *P < 0.001, **P < 0.03 based on two-tailed Student’s t test. B, Fatty acid composition of TAG sn-2 position of wild-type camelina seeds and camelina seeds engineered for expression of CvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown are the means of mol % of each fatty acid and ± sd of the mean for three biological replicates. Asterisks denote statistically significant difference from wild type (or CvFatB1 in the case of 10:0) values at *P < 0.002, **P < 0.02 based on two-tailed Student’s t test. Wt, wild type.

Figure 6.

ESI-MS/MS profiling of 10:0/10:0 DAG-containing TAG molecular species in seeds of camelina lines engineered for expression of CvFatB1 alone or with combinations of CpuDGAT1 and CvLPAT2. TAG species from camelina seeds engineered for expression of CvFatB1 alone (A), with CpuDGAT1 (B), or with CvLPAT2 and CpuDGAT1 (C) were analyzed by ESI-MS/MS. Shown are precursor 383.3 m/z precursor scans of TAG species containing 10:0/10:0 DAGs. The labeled TAG species indicate fatty acid composition, but the stereospecific arrangement cannot be determined from the analyses. Asterisks indicate unknown compounds.

Figure 7.

Fatty acid composition of DAG and PC in mature seeds of wild-type and engineered camelina lines expressing CvFatB1 alone or in combinations with CvLPAT2 and CpuDGAT1. A, Fatty acid composition of DAG in wild-type camelina seeds and camelina seeds engineered for expression of CvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). B, Fatty acid composition of PC in wild-type camelina seeds and camelina seeds engineered for expression of CvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown are the means of mol % of each fatty acid and ± sd of the mean for three biological replicates. Asterisks denote statistically significant difference from wild-type values or CvFatB1 for 10:0 values at *P < 0.01, **P < 0.05 based on two-tailed Student’s t test. Wt, wild type.

Figure 8.

Acyl-CoA species in camelina developing seeds engineered for expression of CvFatB1 alone and with CpuDGAT1 and CvLPAT2+CpuDGAT1. Acyl-CoA species of camelina developing seeds at 10, 17, 22, and 30 DAF from wild-type, and transgenic lines expressing CvFatB1 alone, with CpuDGAT1, and with CvLPAT2+CpuDGAT1 were analyzed by LC-MS/MS. The data are means of mol % of each acyl-CoA species ± sd of three biological replicates. Asterisks denote statistically significant difference from wild type (or CvFatB1 in the case of 10:0) values at *P < 0.02, **P < 0.05 based on two-tailed Student’s t test. A, Acyl-CoA species of camelina seeds at 10 DAF. B, Acyl-CoA species of camelina seeds at 17 DAF. C, Acyl-CoA species of camelina seeds at 22 DAF. D, Acyl-CoA species of camelina seeds at 30 DAF. Wt, wild type.

Figure 9.

Seed weight, germination efficiency, and total fatty acid content of wild-type camelina seeds and seeds of engineered camelina lines expressing CvFatB1 alone or in combinations with CvLPAT2 and CpuDGAT1. Shown are 100-seed weight (A), germination efficiency (B), and total fatty acid content (C) of mature seeds from wild-type camelina and camelina seeds engineered for expression of CvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+ CpuDGAT1). Values shown for 100-seed weight are the means and ± sd of the mean for four biological replicates. Values for seed fatty acid content are the means and ± sd of the mean for three biological replicates. Asterisks indicate statistically significant difference (*P < 0.005, **P < 0.025) as compared to the wild type based on two-tailed Student’s t test. Values for germination efficiency are the means and ± sd of the mean for three biological replicates. Asterisks denote statistically significant difference (*P < 0.005, ***P < 0.05) in germination efficiency of transgenic seeds as compared to the CvFatB1+ CvLPAT2+CpuDGAT1 seeds based on two-tailed Student's t test. Wt, wild type.

Figure 10.

A proposed pathway for 10:0-rich TAG assembly in transgenic camelina seeds coexpressing CvLPAT2 and CpuDGAT1. Acyl-CoA species of 10:0 and 18:1 are sequentially esterified to glycerol-3-P (G3P) at sn-1 and sn-2 positions by GPAT and LPAT, forming LPA and PA. The release of P from PA by phosphatidic acid phosphatase forms DAG. The subsequent acylation of DAG at sn-3 catalyzed by DGAT forms TAG. DAG can be converted to PC via the action of DAG/CPT and/or PDCT. DAG molecules containing 10:0 at the sn-2 position arise from the specialized activity of the Cuphea LPAT CvLPAT2. DAGs containing 10:0 at the sn-2 position are selectively used for acylation with 10:0-CoA at the sn-3 position by activity of the specialized Cuphea diacylglycerol acyltransferase CpuDGAT1 to form TAG species enriched in 10:0. DAG molecules containing 10:0 appear to be poor substrates for PC synthesis, or 10:0 is rapidly removed from PC formed from 10:0-DAG species. As such, 10:0 accumulation in TAG appears to proceed primarily by the Kennedy pathway rather than through PC via activity of enzymes including PDCT and phospholipid/diacylglycerol acyltransferase, which catalyzes the transfer of fatty acids at the sn-2 position of PC to the sn-3 position of DAG producing TAG and lysophosphatidylcholine. The PC route of fatty acid flux for TAG synthesis involves primarily flux of 18:1 for desaturation to 18:2 and 18:3. LPC, lysophosphatidylcholine; PAP, phosphatidic acid phosphatase; PDAT, phospholipid/diacylglycerol acyltransferase.