Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Abstract

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

Fig 1. Comparison of the EgDGAT1-1 sequence with biochemically-characterized plant DGAT1 proteins.

The EgDGAT1-1 amino acid sequence was aligned, using Clustal Omega [31], with five biochemically-characterized plant DGAT1 proteins: Nicotiana tabacum (GenBank accession number AAF19345), V. fordii (ABC94471), Linum usitatissimum (AHA57450) A. thaliana (AAF19262), and Tropaeolum majus (AAM03340). Identical residues are highlighted in black and similar residues are shaded. Transmembrane domains predicted by the TMpred program are underlined (hatched line). Several conserved motifs are boxed, including the acyl-CoA binding site [3, 45], active site [3, 45], typical SnRK1 protein kinase targeting motif [45, 46], thiolase acyl-enzyme intermediate binding motif [45, 46], FA binding protein signature [45], DAG-binding site [45, 46] and ER retrieval motif [11]. A leucine zipper motif [6, 47] is highlighted by vertical arrows. The 41 invariant residues among 55 DGAT1 sequences from animals, plants and fungi are shown with asterisks [48].

Fig 2. Separation of the lipid classes from the recombinant JMY1877 strains by HPTLC.

JMY1877 recombinant strains transformed with the empty cassette or expressing AtDGAT1 or EgDGAT1-1 were grown for 18 h. Lipids corresponding to 0.4 mg dry cell lysate or lipids from the standard (four micrograms each) were separated on a silica plate. This experiment is representative of three independent cultures. A vertical arrow indicates the direction of migration and a horizontal arrow points out the position of TAG spots in strains expressing AtDGAT1 or EgDGAT1-1. O: origin of migration; PLs: phospholipids; FFAs: free fatty acids; TAGs: triacylglycerols; FAMEs: fatty acid methyl esters.

Fig 3. FA content (A) and composition (B) of yeast lipids.

JMY1877 recombinant strains transformed with the empty cassette or expressing AtDGAT1 or EgDGAT1-1 were grown for 18 h. FAs from twenty-five milligrams of dry cell lysate were transmethylated. The resulting FAMEs from three independent cultures were identified and quantified by GC-FID. (A) FA content values are expressed in percentage of dry cell weight per strain. Asterisks indicate statistically significant differences according to a t-test (*P<0.05; **P<0.01). (B) FA composition values are expressed as relative amount of each FA per strain. Asterisks indicate statistically significant differences according to a t-test (*P<0.05; **P<0.01; ***P<0.001).

Fig 4. Microscopic observation (A) and lipid classes (B) of neosynthesized lipid droplets.

(A) JMY1877 recombinant strains transformed with the empty cassette or expressing AtDGAT1 or EgDGAT1-1 were grown for 18 h. Cells were observed under the microscope with phase contrast (Nomarski) or fluorescence (Nile Red). (B) Separation of floating layer lipids by HPTLC. Lipids extracted from the 150,000 g floating layer of each recombinant JMY1877 strain or lipids from the standard (four micrograms each) were separated on silica plates. A vertical arrow indicates the direction of migration. O: origin of migration; PLs: phospholipids; FFAs: free fatty acids; TAGs: triacylglycerols; FAMEs: fatty acid methyl esters.

Fig 5. Microscopic observation of JMY1877 transformants grown in 2% LAME supplemented YP medium.

JMY1877 recombinant strains transformed with the empty cassette or expressing AtDGAT1 or EgDGAT1-1 were grown for 18 h in YPL medium. Cells were observed under the microscope with phase contrast (Nomarski) or fluorescence (Nile Red).

Fig 6. TAG profiling of recombinant yeast strains grown in 2% LAME supplemented YP.

JMY1877 recombinant strains expressing AtDGAT1 or EgDGAT1-1 were grown for 18 h in YPL medium. Lipids were extracted from thirty milligrams of dry cell lysate. TAGs from three independent cultures were separated by SPE and composition was analyzed by UHPLC-HRMS/MS. Values are expressed as the relative amount of each TAG species per strain. For each species the overall number of carbon atoms of the three esterified FA chains and the overall number of double bonds is specified. Asterisks indicate statistically significant differences according to a t-test (*P<0.05; **P<0.01; ***P<0.001).

Fig 7. Functional annotation of the microsomal proteins present in the JMY1877 transformants.

Proteins in 100,000 g microsomes from three independent transformants of the control strain or the EgDGAT1-1 expressing strain were digested in gel. Peptides were separated by liquid chromatography and analyzed with a LTQ Orbitrap mass spectrometer using a nano-electrospray interface. Proteins found in the three transformants of each strain were annotated using the Génolevures annotated sequence database [39] and manually curated into twelve functional classes. Six hundred and twenty-five of the 744 proteins could be sorted into twelve functional classes.

Fig 8. Comparison of the N-terminal sequence of EgDGAT1-1 with several plant DGAT1.

The EgDGAT1-1 sequence was aligned, using Clustal Omega [31], with eleven plant DGAT1 proteins: L. communis (AHN93288), P. dactylifera (XP_008793203), P. dactylifera (XP_008793202), P. dactylifera (XP_008806896), P. dactylifera (XP_008806906), N. tabacum (AAF19345), V. fordii (ABC94471), L. usitatissimum (AHA57450), T. majus (AAM03340), A. thaliana (AAF19262), B. napus (AAD45536). The name of the plants able to store lauric acid in their seeds is underlined. The variable N-terminal region is framed with dashed lines. Conserved motif important for acyl-CoA binding [3, 45] and the FA binding protein signature [45] are boxed with straight lines.