Concurrent biodegradation of tylosin and tilmicosin by a novel Paracoccus versutus 4-B-2.

The co-contamination of macrolide antibiotics tylosin (TYL) and tilmicosin (TLM) poses a persistent environmental threat, exacerbating the spread of antimicrobial resistance. Effective bioremediation has been hampered by the lack of microorganisms capable of degrading both compounds simultaneously. Here, we report the isolation of Paracoccus versutus 4-B-2, a novel bacterial strain that achieves complete co-degradation of TYL and TLM within 144 h, with first-order degradation half-lives of 1.28 days for TLM and 1.47 days for TYL, representing a 1.6-fold enhancement over previously reported strains. Pathway analysis revealed that the initial and rate-limiting step is hydrolysis of the macrolide lactone bond, catalyzed by a novel extracellular MBL-fold metallohydrolase, Est-4151. This enzyme exhibits optimal activity at 40°C and pH 8.0, retains > 50% activity across a broad pH range (6.0-10.0), and is strongly inhibited by EDTA and PMSF, indicating which it is a serine-dependent metalloenzyme. Structural modeling revealed a conserved H96-D98-H163 motif coordinating a catalytic zinc ion, consistent with MBL-fold hydrolases. This enzyme reduced the antibacterial activity of degradation products by over 20%. Transcriptomic profiling confirmed the substrate-inducible expression of gene4151 (encoding Est-4151) and upregulation of redox, deglycosylation and hydrolysis enzymes. The strain demonstrates robust environmental applicability, tolerating high antibiotic concentrations (500 mg/L) and elevated sulfate levels (6% Na₂SO₄). Moreover, it retained significant degradation activity in diluted raw sewage, achieving a maximum degradation efficiency of 21.43% in tryptone-supplemented sewage, with biomass (OD₆₀₀) reaching 2.1. Our work identifies a unique dual-degrading biocatalyst and provides an enzymatic strategy centered on lactone ring cleavage to mitigate antibiotic persistence and resistance selection in contaminated wastewater.