Mutations Elevate an Underground Pathway to a Physiologically Relevant Protopathway.

Underground metabolic pathways-leaks in the metabolic network caused by promiscuous enzyme activities and nonenzymatic transformations-can provide the starting point for emergence of novel protopathways if a mutation or environmental change increases flux to a physiologically significant level. This early stage in pathway evolution, in which promiscuous enzymes are still serving their native functions and proper regulation has not yet emerged, is typically hidden from our view. We previously used laboratory evolution to evolve a novel four-step protopathway in ΔpdxB E. coli, which lacks an enzyme required for synthesis of pyridoxal 5'-phosphate (PLP). By sequencing population genomic DNA from samples archived during the evolution experiment, we have identified mutations that rose and fell in abundance in the population leading to JK1, the dominant clone after 150 population doublings. We have identified the order in which the four mutations arose in JK1 and the physiological effect of each mutation. The first mutation increases the rate of PLP synthesis. The second mutation did not impact PLP synthesis but rather created a cheater that thrived in the population by scavenging nutrients released from the fragile parental cells. Notably, the dominant lineages at the end of the experiment all derived from this cheater strain. The third mutation in JK1 destroyed a PLP phosphatase, which preserves precious PLP. Finally, the fourth mutation improved growth in glucose after the PLP synthesis problem had been solved. Together, these mutations resulted in restoration of PLP synthesis and a 32-fold increase in growth rate.