Enhancement of ketocarotenoid production via heterologous expression of orange protein from Ipomoea batatas in indigenous microalga Ettlia sp.

Astaxanthin, one of the most powerful natural antioxidants, is used in high-value industries such as those of cosmetics, nutraceuticals, and food products derived from microalgae. In this study, Ettlia sp. mutants were generated by expressing two types of heterologous orange proteins, IbOr and IbOr-R96H, in which the 96^th arginine of IbOr was substituted with histidine derived from Ipomoea batatas to enhance astaxanthin production. The Ett-IbOr-R96H mutant showed a 2.4-fold increase in β-carotene content compared to the wildtype (4.59 mg g⁻¹DCW), reaching 10.82 mg g⁻¹ under high-light conditions via two-phase cultivation. Under the stress treatment combination of high-light intensity and nitrogen deprivation, total carotenoid content increased to 17.24 mg L⁻¹ and 21.94 mg L⁻¹ in Ett-IbOr and Ett-IbOr-R96H, respectively. The astaxanthin and canthaxanthin contents in Ett-IbOr-R96H was 4.89 mg L⁻¹ and 0.47 mg L⁻¹, respectively, which were 1.8- and 1.5-fold higher, respectively, than those in Ett-IbOr. Additionally, photosynthetic efficiency (Fv/Fm) recovered in Ett-IbOr-R96H under dual-stress conditions compared to the wildtype while reactive oxygen species levels decreased throughout the cultivation period. Our findings suggest that the heterologous IbOr expression in Ettlia sp. may be an effective approach for enhancing the production of ketocarotenoids and improving stress resistance for industrial applications.