02194nam a2200265Ia 4500003001000000005001700010040000900027245008700036490007600123520134800199650001301547650000901560650001801569650001601587650002901603700001401632700001501646700001301661700001201674700001301686700001601699700001601715856015601731008004101887MX-MdCICY20260521091311.0  cCICY10aCsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production0 vJournal of Agricultural and Food Chemistry, 69(39), p.11626-11636, 20213 aCrocetin, 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.14aCROCETIN14aCCD214aACCESS TUNNEL14aENGINEERING14aSACCHAROMYCES CEREVISIAE12aLiang, N.12aYao, M. D.12aWang, Y.12aLiu, J.12aFeng, L.12aWang, Z. M.12aYuan, Y. J.40uhttps://drive.google.com/file/d/1V18cw3FxEyDWbqPJROqkMtdCifhfFWyX/view?usp=drivesdkzPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx250602s9999    xx |||||s2   |||| ||und|d