Combinatorial Engineering of Upper Pathways and Carotenoid Cleavage Dioxygenase in Escherichia coli for Pseudoionone Production

De Novo Synthesis of Dihydro-β-ionone through Metabolic Engineering and Bacterium-Yeast Coculture

“…Fe 2+ ion is a cofactor of CCD1 and is located at the active site and involved in catalytic function and therefore this was also evaluated as previous studies had reported its positive effects for de novo synthesis of psi-ionone and functional expression of CCD1 in E. coli. 32,35 As expected, a minor increase in β-ionone production was observed at 0.01 mM Fe 2+ ion, similar to the optimal concentration used for psi-ionone production in the previous study. 32 Therefore, the type and concentration of carbon sources played a greater role in the increase in β-ionone production in comparison to the culture conditions.…”

“…32,35 As expected, a minor increase in β-ionone production was observed at 0.01 mM Fe 2+ ion, similar to the optimal concentration used for psi-ionone production in the previous study. 32 Therefore, the type and concentration of carbon sources played a greater role in the increase in β-ionone production in comparison to the culture conditions. The IONE-GST-Ph strain finally produced 40 mg/ L β-ionone at the shake-flask level, a comparable yield with the previously reported highest yield in E. coli (32.4 mg/L βionone in a shake flask).…”

“…These CCD1 enzymes were screened previously in E. coli accumulating lycopene (33 mg/L), and the psi-ionone production made minimal difference (0.75, 0.78, and 0.83 mg/L of psi-ionone obtained by PhCCD1, MnCCD1, and HaCCD1, respectively, with not production in AtCCD1). 32 Here, we suspected that lycopene cyclase (CrtY) and not CCD1 was the primary driving force for lycopene conversion, leading to lycopene preferentially being converted to β-carotene rather than toward psi-ionone. The main rate-limiting reasons of CCD1 in the βionone biosynthetic pathway were attributed to both low efficiency and substrate promiscuity.…”

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

“…For example, Fan et al compared the effects of MnCCD1 expression with T7 and Tac promoters and identified that the tac promoter (∼1.5-fold improvement) was more suitable than the T7 promoter in this system for the production of pseudoionone. 81 In addition, Chen et al constructed RBS libraries with a color-based colony screening strategy and obtained one with moderate translation initiation for producing a high proportion of ε-carotene in total carotenoids. Then, a RBS with a ∼2-fold translation initiation rate was adopted to control the expression of the fusion enzyme (OfCCD1 fused with lycopene cyclase), enabling improved production of α-ionone with a 20% improvement in E. coli.…”

“…Genetic parts like promoters, ribosome binding sites (RBSs), and gene copy numbers are widely used to enhance C13-apocarotenoid production in engineered hosts by fine-tuning the metabolic flux of the CCD1 module (Figure A). For example, Fan et al compared the effects of MnCCD1 expression with T7 and Tac promoters and identified that the tac promoter (∼1.5-fold improvement) was more suitable than the T7 promoter in this system for the production of pseudoionone . In addition, Chen et al constructed RBS libraries with a color-based colony screening strategy and obtained one with moderate translation initiation for producing a high proportion of ε-carotene in total carotenoids.…”