Engineered Chlamydomonas reinhardtii Strains for Enhanced Astaxanthin Production

Microalgae have evolved a diverse carotenoid profile, enabling efficient light harvesting and photoprotection. Previous studies have demonstrated the feasibility of genome editing in the green algal model species <i>Chlamydomonas reinhardtii</i>, leading to significant modifications in carotenoid accumulation. By overexpressing a fully redesigned β-carotene ketolase (bkt), the metabolic pathway of <i>C. reinhardtii</i> was successfully redirected toward astaxanthin biosynthesis, a high-value ketocarotenoid with exceptional antioxidant properties, naturally found in only a few microalgal species. In this study, a tailor-made double knockout targeting lycopene ε-cyclase (LCYE) and zeaxanthin epoxidase (ZEP) was introduced as a background for bkt expression to ensure higher substrate availability for bkt enzyme. The increased zeaxanthin availability resulted in a 2-fold increase in ketocarotenoid accumulation compared to the previously engineered bkt1 or bkt5 strain in the UVM4 background. Specifically, the best Δzl-<i>bkt</i>-expressing lines reached 2.84 mg/L under low light and 2.58 mg/L under high light, compared to 1.74 mg/L and 1.26 mg/L, respectively, in UVM4-<i>bkt</i> strains. These findings highlight the potential of rationally designed microalgal host strains, developed through genome editing, for biotechnological applications and high-value compound production.