The effects of high light and heterologous expression of β-carotene ketolase on the metabolism carotenoids in Chlamydomonas reinhardtii

rui Mei Haihong Yang Chunli Guo Zeyu Hong Zhangli HU Yan WU Danqiong Huang Chaogang WANG ^*

• Shenzhen University, Shenzhen, China

Both high light stress and overexpression of β-carotene ketolase have significant effects on the synthesis of carotenoids in Chlamydomonas reinhardtii. As a promising platform for carotenoid production, C. reinhardtii needs further research and technological innovation to address challenges such as environmental interference, exogenous gene expression, and metabolic regulation, in order to achieve efficient and sustainable production of carotenoids. In this study, the regulation of carotenoids metabolism in C. reinhardtii was time-dependently investigated through high light exposure and heterologous expression of β-carotene ketolase. Results suggested that high light (500 µmol/m 2 /s) negatively regulated the accumulation of β-carotene, positively induced the accumulation zeaxanthin, echinenone, and canthaxanthin, and continuously promoted the accumulation of zeaxanthin and canthaxanthin in C. reinhardtii followed by the extension of high light stress.Metabolomics analysis suggested that high light stress promoted the biosynthesis of carotenoids, improved the intermediates associated with astaxanthin synthesis pathway, and promoted the conversion of β-carotene to downstream substances. Aiming to improve the canthaxanthin production in C. reinhardtii, several strategies were carried out to overexpress β-carotene ketolase genes from different sources, including strong promoters, insertion introns and chloroplast conduction peptides.It was found that β-carotene, echinenone, and canthaxanthin were all significantly increased in transformed C. reinhardtii overexpressing β-carotene ketolase. Among them, the highest canthaxanthin content was found in pH124-CrtO, having 7 times of that in the wild type. Besides, the carotenoids metabolomics analysis also found the promoted abscisic acid pathway and astaxanthin pathway in transformed C. reinhardtii. Results in this study provide a new research scheme for manipulating the carotenoids metabolism and promoting the synthesis of high value carotenoids in C. reinhardtii.