Characterization of Auxenochlorella protothecoides acyltransferases and potential of their protein interactions to promote the enrichment of oleic acid.

ABSTRACT

BACKGROUND: After centuries of heavy reliance on fossil fuel energy, the world suffers from an energy crisis and global warming, calling for carbon emission reduction and a transition to clean energy. Microalgae have attracted much attention as a potential feedstock for biofuel production due to their high triacylglycerol content and CO2 sequestration ability. Many diacylglycerol acyltransferases (DGAT) species have been characterized, which catalyze the final committed step in triacylglycerol biosynthesis. However, the detailed structure-function features of DGATs and the role of the interactions among DGAT proteins in lipid metabolism remained largely unknown. RESULTS: In this study, the three characterized DGATs of Auxenochlorella protothecoides 2341 showed distinct structural and functional conservation. Functional complementation analyses showed that ApDGAT1 had higher activity than ApDGAT2b in yeast and model microalgae, and ApDGAT2a had no activity in yeast. The N-terminus was not essential to the catalysis function of ApDGAT1 but was crucial to ApDGAT2b as its enzyme activity was sensitive to any N-terminus modifications. Similarly, when acyl-CoA binding proteins (ACBPs) were fused to the N-terminus of ApDGAT1 and ApDGAT2b, zero and significant activity changes were observed, respectively. Interestingly, the ApACBP3 + ApDGAT1 variant contributed to higher oil accumulation than the original DGAT1, and ApACBP1 + ApDGAT1 fusion boosted oleic acid content in yeast. Overexpression of the three DGATs and the variation of ApACBP3 + ApDGAT1 increased the content of C181 of Chlamydomonas reinhardtii CC-5235. Significantly, ApDGAT1 interacted with itself, ApDGAT2b, and ApACBP1, which indicated that these three lipid metabolic proteins might have been a part of a dynamic protein interactome that facilitated the enrichment of oleic acid. CONCLUSIONS: This study provided new insights into the functional and structural characteristics of DGATs and elucidated the importance of these physical interactions in potential lipid channeling.