Characterization of type i and type II diacylglycerol acyltransferases from the emerging model alga Chlorella zofingiensis reveals their functional complementarity and engineering potential

Abstract

Background: The green alga Chlorella zofingiensis has been recognized as an industrially relevant strain because of its robust growth under multiple trophic conditions and the potential for simultaneous production of triacylglycerol (TAG) and the high-value keto-carotenoid astaxanthin. Nevertheless, the mechanism of TAG synthesis remains poorly understood in C. zofingiensis. Diacylglycerol acyltransferase (DGAT) is thought to catalyze the committed step of TAG assembly in the Kennedy pathway. C. zofingiensis genome is predicted to possess eleven putative DGAT-encoding genes, the greatest number ever found in green algae, pointing to the complexity of TAG assembly in the alga. Results: The transcription start site of C. zofingiensis DGATs was determined by 5′-rapid amplification of cDNA ends (RACE), and their coding sequences were cloned and verified by sequencing, which identified ten DGAT genes (two type I DGATs designated as CzDGAT1A and CzDGAT1B, and eight type II DGATs designated as CzDGTT1 through CzDGTT8) and revealed that the previous gene models of seven DGATs were incorrect. Function complementation in the TAG-deficient yeast strain confirmed the functionality of most DGATs, with CzDGAT1A and CzDGTT5 having the highest activity. In vitro DGAT assay revealed that CzDGAT1A and CzDGTT5 preferred eukaryotic and prokaryotic diacylglycerols (DAGs), respectively, and had overlapping yet distinctive substrate specificity for acyl-CoAs. Subcellular co-localization experiment in tobacco leaves indicated that both CzDGAT1A and CzDGTT5 were localized at endoplasmic reticulum (ER). Upon nitrogen deprivation, TAG was drastically induced in C. zofingiensis, accompanied by a considerable up-regulation of CzDGAT1A and CzDGTT5. These two genes were probably regulated by the transcription factors (TFs) bZIP3 and MYB1, as suggested by the yeast one-hybrid assay and expression correlation. Moreover, heterologous expression of CzDGAT1A and CzDGTT5 promoted TAG accumulation and TAG yield in different hosts including yeast and oleaginous alga. Conclusions: Our study represents a pioneering work on the characterization of both type I and type II C. zofingiensis DGATs by systematically integrating functional complementation, in vitro enzymatic assay, subcellular localization, yeast one-hybrid assay and overexpression in yeast and oleaginous alga. These results (1) update the gene models of C. zofingiensis DGATs, (2) shed light on the mechanism of oleaginousness in which CzDGAT1A and CzDGTT5, have functional complementarity and probably work in collaboration at ER contributing to the abundance and complexity of TAG, and (3) provide engineering targets for future trait improvement via rational manipulation of this alga as well as other industrially relevant ones.