Directed evolution of TetR for constructing sensitive and broad-spectrum tetracycline antibiotics whole-cell biosensor.

Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 107 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2-22 of TerR was isolated and assembled as a TCs biosensor. The epS2-22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2-22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2-22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2-22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2-22 biosensors have the potential for wider applications in real-world TCs detection.

Directly Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane Production.

Biosynthetic alkane using acyl-ACP aldehyde reductase (AAR) and aldehyde-deformylating oxygenase (ADO) from cyanobacteria is considered a promising alternative for the production of biofuels and chemical feedstocks. However, the lack of suitable screening methods to improve the catalytic efficiency of AAR and ADO has hindered further improvements in alkane production. Herein, a novel alkane biosensor was developed based on transcriptional factor AlkS by directed evolution, which shows sensitive dynamic response curves for exogenous long-chain alkanes as well as in situ monitoring of endogenously produced alkanes. The evolved biosensor enables high-throughput screening of alkane-producing strains from the AAR and ADO mutant library, which led to a 13-fold increase in the production of long-chain alkanes, including a 22-fold increase of C15. This study is the first to improve the alkane production through biosensors, which provides a good reference for the establishment of microbial cell factories for alkane production.