Engineering regulatory networks for complex phenotypes in E. coli

• Published in: Nature communications 2020-08, Vol.11 (1), p.4050-13, Article 4050
• Main Authors: Liu, Rongming, Liang, Liya, Freed, Emily F., Choudhury, Alaksh, Eckert, Carrie A., Gill, Ryan T.
• Format: Article
• Language: English
• Subjects: 38; 38/35; 38/43; 38/47; 631/1647/2163; 631/1647/338/318; 631/1647/338/552; 631/337/4041/3196; BASIC BIOLOGICAL SCIENCES; Cellular communication; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; E coli; Engineering; Environmental regulations; Gene Editing - methods; Gene mapping; Gene Regulatory Networks - genetics; Gene Regulatory Networks - physiology; Genes; Humanities and Social Sciences; isobutanol; isopropanol; large scale saturation; Libraries; Mapping; Metabolic Engineering - methods; Metabolism; multidisciplinary; mutagenesis library; Mutation; Networks; Phenotypes; Regulators; regulatory networks; Science; Science (multidisciplinary); Synthetic Biology - methods; Toolkits; Transcription factors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-17721-4

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli , with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts. Regulatory networks respond to environmental and genetic perturbations by reprogramming metabolism. Here the authors screen a library of 82 regulators with 110,120 mutations to map the regulatory network of 4000 genes.