Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up

• Published in: Microbial cell factories Vol. 21; no. 1; pp. 246 - 9
• Main Authors: Huang, Ching-Ning, Lim, Xiaohui, Ong, Leonard, Lim, Chinchin, Chen, Xixian, Zhang, Congqiang
• Format: Journal Article
• Language: English
• Published: England BioMed Central 23.11.2022; BMC
• Subjects: Alkyl hydroperoxide reductase; Alkyl hydroperoxide reductases; Apocarotenoids; Bioreactors; Biosynthesis; Carotenoid cleavage dioxygenases (CCDs); Carotenoids; Cosmetics; Dioxygenase; E coli; Enzymes; Fermentation; Flasks; Genes; Hydrogen Peroxide; Hydrogen peroxide (H2O2); Hypotheses; Ionone; Lycopene; Metabolic Engineering; Metabolic pathways; Microorganisms; Norisoprenoids - metabolism; Oxidative Stress; Reactive oxygen species (ROSs); Reductases; Robustness; Scaling up; Strain; Tubes; Vitamin A; Hydrogen peroxide (H2O2); Apocarotenoids; Alkyl hydroperoxide reductases; Reactive oxygen species (ROSs); Carotenoid cleavage dioxygenases (CCDs); Ionone
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01968-1

α-Ionone is highly valued in cosmetics and perfumery with a global usage of 100-1000 tons per year. Metabolic engineering by microbial fermentation offers a promising way to produce natural (R)-α-ionone in a cost-effective manner. Apart from optimizing the metabolic pathways, the approach is also highly dependent on generating a robust strain which retains productivity during the scale-up process. To our knowledge, no study has investigated strain robustness while increasing α-ionone yield. Built on our previous work, here, we further increased α-ionone yield to 11.4 mg/L/OD in 1 mL tubes by overexpressing the bottleneck dioxygenase CCD1 and re-engineering the pathway, which is  > 65% enhancement as compared to our previously best strain. However, the yield decreased greatly to 2.4 mg/L/OD when tested in 10 mL flasks. Further investigation uncovered an unexpected inhibition that excessive overexpression of CCD1 was accompanied with increased hydrogen peroxide (H O ) production. Excessive H O broke down lycopene, the precursor to α-ionone, leading to the decrease in α-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of α-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved α-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H O . The strain exhibited improved robustness and produced  ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature. Our study provides an insight on the importance of mediating the oxidative stress to improve strain robustness and microbial production of α-ionone during scaling up. This new strategy may be inspiring to the biosynthesis of other high-value apocarotenoids such as retinol and crocin, in which oxygenases are also involved.