02273nam a2200301Ia 4500 MX-MdCICY 20260521091311.0 CICY B-12287 CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production Journal of Agricultural and Food Chemistry, 69(39), p.11626-11636, 2021 Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD)is the critical enzyme to synthesize crocetin. So, in this study, we used "hybrid-tunnel" engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the ?-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (kcat/Km)toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications. CROCETIN CCD2 ACCESS TUNNEL ENGINEERING SACCHAROMYCES CEREVISIAE Liang, N. Yao, M. D. Wang, Y. Liu, J. Feng, L. Wang, Z. M. Yuan, Y. J. https://drive.google.com/file/d/1V18cw3FxEyDWbqPJROqkMtdCifhfFWyX/view?usp=drivesdk Para ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx Loc REF1 250602s9999 xx |||||s2 |||| ||und|d 22436 22436 0 0 Loc 0 0 F1 CICY CICY RE 2025-06-25 0 B-12287 2025-06-25 15:38:50 2025-06-25 REF1