Li Huimin, Zhang Wei, Han Yuying, Tang Guangyao, Lu Fuping, Qin Hui-Min
Key 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.
Key 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.
Biosens Bioelectron. 2025 Mar 1;271:117072. doi: 10.1016/j.bios.2024.117072. Epub 2024 Dec 17.
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.
色氨酸羟化酶（TPH）用于5-羟色氨酸（5-HTP）生物合成的改造近来成为研究热点。在本研究中，我们建立了基于液滴的超高通量微流控筛选平台（DTSP）以改善TPH的工业特性，同时构建了一种用于检测L-色氨酸（L-Trp）的细菌生物传感器以提高灵敏度。启动子pJ23111使生物传感器的L-Trp浓度与荧光输出之间呈现出强负相关。随后利用优化后的DTSP平台，通过迭代饱和诱变（ISM）筛选出优良的TPH突变体。M4-1变体（D129L/Q132M/P103A/T236K）与亲本CviPAH相比，催化活性提高了4.25倍。此外，它还表现出优异的热稳定性，在45℃下半衰期增加了3.2倍。我们的研究为基于微滴筛选平台的细菌生物传感器开发提供了参考。
