BackgroundThe 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.ResultsThe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1366-2) contains supplementary material, which is available to authorized users.

BackgroundChromochloris zofingiensis is emerging as an industrially relevant alga given its robust growth for the production of lipids and astaxanthin, a value-added carotenoid with broad applications. Nevertheless, poor understanding of astaxanthin synthesis has limited engineering of this alga for rational improvements.ResultsTo reveal the molecular mechanism underlying astaxanthin accumulation in C. zofingiensis, here we conducted an integrated analysis by combining the time-resolved transcriptomes and carotenoid profiling in response to nitrogen deprivation (ND). A global response was triggered for C. zofingiensis to cope with the ND stress. Albeit the little variation in total carotenoid content, individual carotenoids responded differentially to ND: the primary carotenoids particularly lutein and β-carotene decreased, while the secondary carotenoids increased considerably, with astaxanthin and canthaxanthin being the most increased ones. The carotenogenesis pathways were reconstructed: ND had little effect on the carbon flux to carotenoid precursors, but stimulated astaxanthin biosynthesis while repressing lutein biosynthesis, thereby diverting the carotenoid flux from primary carotenoids to secondary carotenoids particularly astaxanthin. Comparison between C. zofingiensis and Haematococcus pluvialis revealed the distinctive mechanism of astaxanthin synthesis in C. zofingiensis. Furthermore, potential bottlenecks in astaxanthin synthesis were identified and possible engineering strategies were proposed for the alga.ConclusionsCollectively, these findings shed light on distinctive mechanism of carotenogenesis for astaxanthin biosynthesis in C. zofingiensis, identify key functional enzymes and regulators with engineering potential and will benefit rational manipulation of this alga for improving nutritional traits.

We find that the DREB subfamily transcription factor, CmERF053, has a novel function to regulate the development of shoot branching and lateral root in addition to affecting abiotic stress. Dehydration-responsive element binding proteins (DREBs) are important plant transcription factors that regulate various abiotic stresses. Here, we isolated an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor from chrysanthemum (Chrysanthemum morifolium 'Jinba'), CmERF053, the expression of which was rapidly up-regulated by main stem decapitation. Phylogenetic analysis indicated that it belongs to the A-6 group of the DREB subfamily, and the subcellular localization assay confirmed that CmERF053 was a nuclear protein. Overexpression of CmERF053 in Arabidopsis exhibited positive effects of plant lateral organs, which had more shoot branching and lateral roots than did the wild type. We also found that the expression of CmERF053 in axillary buds was induced by exogenous cytokinins. These results suggested that CmERF053 may be involved in cytokinins-related shoot branching pathway. In this study, an altered auxin distribution was observed during root elongation in the seedlings of the overexpression plants. Furthermore, overexpress CmERF053 gene could enhance drought tolerance. Together, these findings indicated that CmERF053 plays crucial roles in regulating shoot branching, lateral root, and drought stress in plant. Moreover, our study provides potential application value for improving plant productivity, ornamental traits, and drought tolerance.

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Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid β-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.