| Summary: | Abstract Background Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage. Results Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance. Conclusions We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.
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