Evolution of novel metabolic pathways

The National Institute of General Medical Sciences (NIGMS) awarded a $216,906 Project Grant to The Regents of the University of Colorado, doing business as the University of Colorado Office of Contracts and Grants Division, to conduct research on the "Evolution of Novel Metabolic Pathways" under CFDA Program 93.859 - Biomedical Research and Research Training. The funding will support the University's efforts to study how organisms can evolve new metabolic pathways to access new carbon sources, synthesize beneficial metabolites, and break down toxins, particularly in response to environmental challenges. The research aims to identify factors that make certain evolutionary pathways for novel metabolic functions more likely to emerge. Preliminary work has already led to the identification of multiple strains of E. coli and Salmonella that have evolved alternative pathways for synthesizing an essential cofactor, pyridoxal 5'-phosphate, when the canonical pathway is disrupted. The University will further investigate the specific mechanisms and evolutionary drivers underlying these "serendipitous pathways" across different bacterial species.

Abstract

Since life first emerged, evolution of novel metabolic pathways has been essential to allow organisms to access new carbon sources, synthesize new beneficial metabolites, and break down the toxins of predators, competitors, and foods. In response to an environmental challenge or opportunity, the promiscuous activities of existing enzymes can be patched together in new combinations to create novel pathways. A promiscuous activity is a physiologically irrelevant reaction other than the one an enzyme evolved to catalyze. Pathways patched together from promiscuous activities are called "serenditous pathways," (SPS), because they rely on a fortuitous combination of promiscuous enzymes available in the proteome and mutations that increase flux to physiological levels. There are many potential chemical pathways to synthesize a given metabolite, but are all of them evolutionarily accessible? What factors make a pathway more likely to emerge by evolution? The copley lab has developed a model system to study the emergence of SPS by deleting pdxb, a gene required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP). PLP is only needed in small amounts, so even a little flux through an underground pathway can provide enough PLP for cells to divide, albeit slowly. Even slow growth enables evolution, and in only ~150 generations, multiple strains of pdxb e. Coli were able to restore wild-type levels of PLP synthesis and robust growth. These evolved mutants synthesize PLP by an SP that recruits sera, serc, and thrb to perform promiscuous reactions. While only one strain was fully characterized (JK1), the commonalities of the specific genes that were mutated in eight parallel lineages suggest that most, if not all, use the same SP to make PLP. We expect that different species will produce different solutions to deletion of pdxb because their reservoir of promiscuous activities differs. The copley lab has evolved pdxb mutants of several bacteria, including s. Enterica. In aim 1, i will determine how evolved strains of pdxb s. Enterica make PLP. Preliminary results reveal that the previously characterized SP is not used in some of the evolved strains. I will determine whether the abilities of orthologous pairs of enzymes to catalyze newly important promiscuous reactions differ in ways that favor one SP over another in different bacteria. What makes the pathway in JK1 the most likely to emerge in e. Coli? To address this question, i will identify other possible PLP synthesis pathways by deleting sera or thrb in pdxb e. Coli to remove the possibility of the solution in JK1. Successful evolution of sera pdxb and thrb pdxb e. Coli mutants in minimal medium demonstrates that there are alternative solutions to pdxb deletion in e. Coli. In aim 2, i will determine how these evolved mutants make PLP and the mutations that drive PLP synthesis. To understand why the pathway in JK1 evolved repeatedly, i will compare the efficiency, mutational accessibility, and fitness trade-offs of driver mutations of PLP synthesis in the evolved double mutant strains with those of JK1.