Green metabolic engineering of Synechocystis sp. PCC 6803 for efficient production and sustainable extraction of β-cryptoxanthin and zeaxanthin.

backgroundβ-xanthophylls, such as zeaxanthin and β-cryptoxanthin, display significant bioactive properties including anti-oxidant, anti-inflammation and anti-cancer activities. These properties spur their widespread use in food, animal feed, cosmetic and pharmaceutic sectors. However, commercial carotenoids are dominantly manufactured by chemical synthesis, posing environmental pollution. A minor fraction is sourced from plants and algae, a process often hampered by low yields and high costs. Consequently, microbial biosynthesis has emerged as a promising alternative for producing these high-value compounds.

resultsHere, we engineered the photosynthetic microorganism Synechocystis sp. PCC 6803 for efficient production of zeaxanthin and β-cryptoxanthin by two different strategies. In the first strategy, the gene crtO for echinenone biosynthesis and crtD involved in the myxoxanthophyll biosynthesis were inactivated, which increased the amount of zeaxanthin and β-cryptoxanthin by approximately 30% and 60%, respectively, reaching 1.3 mg/g DCW and 0.15 mg/g DCW in the double mutant ΔcrtO/crtD compared to wild type. The second strategy combined the replacement of the native β-carotene hydroxylase with a heterologous, high-efficiency plant enzyme and further improvement of precursor supply. The final strain oxBDI produced 5 mg/g DCW zeaxanthin and 1.5 mg/g DCW β-cryptoxanthin, fivefold and 18-fold higher than wild type, respectively, representing 80% of the total carotenoids produced. In addition, no significant difference was observed in the growth rate between transgenic strains and wild type under standard lab conditions, suggesting the applicability of this biotechnology strategy. Moreover, we explored the use of natural deep eutectic solvents (NADESs) as a green alternative for β-xanthophylls extraction from the engineered cyanobacteria using a simplified procedure. The NADES composed of food-grade menthol and acetic acid (1:1) achieved the highest efficiency, recovering 3.5 mg/g DCW of zeaxanthin and 1.2 mg/g DCW of β-cryptoxanthin, about 50% higher than the conventional solvent acetone, highlighting the strong potential of NADES as a green and safe solvent for carotenoid recovery from cyanobacteria.

conclusionsTaken together, our work reveals an efficient biotechnological strategy for production of zeaxanthin and β-cryptoxanthin, and a novel and efficient NADES-based method for eco-friendly recovery of high-value carotenoids.