Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green 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.
Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii
Perozeni, Federico;Cazzaniga, Stefano;Ballottari, Matteo;
2023-01-01
Abstract
Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.