Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up
Ching-Ning Huang, Xiaohui Lim, Leonard Ong, Chinchin Lim, Xixian Chen, Congqiang Zhang
Abstract
Abstract Background 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. Results 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 2 O 2 ) production. Excessive H 2 O 2 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 2 O 2 . The strain exhibited improved robustness and produced ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature. Conclusion 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.