TET-Yeasate: An engineered yeast whole-cell lysate-based approach for high performance tetracycline degradation.

The widespread of tetracycline (TC) residues in anthropogenic and natural environments pose an immediate threat to public health. Herein, we established the TET-Yeasate, an approach based on whole-cell lysate of engineered yeast, to mitigate the TC contamination in environment. The TET-Yeasate is defined as the biological matrix of whole cell lysate from engineered yeast that containing TC-degradative components (Tet(X), NADPH, Mg2+) and protective macromolecules. The TET-Yeasate was able to efficiently eliminate TC residues in tap water (98.8%), lake water (77.6%), livestock sewage (87.3%) and pharmaceutical wastewater (35.3%) without necessity for exogenous addition of expensive cofactors. The TET-Yeasate was further developed into lyophilized form for ease of storage and delivery. The TET-Yeasate in lyophilized form efficiently removed up to 74.6% TC residue within 0.25 h. In addition, the lyophilization confers promising resilience to TET-Yeasate against adverse temperatures and pH by maintaining degradation efficacy of 85.69%-97.83%. The stability test demonstrated that the biomacromolecules in lysate served as natural protectants that exerted extensive protection on TET-Yeasate during the 14-day storage at various conditions. In addition, 5 potential degradation pathways were elaborated based on the intermediate products. Finally, the analysis indicated that TET-Yeasate enjoyed desirable bio- and eco-safety without introduction of hazardous intermediates and spread of resistance genes. To summary, the TET-Yeasate based on whole cell lysate of engineered yeast provides a cost-effective and safe alternative to efficiently remove TC residues in environment, highlighting the great potential of such whole-cell based methods in environmental decontamination.

Comprehensive analysis of plasmid-mediated tet(X4)-positive Escherichia coli isolates from clinical settings revealed a high correlation with animals and environments-derived strains.

The emergence of novel plasmid-mediated high-level tigecycline resistance genes tet(X) in the Enterobacteriaceae has increased public health risk for treating severe bacterial infections. Despite growing reports of tet(X)-positive isolates detected in animal sources, the epidemiological association of animal- and environment-derived isolates with human-derived isolates remains unclear. Here, we performed a comprehensive analysis of tet(X4)-positive Escherichia coli isolates collected in a hospital in Guangdong province, China. A total of 48 tet(X4)-positive E. coli isolates were obtained from 1001 fecal samples. The tet(X4)-positive E. coli isolates were genetically diverse but certain strains that belonged to ST48, ST10, and ST877 etc. also have clonally transmitted. Most of the tet(X4) genes from these patient isolates were located on conjugative plasmids that were successfully transferred (64.6%) and generally coexisted with other antibiotic resistance genes including aadA, floR, blaTEM and qnrS. More importantly, we found the IncX1 type plasmid was a common vector for tet(X4) and was prevalent in these patient-derived strains (31.3%). This plasmid type has been detected in animal-derived strains from different species in different regions demonstrating its strong transmission ability and wide host range. Furthermore, phylogenetic analysis revealed that certain strains of patient and animal origin were closely related indicating that the tet(X4)-positive E. coli isolates were likely to have cross-sectorial clonal transmission between humans, animals, and farm environments. Our research greatly expands the limited epidemiological knowledge of tet(X4)-positive strains in clinical settings and provides definitive evidence for the epidemiological link between human-derived tet(X4)-positive isolates and animal-derived isolates.

Reducing tetracycline antibiotics residues in aqueous environments using Tet(X) degrading enzymes expressed in Pichia pastoris.

Tetracycline antibiotics (TCs) are massively produced and consumed in various industries resulting in large quantities of residuals in the environment. In this study, to achieve safe and efficient removal of residual TCs, a Pichia pastoris (P. pastoris) was gained to stably express glycosylated TCs degrading enzyme Tet(X) followed codon and expression parameter optimization of tet(X4). As expected, glycosylated Tet(X) still maintains efficient capacity of degrading TCs. The expressed Tet(X) maintained efficient TCs degrading ability over a pH range of 6.5 - 9.5 and temperature range of 17 - 47 °C. We tested this recombinant protein for its ability to degrade tetracycline in pond water and sewage models of tetracycline removal at starting levels of 10 mg/L substrate. 80.5 ± 3.8% and 26.2 ± 2.6% of tetracycline was degraded within 15 min in the presence of 0.2 µM Tet(X) and 50 µM NADPH, respectively. More importantly, the direct use of a Tet(X) degrading enzymes reduces the risk of gene transmission during degradation. Thus, the Tet(X) degrading enzyme expressed by P. pastoris is an effective and safe method for treating intractable TCs residues.

Evolutionary Trajectory of the Tet(X) Family: Critical Residue Changes towards High-Level Tigecycline Resistance.

The emergence of the plasmid-mediated high-level tigecycline resistance mechanism Tet(X) threatens the role of tigecycline as the "last-resort" antibiotic in the treatment of infections caused by carbapenem-resistant Gram-negative bacteria. Compared with that of the prototypical Tet(X), the enzymatic activities of Tet(X3) and Tet(X4) were significantly enhanced, correlating with high-level tigecycline resistance, but the underlying mechanisms remain unclear. In this study, we probed the key amino acid changes leading to the enhancement of Tet(X) function and clarified the structural characteristics and evolutionary path of Tet(X) based upon the key residue changes. Through domain exchange and site-directed mutagenesis experiments, we successfully identified five candidate residues mutations (L282S, A339T, D340N, V350I, and K351E), involved in Tet(X2) activity enhancement. Importantly, these 5 residue changes were 100% conserved among all reported high-activity Tet(X) orthologs, Tet(X3) to Tet(X7), suggesting the important role of these residue changes in the molecular evolution of Tet(X). Structural analysis suggested that the mutant residues did not directly participate in the substrate and flavin adenine dinucleotide (FAD) recognition or binding, but indirectly altered the conformational dynamics of the enzyme through the interaction with adjacent residues. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and UV full-wavelength scanning experiments confirmed that each mutation led to an increase in activity without changing the biochemical properties of the Tet(X) enzyme. Further phylogenetic analysis suggested that Riemerella anatipestifer served as an important incubator and a main bridge vector for the resistance enhancement and spread of Tet(X). This study expands the knowledge of the structure and function of Tet(X) and provides insights into the evolutionary relationship between Tet(X) orthologs.IMPORTANCE The newly emerged tigecycline-inactivating enzymes Tet(X3) and Tet(X4), which are associated with high-level tigecycline resistance, demonstrated significantly higher activities in comparison to that of the prototypical Tet(X) enzyme, threatening the clinical efficacy of tigecycline as a last-resort antibiotic to treat multidrug-resistant (MDR) Gram-negative bacterial infections. However, the molecular mechanisms leading to high-level tigecycline resistance remain elusive. Here, we identified 5 key residue changes that lead to enhanced Tet(X) activity through domain swapping and site-directed mutagenesis. Instead of direct involvement with substrate binding or catalysis, these residue changes indirectly alter the conformational dynamics and allosterically affect enzyme activities. These findings further broaden the understanding of the structural characteristics and functional evolution of Tet(X) and provide a basis for the subsequent screening of specific inhibitors and the development of novel tetracycline antibiotics.