The algal lipids-based biodiesel, albeit having advantages over plant oils, still remains high in the production cost. Co-production of value-added products with lipids has the potential to add benefits and is thus believed to be a promising strategy to improve the production economics of algal biodiesel. Chromochloris zofingiensis, a unicellular green alga, has been considered as a promising feedstock for biodiesel production because of its robust growth and ability of accumulating high levels of triacylglycerol under multiple trophic conditions. This alga is also able to synthesize high-value keto-carotenoids and has been cited as a candidate producer of astaxanthin, the strongest antioxidant found in nature. The concurrent accumulation of triacylglycerol and astaxanthin enables C. zofingiensis an ideal cell factory for integrated production of the two compounds and has potential to improve algae-based production economics. Furthermore, with the advent of chromosome-level whole genome sequence and genetic tools, C. zofingiensis becomes an emerging model for studying lipid metabolism and carotenogenesis. In this review, we summarize recent progress on the production of triacylglycerol and astaxanthin by C. zofingiensis. We also update our understanding in the distinctive molecular mechanisms underlying lipid metabolism and carotenogenesis, with an emphasis on triacylglycerol and astaxanthin biosynthesis and crosstalk between the two pathways. Furthermore, strategies for trait improvements are discussed regarding triacylglycerol and astaxanthin synthesis in C. zofingiensis.

Background: Chromochloris 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. Results: To 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. Conclusions: Collectively, 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.

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. 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. 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.

Background: 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). 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. Results: To 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. Conclusions: Taken 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.

Inflammatory bowel disease remains a significant challenge in clinical settings. This study investigated the therapeutic potential of sea cucumber ovum hydrolysates (SCH) in a dextran sulfate sodium (DSS)-induced colitis mouse model. SCH, defined by its elevated stability and solubility, with a molecular weight below 1000 Da, significantly alleviated DSS-induced colitis, as evidenced by enhanced splenic index, reduced colonic damage, and diminished serum pro-inflammatory cytokines. Furthermore, macrogenomic analysis demonstrated that SCH increased beneficial gut microbes and decreased pro-inflammatory bacteria. Furthermore, metabolomic analysis of colonic tissues identified elevated levels of anti-inflammatory metabolites, such as Phenyllactate, 2-Hydroxyglutarate, and L-Aspartic acid, in colitis mice after oral administration of SCH. In conclusion, SCH represents a promising candidate for the treatment of colitis.

Inflammatory bowel disease continues to pose substantial therapeutic challenges in modern gastroenterology. This study systematically evaluated the anti-colitis efficacy of Cucumaria frondosa tentacles hydrolysates (CFTHs) using a dextran sulfate sodium (DSS)-induced murine colitis model. Characterized by enhanced stability and solubility with molecular weights below 1000 Da, administration of CFTHs demonstrated a significant mitigation in colitis pathology. Therapeutic outcomes included an improved splenic index, attenuated colonic mucosal damage, and substantial decreases in serum pro-inflammatory cytokines. Relative to the DSS group, the MPO value in the CFTHs-H group decreased by 27.6%, and the IL-6 value exhibited a reduction of 33%. Metagenomic profiling revealed that CFTHs mediated gut microbiota modulation, particularly the enrichment of beneficial Bacteroidetes and suppression of pro-inflammatory Proteobacteria. Metabolomic analysis identified elevated colonic concentrations of anti-inflammatory metabolites such as gamma-linolenic acid and prostaglandin I2, suggesting a microbiome–metabolome crosstalk in the therapeutic mechanism. These multi-omics findings in a murine model suggest that CFTHs may represent a promising candidate for future studies as a nutraceutical intervention for inflammatory bowel disorder. This intervention may operate through mechanisms that include simultaneous immunomodulation, microbiota restoration, and metabolic reprogramming.