Complete remediation of a mixture of 5 acetophenone derivatives by multi-modular engineered Escherichia coli: Avoiding intermediate toxicity from incomplete degradation

In natural environments, numerous organic compounds possessing low intrinsic toxicity can be transformed through microbial incomplete degradation into highly toxic intermediates that persist and accumulate, causing significant environmental pollution and ecological deterioration. Acetophenone, hydroxyacetophenone, and their derivatives, as common industrial intermediates, represent those low-toxicity precursor compounds. We discovered that Baeyer-Villiger monooxygenases (BVMO) widely existed in microorganism, show catalytic activity to various acetophenone derivatives. The catalytic products are readily hydrolyzed into phenols, which accumulate and cause significant biotoxicity due to their limited subsequent metabolic pathways. To address the toxic intermediate accumulation issue, we constructed a multi-module engineered Escherichia coli strain BL-all by integrating 14 structurally optimized genes into 4 catabolic modules using synthetic biology approaches. BL-all demonstrated remarkable biodegradation capability, completely degrading five acetophenone derivatives including acetophenone, p-hydroxyacetophenone, p-hydroxypropiophenone, p-hydroxybenzaldehyde, and o-hydroxyacetophenone within 24 h and a mixture of all these five derivatives (each at 1 mM), within 48 h. These compounds were utilized as sole carbon sources for growth. Their incorporation into the bacterial tricarboxylic acid (TCA) cycle was definitively confirmed through 13 C-labeled isotopic tracing. Zebrafish toxicity tests simulated a miniature ecological environment, demonstrating the effective detoxification capability of BL-all, in contrast to the severe biotoxicity including deformities and developmental abnormalities caused by incomplete degradation. All tested acetophenone derivatives were completely degraded by BL-all in natural water. Its degradation capability may extend beyond the five tested substrates due to the broad substrate range of BVMO, suggesting its potential for remediating complex multi-pollutant environments.