Refactoring of a synthetic raspberry ketone pathway with EcoFlex

Background: A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (similar to(sic)20,000 kg(-1)) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results: By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10 beta, as a routine cloning host. The use of E. coli DH10 beta facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions: Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

Automated summary

Through a design-build-test-learn cycle approach in synthetic biology, the raspberry ketone biosynthetic pathway was successfully optimized from a low level of productivity to achieve a significant improvement in production. The use of E. coli DH10 beta and a novel color-based phenotypic screen facilitated the optimization process and screening of productive clones. This study demonstrates the capability of fine-tuning chemical pathways using EcoFlex and introduces newly characterised promoter tools for gene expression in E. coli.