Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis

Abstract

Synthesis of β-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 β-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 βionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro. MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-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 % ethanol could increase enzyme activity at above 15 %. However, an obvious decline was observed on enzyme activity as the concentration of Fe 2 + increased (0-1 mM). The V max for β-apo-8'-carotenal was 72.5 U/mg, while the K m 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.

Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of β-Ionone

“…In order to obtain CCD1 that are more compatible with the C. tropicalis expression system, we investigated different CCD1 from Petunia hybrida, Morus notabilis, and Helianthus annuus (PhCCD1, MnCCD1 and HaCCD1 to optimize β-ionone synthesis in C. tropicalis.…”

Identification of the carotenoid cleavage dioxygenase genes and functional analysis reveal DoCCD1 is potentially involved in beta-ionone formation in Dendrobium officinale

“…The possible explanation was the volatile ability of products was too low or the products were catabolized by the strains ( Auldridge et al, 2006b ). Previous studies have shown that CCD1 can cleave the substrates at the 9, 10 (9′, 10′) bond sites, such as CCD1 from A. thaliana ( Schwartz et al, 2001 ), tea plant ( Wang et al, 2020a ), Morus notabilis ( Qi et al, 2021 ), Petunia hybrida ( Simkin et al, 2004b ), Lycopersicon esculentum ( Simkin et al, 2004a ), Rosa damascena ( Huang et al, 2009 ), Osmanthus fragrans ( Baldermann et al, 2010 ). When beta-carotene was the substrate for CCD1, the cleavage at the 9, 10 (9′, 10′) bond sites can produce beta-ionone ( Schwartz et al, 2001 ), which can influence the fragrance and flavors of flowers, fruits, and essential oils ( Schwartz et al, 2001 ; Simkin et al, 2004a , b ; Brandi et al, 2011 ).…”

“…Other CCD1 enzymes from Arabidopsis, Bixa Orellana, Zea mays , and Lycopersicon esculentum can also cleave lycopene at 5, 6 and/or 5′, 6′ bond sites to form MHO ( Vogel et al, 2008 ). However, MnCCD1 failed to produce MHO but produced pseudoionone due to the cleavage at 9, 10 (9′, 10′) sites ( Qi et al, 2021 ). CCD1 from carrot didn’t use lycopene as a substrate ( Yahyaa et al, 2013 ).…”

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review

“…Interestingly, the Fe 2+ ion seems not to be an essential constituent in CCD1's reaction mix for in vitro characterization. 44,45 Additionally, other metal ions show no obvious regularity on CCD1. It has been reported that most metal ions showed a slight enhancement effect on the activity of MnCCD1 and HaCCD1, while a significant inhibitory effect was observed on PhCCD1.…”