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

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 (H2O2) production. Excessive H2O2 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 H2O2. 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.

Microaerobic Fermentation Enables High-Titer Biosynthesis of the Rose Monoterpenes Geraniol and Geranyl Acetate in Escherichia coli

“…Next, we evaluated strain performance in 125 mL shaking flasks. We observed that mixing and oxygen supply had dramatic effects on the monoterpene yields, similar to a recent study on α-ionone biosynthesis . With the decrease of shaking speed from 300 to 100 rpm, the geraniol yield was increased by over 6-fold from 62 to 402 mg/L, in contrast, geranial production only increased by 86% (Figure A).…”

“…This observation was similar to a recent study on α-ionone biosynthesis in E. coli in which lower mixing also led to high yield of α-ionone in flasks. 32 High mixing often leads to high supply of oxygen, which might induce the oxidation of geraniol to geranial. This hypothesis was supported by the results in Figure 5A.…”

“…We observed that mixing and oxygen supply had dramatic effects on the monoterpene yields, similar to a recent study on α-ionone biosynthesis. 32 With the decrease of shaking speed from 300 to 100 rpm, the geraniol yield was increased by over 6-fold from 62 to 402 mg/L, in contrast, geranial production only increased by 86% (Figure 5A). In other words, lower shaking speed increased both overall monoterpene bioproduction and the geraniol percentage in flasks by reducing the oxidation from geraniol to geranial.…”