Polyethylene and polystyrene oxidation by host and microbial oxidoreductases in Zophobas atratus.

INTRODUCTION: Insect-mediated oxidation is a promising strategy for degrading hydrocarbon-based plastics. There is evidence for better long-term ingestion and sustained intestinal oxidation of polyethylene (PE) and polystyrene (PS) in the plastivore Zophobas atratus than in Galleria mellonella. However, the mechanisms underlying intestinal oxidation of PE and PS remain unclear. OBJECTIVES: Host and microbial enzymes that mediate intestinal PE and PS oxidation in Z. atratus were evaluated. METHODS: Long-term feeding assays were performed using G. mellonella and Z. atratus to compare PE and PS ingestion and oxidation activities. Germ-free Z. atratus larvae were generated using antibiotic treatment to evaluate host-derived oxidation. Transcriptome-based reverse genetics with heterologous expression in Pichia pastoris was used to identify candidate intestinal PE-oxidizing enzymes. The Z. atratus gut microbiome was enriched in media containing PE or PS as the sole carbon source and plastic-oxidizing bacteria were isolated. Candidate microbial enzymes were screened using a single-gene mutant library of a phylogenetic neighbor and validated by heterologous expression in Escherichia coli. RESULTS: Z. atratus exhibited higher ingestion and intestinal oxidation of PE and PS than G. mellonella. Antibiotic treatment significantly reduced PE and PS oxidation in Z. atratus; however, the residual activity indicated contributions from both gut microbes and host-derived enzymes. Transcriptome-based reverse genetics using P. pastoris expression identified CYP6k1-ZP10 as a host PE-oxidizing enzyme, which was validated by RNA interference. Host enzymes involved in PS oxidation were not detected. Instead, Klebsiella variicola was isolated as the dominant gut bacterium capable of oxidizing both plastics. Functional screening revealed an FMN-dependent monooxygenase responsible for PE and PS oxidation. CONCLUSION: These findings demonstrate the dual contribution of host- and microbe-derived oxidoreductases to PE and PS oxidation in Z. atratus.