Genetic Context Significantly Influences the Maintenance and Evolution of Degenerate Pathways

Understanding the evolution of novel physiological traits is highly relevant for expanding the characterization and manipulation of biological systems. Acquisition of new traits can be achieved through horizontal gene transfer (HGT). Here, we investigate drivers that promote or deter the maintenance of HGT-driven degeneracy, occurring when processes accomplish identical functions through nonidentical components. Subsequent evolution can optimize newly acquired functions; for example, beneficial alleles identified in an engineered Methylorubrum extorquens strain allowed it to utilize a “Foreign” formaldehyde oxidation pathway substituted for its Native pathway for methylotrophic growth. We examined the fitness consequences of interactions between these alleles when they were combined with the Native pathway or both (Dual) pathways. Unlike the Foreign pathway context where they evolved, these alleles were often neutral or deleterious when moved into these alternative genetic backgrounds. However, there were instances where combinations of multiple alleles resulted in higher fitness outcomes than individual allelic substitutions could provide. Importantly, the genetic context accompanying these allelic substitutions significantly altered the fitness landscape, shifting local fitness peaks and restricting the set of accessible evolutionary trajectories. These findings highlight how genetic context can negatively impact the probability of maintaining native and HGT-introduced functions together, making it difficult for degeneracy to evolve. However, in cases where the cost of maintaining degeneracy was mitigated by adding evolved alleles impacting the function of these pathways, we observed rare opportunities for pathway coevolution to occur. Together, our results highlight the importance of genetic context and resulting epistasis in retaining or losing HGT-acquired degenerate functions.