CRISPR-Enhanced Microbial Consortia for Pollutant Degradation: Molecular Dynamics-Guided Enzyme Stabilization and Metabolic Pathway Optimization

Abstract

Novel research is proposed to optimize a pollutant-degrading microbial consortium by employing two state-of-the-art technologies. These technologies will incorporate CRISPR-Cas9-based genome editing in conjunction with molecular dynamics (MD) guided enzyme stabilization methods into one unified product. Both technologies, when used in conjunction, make for a robust and scalable way to engineer a stable, predictable, and efficient microbial system. However, most of the current techniques fail to address the unstable nature of the enzyme structure, which limits their use in real-world applications, and MD provides a detailed method for assessing enzyme stability under variable environmental stresses. MD is performed at the atomic level and can identify the regions of the target enzyme (e.g., cytochrome P450 monooxygenases) with the most structural instability, due to stress from variable pH and salinity conditions. Once an enzyme's stress point is identified, CRISPR-Cas9 is then utilized to design the specific modifications (rigidifying modifications) that enhance the rigidity of the structure while maintaining the enzyme's catalytic property or activity. Additionally, metabolic pathways containing the enzymes of interest will be engineered into the microbe consortium and then evaluated for synergistic interactions and degradation rates using simulated field conditions. The closed-loop approach uniquely combines MD with consortium engineering by providing a system where the MD-derived information will directly affect the genetic modifications of the enzyme(s) and therefore influence the optimization of the metabolic pathways/consortia. Results of experimental testing have shown a 30% increase in the resilient nature of the engineered consortia compared to non-engineered consortia (wild-type), demonstrating the potential of this approach for the construction of scalable environmental bioremediation strategies. Overall, this study significantly advances the current tech