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

Abstract

Monoterpenes are commercially important flavors and fragrances with high demand. Microbial production of monoterpenes is more sustainable than plant extraction, however, it is restricted by high product toxicity/volatility and inefficient monoterpene synthases. Hence, most reported monoterpene titers are still low for commercialization. To overcome these challenges, we utilized the rose NUDIX hydrolase instead of geraniol synthase to produce geraniol in E. coli. The supply of the monoterpene precursor, geraniol pyrophosphate (GPP), was enhanced by the mevalonate pathway optimization and screening/engineering of various GPP synthases from plants, yeasts, and bacteria. Furthermore, geraniol production was improved by deleting the competing pathway genes (tnaA, yjgB, and ackA-pta) and optimizing the bioprocess. The final strain produced 1.05 g/L monoterpenes in total including 0.91 g/L geraniol in flasks. Moreover, the geraniol strain was reprogrammed to produce geranyl acetate, reaching ∼4.1 g/L in flasks from 20 g/L glycerol (∼66% of theoretic yield). We observed that microaerobic fermentation is critical to achieve high-yield production of geraniol and geranyl acetate. By controlling the redox potential at −190 mV in 5 L bioreactors, our strain produced ∼19 g/L geranyl acetate in 100 h, with a yield of 0.12 g/g-glycerol.