Diamines serve as major platform chemicals that can be employed to a variety of industrial scenarios, particularly as monomers for polymer synthesis. High-throughput sensors for diamine biosynthesis can greatly improve the biological production of diamines. Here, we identified and characterized a transcription factor-driven biosensor for putrescine and cadaverine in Corynebacterium glutamicum. The transcriptional TetR-family regulatory protein CgmR (CGL2612) is used for the specific detection of diamine compounds. This study also improved the dynamic range and the sensitivity to putrescine by systematically optimizing genetic components of pSenPut. By a single cell-based screening strategy for a library of CgmR with random mutations, this study obtained the most sensitive variant CgmRI152T, which possessed an experimentally determined limit of detection (LoD) of ≤0.2 mM, a K of 11.4 mM, and a utility of 720. Using this highly sensitive putrescine biosensor pSenPutI152T, we demonstrated that CgmRI152T can be used as a sensor to detect putrescine produced biologically in a C. glutamicum system. This high sensitivity and the range of CgmR will be an influential tool for rewiring metabolic circuits and facilitating the directed evolution of recombinant strains toward the biological synthesis of diamine compounds.

2-Oxobutyrate (2-OBA), as a toxic metabolic intermediate, generally arrests the cell growth of most microorganisms and blocks the biosynthesis of target metabolites. In this study, we demonstrated that using the acetate bypass to replace the pyruvate dehydrogenase complex (PDHc) in Escherichia coli could recharge the intracellular acetyl-CoA pool to alleviate the metabolic toxicity of 2-OBA. Furthermore, based on the crystal structure of pyruvate oxidase (PoxB), two candidate residues in the substrate-binding pocket of PoxB were predicted by computational simulation. Site-directed saturation mutagenesis was performed to attenuate 2-OBA-binding affinity, and one of the variants, PoxBF112W, exhibited a 20-fold activity ratio of pyruvate/2-OBA in substrate selectivity. PoxBF112W was employed to remodel the acetate bypass in E. coli, resulting in l-threonine (a precursor of 2-OBA) biosynthesis with minimal inhibition from 2-OBA. After metabolic detoxification of 2-OBA, the supplies of intracellular acetyl-CoA and NADPH (nicotinamide adenine dinucleotide phosphate) used for l-threonine biosynthesis were restored. Therefore, 2-OBA is the substitute for pyruvate to engage in enzymatic reactions and disturbs pyruvate metabolism. Our study makes a straightforward explanation of the 2-OBA toxicity mechanism and gives an effective approach for its metabolic detoxification.

Objective. To assess the treatment outcome and postoperative quality of life of patients with gallbladder stones and chronic cholecystitis after open cholecystectomy and laparoscopic cholecystectomy. Methods. Between 2018 and 2020, 108 patients with gallbladder stones and chronic cholecystitis treated in our hospital were assessed for eligibility and randomly recruited. They were concurrently assigned (1 : 1) to receive either open cholecystectomy (control group) or laparoscopic cholecystectomy (study group). Outcome measures include surgical indices, inflammatory response, postoperative complications, and quality of life of patients. Results. Laparoscopic cholecystectomy was associated with a shorter duration of surgery, intraoperative bleeding, time to first postoperative bowel movement, and postoperative hospital stay versus open cholecystectomy ( P < 0.05 ). The levels of inflammatory factors of all eligible patients were comparable before cholecystectomy ( P > 0.05 ). The patients given laparoscopic cholecystectomy showed lower levels of C-reactive protein (CRP), interleukin (IL)-6, and IL-8 versus those given open cholecystectomy ( P < 0.05 ). Laparoscopic cholecystectomy resulted in a significantly lower incidence of complication (3.56%) versus open cholecystectomy (24.07%) ( P < 0.05 ). The patients had significantly higher physical, psychological, and social function scores after laparoscopic cholecystectomy versus open cholecystectomy ( P < 0.05 ). Conclusion. Laparoscopic cholecystectomy provides better surgical results, mitigates the inflammatory response, lowers the incidence of complications, and improves the quality of life of patients versus open cholecystectomy, so it is worthy of application in clinical treatment.