Abstract
Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in the synthesis of triacylglycerol (TAG), the most important form of energy storage in plants. Some residues have previously been proven to be crucial for DGAT1 activity. In this study, we used site-directed mutagenesis of the CeDGAT1 gene from Chlorella ellipsoidea to alter 16 amino acids to investigate effects on DGAT1 function. Of the 16 residues (L482R, E542R, Y553A, G577R, R579D, Y582R, R596D, H603D, H609D, A624R, F629R, S632A, W650R, A651R, Q658H, and P660R), we newly identified 5 (L482, R579, H603, A651, and P660) as being essential for DGAT1 function and 7 (E542, G577, R596, H609, A624, S632, and Q658) that significantly affect DGAT1 function to different degrees, as revealed by heterologous expression of the mutants in yeast strain INVSc1. Importantly, compared with CeDGAT1, expression of the mutant CeDGAT1Y553A significantly increased the total fatty acid and TAG contents of INVSc1. Comparison among CeDGAT1Y553A, GmDGAT1Y341A, AtDGAT1Y364A, BnDGAT1Y347A, and BoDGAT1Y352A, in which tyrosine at the position corresponding to the 553rd residue in CeDGAT1 is changed into alanine, indicated that the impact of changing Y to A at position 553 is specific for CeDGAT1. Overall, the results provide novel insight into the structure and function of DGAT1, as well as a mutant gene with high potential for lipid improvement in microalgae and plants.
Highlights
Triacylglycerols (TAGs) are important storage lipids that can be used for edible oil and/or be processed for biodiesel
G577, R579, and Y582 are located in domain II and H603 and H609 in domain III, both of which are predicted to be inside the endoplasmic reticulum (ER) lumen
L482 is found on the stem of the 4th transmembrane domain (TMD) and R596 on the loop between the 5th and the 6th TMDs, which are inside the ER; E542 was on the stem of the 5th TMD, which is outside the ER; and the other sites are all within the TMD (Figure 1)
Summary
Triacylglycerols (TAGs) are important storage lipids that can be used for edible oil and/or be processed for biodiesel. In algae and higher plants, TAGs are mainly synthesized by diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme of the TAG synthesis pathway [1]. DGAT1 and DGAT2, which are mainly associated with the endoplasmic reticulum (ER), are microsomal enzymes; they catalyze similar biochemical reactions, their protein sequence similarity is extremely low. DGAT1 has a wider role in TAG synthesis, whereas DGAT2 activity is more related to the accumulation of specific fatty acids [2]. Bifunctional WS/DGAT predominantly catalyzes the synthesis of wax esters and minor amounts of TAGs in Arabidopsis thaliana (WSD1) [3]. Cytoplasmic DGAT3 has been reported to be involved in wax synthesis in a variety of plants [4,5], but its specific role in TAG synthesis remains unclear
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