25Survival under excess light conditions requires the light-induced accumulation of protein 26 LHCSR3 and other photoprotection factors, to enable efficient energy-dependent 27 quenching in the green microalga Chlamydomonas reinhardtii. Here, we demonstrate 28 that the high light-tolerant phenotype of mutant hit1 is caused by a de-repression of 29 promoters belonging to photoprotection genes, which in turn results from an inactivation 30 of the E3 ubiquitin ligase substrate adaptor LRS1. Transcriptome analyses of hit1 31 revealed massive alterations of gene expression modulation as a consequence of 32 perturbed LRS1 function, indicating its role as a crown regulator. In conjunction with 33 random forest-based network modeling, these transcriptome analyses predicted that 34 LRS1 controls photoprotection gene expression via an algal HY5 homolog as its prime 35 transcription factor target. CrHY5 binds to T-box elements present in the promoters of 36 these genes and its inactivation in the hit1 mutant via CRISPR-Cas9 genome editing, 37 confirmed the regulatory connection between LRS1 and CrHY5, predicted by the 38 network analysis. 39 40 41 42 43 44 45 46 47 48 54 Light provides the energy driving photosynthesis and it is an important cue controlling 55 many physiological processes in phototrophic organisms. In the seed plant Arabidopsis 56 thaliana the E3 ubiquitin ligase substrate adaptor COP1/SPA is a master switch of light 57 signaling, by repressing the accumulation of various transcription factors required for the 58 induction of light-dependent processes like photomorphogenesis or flowering in 59 darkness. Repression is achieved by ubiquitination of target transcription factors and 60

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Background The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. Results Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g−1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g−1 DCW), and lipids (450.09 ± 25.48 mg g−1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g−1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g−1 lutein of oil and 7.69 ± 1.03 mg g−1 zeaxanthin of oil was produced. Conclusion Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

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