Construction of Bacterial Cells with an Active Transport System for Unnatural Amino Acids

• Published in: ACS synthetic biology 2019-05, Vol.8 (5), p.1195-1203
• Main Authors: Ko, Wooseok, Kumar, Rahul, Kim, Sanggil, Lee, Hyun Soo
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Bacteria - metabolism; Biological Transport, Active; Fluorescence Resonance Energy Transfer; Genetic Code; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Mutagenesis, Site-Directed; Periplasmic Binding Proteins - genetics; Periplasmic Binding Proteins - metabolism; Protein Engineering - methods
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.9b00076

Engineered organisms with an expanded genetic code have attracted much attention in chemical and synthetic biology research. In this work, engineered bacterial organisms with enhanced unnatural amino acid (UAA) uptake abilities were developed by screening periplasmic binding protein (PBP) mutants for recognition of UAAs. A FRET-based assay was used to identify a mutant PBP (LBP-AEL) with excellent binding affinity (K d ≈ 500 nM) to multiple UAAs from 37 mutants. Bacterial cells expressing LBP-AEL showed up to 5-fold enhanced uptake of UAAs, which was determined by genetic incorporation of UAAs into a green fluorescent protein and measuring UAA concentration in cell lysates. To the best of our knowledge, this work is the first report of engineering cellular uptake of UAAs and could provide an impetus for designing advanced unnatural organisms with an expanded genetic code, which function with the efficiency comparable to that of natural organisms. The system would be useful to increase mutant protein yield from lower concentrations of UAAs for industrial and large-scale applications. In addition, the techniques used in this report such as the sensor design and the measurement of UAA concentration in cell lysates could be useful for other biochemical applications.