Refactoring of a synthetic raspberry ketone pathway with EcoFlex

Microbial Cell Factories

Karen M. Polizzi¹  Yonek B. Hleba¹  Paul S. Freemont¹  Simon J. Moore¹  David Bell¹  Sarah Bischoff² 

[1] Centre for Synthetic Biology and Innovation, Imperial College London;School of Biosciences, University of Kent;

关键词: Synthetic biology;    Fine chemicals;    Tyrosine;    Raspberry ketone;    Golden Gate;    E. coli;   

DOI  :  10.1186/s12934-021-01604-4

来源: DOAJ

【 摘 要 】

Abstract 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 (~ £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 DH10β, as a routine cloning host. The use of E. coli DH10β 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.

【 授权许可】

Unknown