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245	1	0	_aMolecular cloning, expression, and biochemical characterization of carotenoid cleavage dioxygenase 1 (LbCCD1) from Lycium barbarum
490	0		_aMolecular Catalysis, 569, p.114561, 2024
500			_aArtículo
520	3		_aCarotenoid cleavage dioxygenases (CCDs) play a pivotal role in the biosynthesis of volatile apocarotenoids, which have significant industrial applications due to their aromatic and bioactive properties. This study focuses on the molecular cloning and biochemical characterization of the LbCCD1 from Lycium barbarum. The LbCCD1 gene was successfully cloned and heterologously expressed in Escherichia coli and several carotenoids were using as substrates for investigate its specificity by in vitro and in vivo experiment. The LbCCD1 protein was able to cleave a variety of carotenoids including β-carotene, zeaxanthin, astaxanthin, and β-apo-8'-carotenal, at the 9, 10 (9', 10') double bond to produce β-ionone, 3‑hydroxy-4-oxo-β-ionone, and 3‑hydroxy-β-ionone, respectively in vitro. LbCCD1 could also cleave zeaxanthin and β-carotene at the 9, 10 (9', 10') double bond to produce β-ionone, respectively, in E. coli accumulating carotenoids. Interestingly, LbCCD1 did not exhibit cleavage activity on lycopene either in vivo or in vitro unlike other CCD1 family enzymes. In the previous experiment, it was confirmed that LbCCD1 exhibits cleavage activity towards β-apo-8'-carotenal in vitro, so we used β-apo-8'-carotenal as the substrate for characterizing the enzymatic properties. The expression of LbCCD1 was optimized at such conditions (temperature 24 ◦C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of LbCCD1 revealed the optimal activities were at pH 9 and 55 ◦C. The addition of 10 percent ethanol could increase enzyme activity to above 15 percent. However, the concentration of Fe2+ has a minimal effect on enzyme activity. The Vmax for β-apo-8'-carotenal was 8.6 U/mg, while the Km was 0.27 mM. To preliminarily verify the potential of LbCCD1 as a biological component for β-ionone production. By introducing LbCCD1 into the β-carotene-high-producing chassis cell and optimizing the conditions (temperature 30 ◦C, IPTG 0.01 mM, Fe2+ concentration 0.05 mM), the β-ionone yield reached 21.45 mg/L. This study focused on one of the CCDs derived from woody plants, which have been relatively underexplored. It lays the groundwork for expanding the CCD enzyme library, identifying suitable CCDs, and investigating the structure-function relationship of CCDs. Furthermore, it sets the stage for engineering novel CCD genes and developing advanced applications of CCDs as biocatalysts and platforms for synthetic biology. These advancements will enable the efficient production of volatile aroma compounds from carotenoids.
650	1	4	_aCAROTENOID CLEAVAGE DIOXYGENASES
650	1	4	_aCCD1 FAMILY
650	1	4	_aLYCIUM BARBARUM
650	1	4	_aBIOCHEMICAL CHARACTERIZATION
650	1	4	_aB-IONONE
700	1	2	_aKe, K.
700	1	2	_aGuo, L.
700	1	2	_aQi, Z.
700	1	2	_aDou, D.
700	1	2	_aMa, H.
700	1	2	_aFang, X.
700	1	2	_aZhao, L.
856	4	0	_uhttps://drive.google.com/file/d/1OYB6yyKaf5-4Dkbe5FgFUECtjmxLchCp/view?usp=drive_link _zPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx
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