Overexpression and characterization of a novel plant carotenoid cleavage dioxygenase 1 from Morus notabilis
Overexpression and characterization of a novel plant carotenoid cleavage dioxygenase 1 from Morus notabilis
- Chemistry & BioDiversity, 19(2), p.e202100735, 2022 .
Synthesis of ?-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating ?-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9?, 10?)to produce ?-ionone, 3-hydroxy-4-oxo-?-ionone, 3-hydroxy-?-ionone, and 3-hydroxy-?-ionone in vitro. MnCCD1 could also cleave lycopene and ?-carotene at the 9, 10 (9?, 10?)bind bond to produce pseudoionone and ?-ionone, respectively, in E. coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28?°C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35?°C. The addition of 10? percent ethanol could increase enzyme activity at above 15? percent. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0-1 mM). The Vmax for ?-apo-8?-carotenal was 72.5 U/mg, while the Km was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.
MORUS NOTABILIS
CAROTENOID CLEAVAGE DIOXYGENASES
CCD1
B-IONONE
Synthesis of ?-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating ?-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9?, 10?)to produce ?-ionone, 3-hydroxy-4-oxo-?-ionone, 3-hydroxy-?-ionone, and 3-hydroxy-?-ionone in vitro. MnCCD1 could also cleave lycopene and ?-carotene at the 9, 10 (9?, 10?)bind bond to produce pseudoionone and ?-ionone, respectively, in E. coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28?°C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35?°C. The addition of 10? percent ethanol could increase enzyme activity at above 15? percent. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0-1 mM). The Vmax for ?-apo-8?-carotenal was 72.5 U/mg, while the Km was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.
MORUS NOTABILIS
CAROTENOID CLEAVAGE DIOXYGENASES
CCD1
B-IONONE