ABSTRACT: Background:Chromochloris zofingiensis, a freshwater alga capable of synthesizing both triacylglycerol (TAG) and astaxanthin, has been receiving increasing attention as a leading candidate producer. While the mechanism of oleaginousness and/or carotenogenesis has been studied under such induction conditions as nitrogen deprivation, high light and glucose feeding, it remains to be elucidated in response to salt stress, a condition critical for reducing freshwater footprint during algal production processes. Results:Firstly, the effect of salt concentrations on growth, lipids and carotenoids was examined for C. zofingiensis, and 0.2 M NaCl demonstrated to be the optimal salt concentration for maximizing both TAG and astaxanthin production. Then, the time-resolved lipid and carotenoid profiles and comparative transcriptomes and metabolomes were generated in response to the optimized salt concentration for congruent analysis. A global response was triggered in C. zofingiensis allowing acclimation to salt stress, including photosynthesis impairment, ROS build-up, protein turnover, starch degradation, and TAG and astaxanthin accumulation. The lipid metabolism involved a set of stimulated biological pathways that contributed to carbon precursors, energy and reductant molecules, pushing and pulling power, and storage sink for TAG accumulation. On the other hand, salt stress suppressed lutein biosynthesis, stimulated astaxanthin biosynthesis (mainly via ketolation), yet had little effect on total carotenoid flux, leading to astaxanthin accumulation at the expense of lutein. Astaxanthin was predominantly esterified and accumulated in a well-coordinated manner with TAG, pointing to the presence of common regulators and potential communication for the two compounds. Furthermore, the comparison between salt stress and nitrogen deprivation conditions revealed distinctions in TAG and astaxanthin biosynthesis as well as critical genes with engineering potential. Conclusions:Our multi-omics data and integrated analysis shed light on the salt acclimation of C. zofingiensis and underlying mechanisms of TAG and astaxanthin biosynthesis, provide engineering implications into future trait improvements, and will benefit the development of this alga for production uses under saline environment, thus reducing the footprint of freshwater.
