Enzyme Recruitment in Metabolic Plasticity and Evolvability

Environmental change drives the evolution of new metabolic pathways. A key step in metabolic plasticity and innovation is enzyme recruitment — when an existing catalyst is enlisted to provide a new function. Enzyme recruitment fuels the evolution of new metabolism and facilitates real-world, health-relevant processes such as antibiotic resistance and pollutant remediation. For example, a degradative pathway for the toxic herbicide atrazine recently emerged via recruitment of two hydrolases from melamine and cyanurate catabolism. Enzyme recruitment is powered by the inherent functional promiscuity of modern enzymes, which allows them to transform multiple substrates and perform multiple chemical reactions. Indeed, a single bacterial proteome is estimated to contain thousands of promiscuous enzymatic activities “lying in wait” for future recruitment. Such widespread promiscuity suggests a multitude of potential solutions to new metabolic challenges. Yet the solution selected by evolution remains unpredictable, in part, because enzyme recruitment occurs amidst a complex, dynamic physiological backdrop. Indeed, the factors that facilitate or constrain the recruitment process remain unknown, representing a major gap in knowledge. This proposal’s objective is to use a combination of classical biochemistry, adaptive laboratory evolution and experimental -omics methods to understand how specific organismal features impact recruitment outcomes. Why is one enzyme’s recruitment favored over another? Investigating this question requires a model system in which multiple promiscuous enzymes are known to provide multiple solutions to the same metabolic challenge. We have identified three distinct model systems in Escherichia coli that will be leveraged to investigate the genetic, biochemical, and cellular factors that impact enzyme recruitment during adaptive laboratory evolution. The goal of this proposal is to investigate three specific questions related to factors governing enzyme recruitment outcomes: (1) How does local genomic structure and/or context shape recruitment? (2) How do functional pleiotropic constraints alter the trajectory and/or outcome of the recruitment process? (3) How does metabolic plasticity enable recruitment of an enzyme that provides an indirect metabolic bypass via synthesis of a “new-to-nature” metabolic intermediate? The results of the proposed work will be impactful to human health, as enzyme recruitment drives events such as bioremediation of anthropogenic chemicals and drug inactivation. This study will also impact synthetic biology, where a major goal is to design new metabolic pathways from extant promiscuous enzymes. Project Number: 1R01GM157172-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Brian Miller | Institution: FLORIDA STATE UNIVERSITY, TALLAHASSEE, FL | Award Amount: $616,401 | Activity Code: R01 | Study Section: Genetic Variation and Evolution Study Section[GVE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11224276