An optimized ChIP-Seq framework for profiling of histone modifications in Chromochloris zofingiensis

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin Immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein crosslinking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

Characterization of fatty acid desaturases reveals stress-induced synthesis of C18 unsaturated fatty acids enriched in triacylglycerol in the oleaginous alga Chromochloris zofingiensis

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.

Epigenomic stability assessment during cryopreservation and physiology among various strains of Chromochloris zofingiensis (Chlorophyceae) and their genetic variability revealed by AFLP and MS-AFLP

Chromochloris zofingiensis (Dönz) Fucíková & L.A.Lewis, due to its production of highly valuable carotenoids such as astaxanthin, is a model organism in biotechnology. Since the recognition of this physiological property, many biotechnological applications have only used a single strain (SAG 211-14 = CCAP 211/14 = UTEX 32 = ATCC 30412) to produce biomass and carotenoids. However, multiple acquisitions of strains putatively belonging to the same species raised the question of the conspecificity of those strains and their properties. In this study, the conspecificity of the available strains, which are deposited axenically in SAG, was tested using SSU and ITS rDNA sequencing and AFLP (EcoRI/PstI) analyses. The comparison of SSU and ITS rDNA sequences as well as the AFLP patterns revealed that the investigated strains formed two very similar groups, (1) SAG 211-14, SAG 4.80, SAG 31.80, and SAG 34.80 and (2) SAG 221-2. All strains belonged to one species, C. zofingiensis, and represented one monophyletic lineage within the so-called DO-group of the Chlorophyceae. The robustness to cryopreservation and the subsequent epigenetic variability was detected using the methylation-sensitive AFLP (EcoRI/MspI and EcoRI/HpaII) among the five Chromochloris strains. All strains showed a high rate of survival (54.4–98.1%) during cryopreservation. The methylation patterns varied between precryo and postcryo in all strains detected among three time points (before, shortly after, and 8 weeks after cryopreservation), showing that the MS-AFLP technique has the potential to detect epigenetic effects occurring in response to cryopreservation and other stresses. Finally, the potential of these five strains for usage in biotechnological applications was proven by growing them in aerated cultures with and without additional carbon dioxide supply. The comparison showed that all strains produced high amounts of biomass and carotenoids under aeration with additional CO2 and were therefore suitable in biotechnology.

Genome-Scale Metabolic Model Accurately Predicts Fermentation of Glucose by Chromochloris zofingiensis

Algae have the potential to be sources of renewable fuels and chemicals. One particular strain, Chromochloris zofingiensis, is of interest due to the co-production of triacylglycerols (TAGs) and astaxanthin, a valuable nutraceutical. To aid in future engineering efforts, we have developed the first genome-scale metabolic model on C. zofingiensis, iChr1925. This model includes 1925 genes, 3481 metabolic reactions and 2778 metabolites. The model was used to predict flux distributions for three different growth conditions: autotrophic, mixotrophic and heterotrophic growth. The model predicted production of fermentation products for growth on glucose due to overflow metabolism and we confirmed this via metabolomics analysis of the spent medium. The metabolic network robustness of Chromochloris zofingiensis was also evaluated by conducting an in silico gene essentiality analysis. This work lays a foundation for future endeavors in the metabolic engineering of this unique organism.

The oleaginous astaxanthin-producing alga Chromochloris zofingiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis

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.