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Refactoring of a Synthetic Raspberry Ketone Pathway with EcoFlex

Moore, Simon J, Hleba, Yonek B, Bischoff, Sarah Lena, Bell,, David, Polizzi, Karen M, Freemont, Paul S (2021) Refactoring of a Synthetic Raspberry Ketone Pathway with EcoFlex. Microbial Cell Factories, . Article Number 116 (2021). E-ISSN 1475-2859. (doi:10.1186/s12934-021-01604-4) (KAR id:88542)

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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 DH10, as a routine cloning host. The use of E. coli DH10 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.

Item Type: Article
DOI/Identification number: 10.1186/s12934-021-01604-4
Uncontrolled keywords: Synthetic biology, fine chemicals, tyrosine, raspberry ketone, Golden Gate, E. coli
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Simon Moore
Date Deposited: 04 Jun 2021 08:32 UTC
Last Modified: 17 Aug 2021 09:13 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/88542 (The current URI for this page, for reference purposes)
Moore, Simon J: https://orcid.org/0000-0002-1968-206X
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