An Engineering Approach for Rewiring Microbial Metabolism

Methods in Enzymology
Sebastian WenkArren Bar-Even

Abstract

The introduction of synthetic pathways into microbes often requires substantial modifications of the host metabolism. Here, we present and discuss key experimental aspects required for modifying microbial central metabolism. We introduce the concept of dividing pathways into metabolic modules, the activity of which can be selected for and optimized in dedicated gene-deletion strains. We provide a comprehensive methodology for systematic pathway implementation in vivo, ranging from gene-deletion methods for the creation of selection strains to cloning strategies that allow fine-tuned expression of individual pathway enzymes in synthetic operons. We further describe pathway testing and validation via high-throughput growth experiments and 13C-labeling measurements. While we focus on Escherichia coli as bacterial host, the holistic approach we present could be easily adapted for the metabolic engineering of other microbes.

Citations

Jul 14, 2020·Biotechnology and Bioengineering·Sebastian WenkArren Bar-Even
Feb 12, 2020·Nature Chemical Biology·Seohyoung KimArren Bar-Even
Aug 25, 2020·ELife·Charles Ar CottonArren Bar-Even
Nov 18, 2020·Nature Communications·Ari SatanowskiArren Bar-Even
Aug 1, 2020·ACS Catalysis·Liliana Calzadiaz-RamirezCarlos G Acevedo-Rocha
Nov 27, 2018·ACS Synthetic Biology·Steffen N LindnerArren Bar-Even
Sep 8, 2021·Nature Communications·Enrico OrsiSteffen N Lindner
Aug 18, 2020·Metabolic Engineering·Nico J ClaassensArren Bar-Even

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