Biosensor-Based Evolution and Elucidation of a Biosynthetic Pathway in Escherichia coli

• Published in: ACS synthetic biology 2017-05, Vol.6 (5), p.837-848
• Main Authors: Liu, Yongfei, Zhuang, Yinyin, Ding, Dongqin, Xu, Yiran, Sun, Jibin, Zhang, Dawei
• Format: Article
• Language: English
• Subjects: Biosensing Techniques - methods; Biosynthetic Pathways - genetics; Biosynthetic Pathways - physiology; Directed Molecular Evolution - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Metabolic Engineering - methods; Phenylalanine - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.6b00328

The successful evolution of metabolite-producing microbes requires a high-throughput screening method to obtain the desired properties within a short time. In this study, we developed a transcription-factor-driven device that combines a metabolite-responsive element and a selection module. This device was able to specifically sense intracellular l-phenylalanine (l-Phe) and convert this signal into an observable phenotype. Applying this device, we successfully improved l-Phe production by screening hyperproducing phenotypes from a ribonucleotide binding site library and a random mutagenesis library. In addition, several site mutations introduced by random mutagenesis were identified and elucidated to facilitate the improvement of l-Phe production. Our results present a paradigm for screening of compounds that are not easily observable to raise the yield of targeted compounds from a large candidate library. This approach may guide further applications in rewiring metabolic circuits and facilitate the directed evolution of recombinant strains.