Mutations Elevate an Underground Pathway to a Physiologically Relevant Protopathway.

Area of Science:

• Metabolic Engineering
• Evolutionary Biology
• Microbial Genetics

Background:

• Metabolic pathway evolution can originate from 'leaky' metabolic networks.
• Promiscuous enzyme activities and nonenzymatic reactions can form novel protopathways.
• Early pathway evolution stages are typically unobservable.

Purpose of the Study:

• To identify mutations and their order during laboratory evolution of a novel metabolic pathway.
• To understand the physiological effects of these mutations in E. coli.
• To characterize the emergence of a cheater strain during evolution.

Main Methods:

• Laboratory evolution of ΔpdxB E. coli lacking pyridoxal 5 '-phosphate (PLP) synthesis.
• Genomic DNA sequencing of archived population samples.
• Analysis of mutation order and physiological impact on growth.

Main Results:

• Identified four sequential mutations leading to a novel four-step protopathway.
• The first mutation enhanced PLP synthesis; the second created a nutrient-scavenging cheater strain.
• Subsequent mutations eliminated a PLP phosphatase and improved glucose metabolism, restoring PLP synthesis and increasing growth 32-fold.

Conclusions:

• Underground metabolic pathways can serve as a source for novel pathway evolution.
• Evolutionary trajectories can involve the emergence and dominance of cheater strains.
• Sequential mutations can restore essential metabolic functions and significantly enhance growth rate.