Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii .

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2- C -methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii , and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii . Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L -1 (4.5 ± 0.1 mg g -1 CDW) in mixotrophic and 23.5 mg L -1 (1.09 mg L -1 h -1 ) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii . This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.