Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA‐free CRISPR‐Cas9 RNP‐mediated mutagenesis

Lutein and zeaxanthin are dietary carotenoids reported to be protective against age‐related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC‐4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock‐out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA‐free CRISPR‐Cas9 ribonucleoproteins in the CC‐4349 strain had a significantly higher zeaxanthin content (56‐fold) and productivity (47‐fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2‐fold) and zeaxanthin (2.2‐fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost‐effective commercial use of microalgal mutants induced by DNA‐free CRISPR‐Cas9 ribonucleoproteins in algal biotechnology for the production of high‐value products. CC‐4349 is the best Chlamydomonas reinhardtii strain for carotenoid production. ZEP knockout mutants of CC‐4349 induced by DNA‐free CRISPR‐Cas9 RNP have a significantly higher zeaxanthin content and productivity than the wild‐type without the reduction in lutein level. Eggs are enriched with macular pigment by feeding hens the mutant‐containing diet. Approaches to circumvent GMO regulations in algal biotechnology are discussed.