000			02273nam a2200301Ia 4500
003			MX-MdCICY
005			20250625153850.0
040			_cCICY
090			_aB-12287
245	1	0	_aCsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production
490	0		_vJournal of Agricultural and Food Chemistry, 69(39), p.11626-11636, 2021
520	3		_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.
650	1	4	_aCROCETIN
650	1	4	_aCCD2
650	1	4	_aACCESS TUNNEL
650	1	4	_aENGINEERING
650	1	4	_aSACCHAROMYCES CEREVISIAE
700	1	2	_aLiang, N.
700	1	2	_aYao, M. D.
700	1	2	_aWang, Y.
700	1	2	_aLiu, J.
700	1	2	_aFeng, L.
700	1	2	_aWang, Z. M.
700	1	2	_aYuan, Y. J.
856	4	0	_uhttps://drive.google.com/file/d/1V18cw3FxEyDWbqPJROqkMtdCifhfFWyX/view?usp=drivesdk _zPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx
942			_2Loc _cREF1
008			250602s9999 xx |||||s2 |||| ||und|d
999			_c46497 _d46497