Enhanced Phenanthrene Degradation by a Synthetically Engineered E. coli Consortium Utilizing the SCRaMbLE for the Evolution of Key Genes

Phenanthrene, a pervasive polycyclic aromatic hydrocarbon (PAH), poses significant environmental and health risks due to its persistence and bioaccumulation. This study presents the development of a synthetic bacterial consortium comprising three engineered Escherichia coli BL21(DE3) strains, each harboring distinct catabolic modules encoded by pET-28(a) plasmids for the complete mineralization of phenanthrene. The consortium was engineered by integrating 17 genes critical for phenanthrene degradation, organized into three distinct modules that harness the collective metabolic potential of separate strains to enhance the degradation process. Each module was tailored to a specific phase of phenanthrene catabolism, ensuring the coordinated and efficient breakdown of the pollutant. Following the identification of rate-limiting enzymes within the up- and downstream degradation modules, the eight corresponding genes were subjected to laboratory evolution using the SCRaMbLE method to bolster the degradation capabilities of the strains. The consortium was then tested in a minimal salt medium (MSM) containing 100 mg/L phenanthrene. Following their evolution, the two SCRaMbLE strains E. coli SCRPHE033 and E. coli SCRKA065 exhibited enhanced degradation rates of 11.5 and 25.18% for their respective substrates. Finally, the comprehensive phenanthrene degradation system, which includes the two SCRaMbLE strains and E. coli CRSA, was subjected to comprehensive optimization, after which it achieved a significant degradation efficiency of 84.33% for 100 mg/L phenanthrene within a 120-hour timeframe. This research demonstrates the potential of synthetic biology in constructing microbial consortia for environmental bioremediation based on the SCRaMbLE method, offering a promising strategy for mitigating PAH pollution.