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

Background: alpha-lonone 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)-alpha-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 alpha-ionone yield. Results: Built on our previous work, here, we further increased alpha-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 (H2O2) production. Excessive H2O2 broke down lycopene, the precursor to alpha-ionone, leading to the decrease in alpha-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of alpha-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved alpha-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H2O2. The strain exhibited improved robustness and produced similar to 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 alpha-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.

Automated summary

This study successfully increased the yield of α-ionone by overexpressing bottleneck enzyme CCD1 and re-engineering the pathway. However, during scale-up, excessive overexpression of CCD1 led to a decrease in α-ionone production due to excessive hydrogen peroxide production. By overexpressing another enzyme, the oxidative stress was reduced and α-ionone yield was improved. This study provides a new strategy for improving strain robustness and microbial production of α-ionone, and may inspire the biosynthesis of other high-value compounds.