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

Abstract

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.

Site-Specific Gene Knock-Out and On-Site Heterologous Gene Overexpression in Chlamydomonas reinhardtii via a CRISPR-Cas9-Mediated Knock-in Method

“…Chlamydomonas reinhardtii is widely used as a model organism and considered to be a potential cell factory to produce value-added compounds (Khan et al, 2018;Salomé and Merchant, 2019). Production of compounds such as zeaxanthin, sesquiterpene, bio-hydrogen, and human epidermal growth factor, have been reported in C. reinhardtii (Torzillo and Seibert, 2013;Lauersen et al, 2016;Baek et al, 2018;Baier et al, 2018a). These reports have increased the attention on the commercial use of C. reinhardtii.…”

Nitric Oxide Remodels the Photosynthetic Apparatus upon S-Starvation in Chlamydomonas reinhardtii

“…In the green alga (Chlamydomonas reinhardtii), the increasing number of available genetic tools allows the more detailed study of both ubiquitous and specific stress responses, such as nutrient limitations (Grossman et al, 2007;Zhao et al, 2009;Schmollinger et al, 2013;Sizova et al, 2013;Baek et al, 2016Baek et al, , 2018Shin et al, 2016;Ferenczi et al, 2017;Greiner et al, 2017;Crozet et al, 2018). These stresses consistently encompass the expression of scavenging enzymes for the missing element and selective degradation processes (Merchant and Helmann, 2012;Saroussi et al, 2017).…”

Potential and Challenges of Improving Photosynthesis in Algae

“…Regarding the endogenous isoprenoids, carotenoids represent commercially successful products from microalgae: β-carotene from D. salina and astaxanthin from H. pluvialis are high-price products mainly used in aquaculture, and as food colouring agents and nutraceuticals [158]. As for zeaxanthin, hyper-accumulating D. salina strains were identified [159], while a ∆ZEP Chlamydomonas strain with a far higher zeaxanthin content than WT (56-fold) was shown to have a commercial potential in the production of eggs fortified with carotenoids [83,160]. In H. pluvialis, the endogenous phytoene desaturase was modified by site-directed mutagenesis, enhancing both the resistance to the herbicide norflurazon and the astaxanthin productivity [161].…”