Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.

Automated summary

This study successfully increased the production of geraniol through strategies such as constructing a platform strain, optimizing protein expression, and utilizing fusion tags, as well as developing a high-throughput screening system to monitor fusion tag variants.