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Updated: May 31, 2026
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Programming a bacterial biosensor for directed evolution of tryptophan hydroxylase via high-throughput droplet
Huimin Li1, Wei Zhang1, Yuying Han1
1Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin, 300457, PR China.
Abstract:
The modification of tryptophan hydroxylase (TPH) for the biosynthesis of 5-hydroxytryptophan (5-HTP) has recently become a focus of research. In this study, we established a droplet-based ultrahigh-throughput microfluidic screening platform (DTSP) to improve the industrial properties of TPH, whereas a bacterial biosensor for L-tryptophan (L-Trp) detection was engineered to improve sensitivity. The promoter pJ23111 achieved a strong negative correlation between the L-Trp concentration and the fluorescence output of the biosensor. The optimized DTSP platform was then utilized to screen superior TPH mutants by employing iterative saturation mutagenesis (ISM). The M4-1 variant (D129L/Q132M/P103A/T236K) demonstrated a 4.25-fold increase of catalytic activity compared to the parental CviPAH. Moreover, it exhibited excellent thermostability, with a 3.2-fold increase of half-life at 45 °C. Our study provides a reference for the development of bacterial biosensors for microdroplet-based screening platforms.
