A globally distributed cyanobacterial nitroreductase capable of conferring biodegradation of chloramphenicol

Background

Aquaculture provides protein for 35% of the global population, contributing over 20% of total protein supply. Growing demand for animal-derived products has driven intensive aquaculture expansion, consuming substantial water resources (e.g., 6.6×10¹⁰ m³ for Chinese pond aquaculture in 2018) and antibiotics (0.04~0.91 kg per ton of animal biomass). Notably, 50~70% of administered antibiotics enter aquatic systems through excretion, exacerbating antibiotic pollution and antimicrobial resistance dissemination.

Aquaculture wastewater, rich in nitrogen, phosphorus, and COD, provides ideal growth conditions for microalgae. Cyanobacteria, as key primary producers, play pivotal roles in global biogeochemical cycles. Studies demonstrate microalgae-based technologies can remove >90% of antibiotics, outperforming conventional sludge treatment. The resulting algal biomass can be repurposed for high-value products or feed, positioning this approach as the most promising wastewater treatment strategy. However, the mechanisms underlying antibiotic metabolism in microalgae remain unclear, hindering engineering applications.

Current research attributes antibiotic degradation to cytochrome P450 (CYP450) and glutathione-S-transferase activity, though direct correlation with removal efficiency lacks verification. Despite confirmed CYP450 genes in Chlorella and other algae genomes, their removal rates for persistent antibiotics like sulfadiazine remain limited to 10-40%, suggesting the existence of unidentified enzymatic degradation systems.

Research Progress

In order to explore whether CYP450s are necessary for degradation of antibiotics and identity other potential enzymes, we performed the inhibiting experiments with taking chloramphenicol (CAP) and Synechocystis sp. as the test chemical and microorganism.

Results indicated that the inhibitory effect of CAP on algal growth gradually weakened with cultivation time, with only high concentrations (≥2 mg/L) showing significant inhibition after 14 days (EC₅₀ = 1.09~2.0 mg/L). Across 0.1~5 mg/L CAP concentrations, Synechocystis sp. achieved removal efficiencies of 41.85%-94.27%, following first-order kinetics (R² = 0.90~0.97). Mass balance analysis revealed biodegradation as the primary removal pathway (contributing 38.38%~86.96%) (Fig. 1), demonstrating the remarkable CAP tolerance and degradation capability of Synechocystis sp.. Further mechanistic investigation revealed that CYP450 plays no decisive role in CAP degradation in Synechocystis sp.. Instead, nitroreductase (NTR) was identified as the key functional enzyme, showing significant positive correlation between its expression level and removal efficiency (R²=0.97). Engineered bacteria expressing NTR achieved 39.8%~94.4% CAP removal within 6 hours (Fig. 2). This discovery provides a novel, high-efficiency biocatalyst for antibiotic pollution remediation. The global distribution and transcription results revealed NTR homologs were ubiquitously distributed throughout three oceanic layers, predominantly in Bacteroidetes, Archaea, Chloroflexi, and Gammaproteobacteria. Transcriptional activity was most pronounced in surface waters, with particularly high expression observed in Bacteroidetes and Gammaproteobacteria (Fig. 3).

The further functional characterization of the NTR protein demonstrated its NADPH-dependent reductase activity, with optimal activity at pH 7.0-8.0 and 45℃. Kinetic analysis revealed a Km of 104.0 µM and Vmax of 3.82 µM/min (R²=0.95). In practical wastewater treatment, NTR degraded 52.68% of CAP within 30 minutes. Molecular interaction studies showed high affinity between NTR and CAP (Kd=2.9 nM), with docking simulations identifying hydrogen bonding with TYR8, ARG10 and GLN133 (binding energy: -3.94 kcal/mol) (Fig. 4). These properties highlight NTR's significant potential for nitroaromatic pollutant treatment, offering an efficient biocatalytic tool for wastewater remediation. The results of CAP transformation mechanism mediated by Synechocystis NTR exhibited that the NTR converts CAP into less toxic amino-CAP through nitro group reduction (Fig. 5). This biotransformation significantly reduces CAP's biological toxicity, demonstrating NTR as an environmentally friendly biocatalyst. Engineered bacteria expressing this enzyme show strong potential for CAP-polluted wastewater remediation.

This study revealed a novel enzyme capable of degrading CAP, advancing sustainable biotechnological solutions to combat antibiotic pollution and addressing pressing environmental challenges in aquaculture and other industries worldwide.

Future Prospects

This study bridges enzymatic discovery with practical applications, offering sustainable biotechnology for antibiotic pollution control: engineered bacteria expressing Synechocystis NTR can rapidly degrade CAP while preventing resistance gene spread. Integrating this targeted biocatalyst into circular aquaculture systems - where treated microalgal biomass serves as feed or biofertilizer - simultaneously reduces environmental footprints and enhances resource efficiency. The findings provide a "planetary health" solution to the growing antimicrobial resistance crisis and advance precision bioremediation tools. However, challenges remain in engineered bacteria applications, including ecological risks (e.g., horizontal gene transfer), technical limitations in long-term stability, and management hurdles in real-time monitoring, necessitating balanced approaches between innovation and biosafety.

Sources: https://spj.science.org/doi/10.34133/research.0692