Genetically engineered bacteria and microalgae expressing a mutant of cytochrome P450 BM3 for efficient Diuron degradation in wastewater treatment

The diverse members of the cytochrome P450 superfamily conduct the metabolism of a wide variety of compounds, both endogenous and xenobiotic, thus offering potential for degrading emerging contaminants such as prescription drugs and pesticides in wastewater treatment and aquaculture. This study demonstrates the expression and activity of the mutant P450 enzyme of CYP102A1 (BM3 MT35) engineered in <i>Bacillus megaterium</i> and <i>Chlamydomonas reinhardtii</i>, harnessed for the degradation of the herbicide Diuron. The P450 BM3 MT35 enzyme was transgenically expressed in <i>Escherichia coli</i> and purified via a His6-tag system, with its activity confirmed by spectral and <i>in vitro</i> metabolism assays. During <i>in vivo</i> experiments, transgenic <i>Bacillus megaterium</i> degraded 23% of Diuron after 24 h, reaching 65% by day 5 in Terrific Broth (TB) media. In synthetic and municipal wastewater, Diuron degradation reached 45% and 15%, respectively. No Diuron metabolism occurred in wild-type (WT) <i>Bacillus megaterium</i> in all the above tested conditions. Additionally, transgenic <i>Chlamydomonas reinhardtii</i> expressing P450 BM3 MT35 in the chloroplast also exhibited enhanced Diuron degradation (52%) compared to WT strains (6%). Overall, our results demonstrate the potential of genetically engineered bacteria and microalgae with cytochrome P450 as a viable strategy for the enhanced treatment of emerging contaminants in wastewater, potentially offering a new, sustainable alternative approach to pollution mitigation.IMPORTANCEWith a growing number and variety of prescription drugs, pesticides, food additives, and chemicals produced, wastewater is being contaminated with an increasing number of emerging pollutants that cannot be eliminated through classical wastewater treatment. New methods should therefore be developed to remove or deactivate these contaminants. Here, we demonstrate that by using genetically engineered bacteria and microalgae expressing a mutated enzyme, it is possible to efficiently metabolize a targeted pesticide, in this case, Diuron. These new findings should open the door to new ways to treat wastewater by developing low-cost and efficient modified microorganisms that will be able to specifically detoxify past and new emerging water contaminants that cannot be eliminated through classical wastewater treatment.