Abstract
BackgroundThe green microalga Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). Nevertheless, it remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis.ResultsTo address these issues, we firstly determined the transcription start sites of 11 putative membrane-bound FAD-coding genes (CzFADs) and updated their gene models. Functional validation of these CzFADs in yeast and cyanobacterial cells revealed that seven are bona fide FAD enzymes with distinct substrates. Combining the validated functions and predicted subcellular compartments of CzFADs and the FA profiles of C. zofingiensis, the FA desaturation pathways in this alga were reconstructed. Furthermore, a multifaceted lipidomic analysis by systematically integrating thin-layer chromatography, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry techniques was conducted, unraveling profiles of polar membrane lipids in C. zofingiensis and major desaturation steps occurring in these lipids. By correlating transcriptional patterns of CzFAD genes and changes of lipids upon abiotic stress conditions, our results highlighted collaboration of CzFADs for C18 UFA synthesis and supported that both de novo FA synthesis and membrane lipid remodeling contributed C18 UFAs to triacylglycerol for storage.ConclusionsTaken together, our study for the first time elucidated the pathways of C18 FA desaturations and comprehensive profiles of polar membrane lipids in C. zofingiensis and shed light on collaboration of CzFADs for the synthesis and enrichment of C18 UFAs in triacylglycerol.
Highlights
Fatty acids (FAs) are building blocks of acyl-lipids that generally comprise polar membrane lipids (PMLs) and neutral storage lipids
Identification of CzFAD genes and bioinformatics analysis Using the Fatty acid desaturase (FAD) from C. reinhardtii and Arabidopsis as the query sequences, Blast against the C. zofingiensis non-redundant protein sequences database revealed the presence of 14 putative FAD-coding genes (Additional file 2: Table S1)
As Cz11g21120 and Cz11g21110 have much shorter coding sequences than the regular FADs and are adjacently located in the same chromosome, they may belong to the same gene locus, but were annotated as two independent ones
Summary
Fatty acids (FAs) are building blocks of acyl-lipids that generally comprise polar membrane lipids (PMLs) and neutral storage lipids. UFAs can be further classified as monounsaturated FAs (MUFAs; with one double bond) and polyunsaturated FAs (PUFAs; with more than one double bond). The desaturation degree of FAs, determined by the action of FA desaturases (FADs), varies across organisms and responds to both abiotic and biotic stresses [7,8,9,10]. The green microalga Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). It remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis
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