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 quan-tities 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 degrees 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 mu M Tet(X) and 50 mu M 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. (c) 2021 Elsevier B.V. All rights reserved.