Proof-of-concept engineering of Escherichia coli expressing a bee-derived cytochrome P450 monooxygenase for...

Bees are unintentionally exposed to pesticides applied to control other insect pests. Although bees possess endogenous detoxification mechanisms, their efficiency may not always be sufficient. To this end, the use of probiotics that can assist in pesticide detoxification may offer an additional layer of protection. In this study, we engineered Escherichia coli BL21(DE3) to heterologously express the bee-derived cytochrome P450 monooxygenase (CYP450), CYP9Q1, from Apis mellifera as a proof-of-concept microbial platform for thiamethoxam biotransformation and future microbiome-assisted pesticide detoxification strategies. We first assessed the toxicity of thiamethoxam, a systemic insecticide, on E. coli, which showed obvious impaired growth at relatively high concentrations (5 and 10 g/L). Subsequently, we computationally modeled the 3D structure and modified the CYP450 to improve its solubility when expressed in E. coli. Molecular docking of the CYP450 and thiamethoxam was also performed to confirm their potential interaction. pRSFDuet-1 plasmid was used to carry the modified CYP450 gene for expression in E. coli and the induction condition was optimized, with 0.5 mM isopropyl 𝛽-D-1-thiogalactopyranoside (IPTG) yielding the most favorable expression level. A whole-cell detoxification assay subsequently showed reduced concentrations of thiamethoxam after 46 h of biocatalytic activity, as monitored by High-Performance Liquid Chromatography (HPLC). Liquid Chromatography-Mass Spectrometry (LC-MS) analysis further revealed the presence of desnitro thiamethoxam corresponding to m/z 247.04105 confirming enzymatic transformation of thiamethoxam. To our knowledge, this study provides the first demonstration of bee CYP9Q1 heterologous expression in E. coli for whole-cell thiamethoxam biotransformation, highlighting its potential as a platform for developing probiotic or gut microbiome-based detoxification strategies to support bee pesticide tolerance.