Multicopy Suppression Underpins Metabolic Evolvability

Abstract

Our understanding of the origins of new metabolic functions is based upon anecdotal genetic and biochemical evidence. Some auxotrophies can be suppressed by overexpressing substrate-ambiguous enzymes (i.e., those that catalyze the same chemical transformation on different substrates). Other enzymes exhibit weak but detectable catalytic promiscuity in vitro (i.e., they catalyze different transformations on similar substrates). Cells adapt to novel environments through the evolution of these secondary activities, but neither their chemical natures nor their frequencies of occurrence have been characterized en bloc. Here, we systematically identified multifunctional genes within the Escherichia coli genome. We screened 104 single-gene knockout strains and discovered that many (20%) of these auxotrophs were rescued by the overexpression of at least one noncognate E. coli gene. The deleted gene and its suppressor were generally unrelated, suggesting that promiscuity is a product of contingency. This genome-wide survey demonstrates that multifunctional genes are common and illustrates the mechanistic diversity by which their products enhance metabolic robustness and evolvability.

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

“…Hits were obtained from every class of toxic compound tested ( Table 1). The hit rate (resistance to 36% of all toxins) was even higher than in our previous study (23), where only 20% of single-gene knockout strains were rescued by noncognate ASKA ORFs. As hypothesized previously (38), we have shown that a bacterium from a nonclinical environment (in this case, a laboratory strain of E. coli) can nevertheless possess a significant reservoir of latent resistance determinants.…”

“…To avoid this source of bias, we used a significantly lower concentration of IPTG (50 μM) to induce protein expression. This amount is sufficient for derepression of the pCA24N T5-lacO promoter, but minimizes fitness differences associated with overexpression of different ORFs (20,23).…”

“…The plasmids of the library were pooled and used to transform 104 conditionally auxotrophic, single-gene deletion strains. Twenty-one of the auxotrophies were specifically suppressed by the overexpression of noncognate E. coli genes (23). The implication was that many proteins are multifunctional; however, the ability of proteome-wide promiscuity to drive an organism's adaptation to new environments remained unknown.…”

Evolving promiscuously

“…The present study, as well as a similar study by Patrick et al (23), fills an important gap and comprehensively shows that multifunctionality and promiscuity in proteins are common and that gene overexpression can allow access to this reservoir of new activities. The multicopy suppression approach provides a powerful methodology to access this reservoir, and it is likely to be an important future tool to define the "promiscuome" in various organisms.…”

A protein constructed de novo enables cell growth by altering gene regulation

“…This suggestion can be tested both genetically and biochemically. The genetic studies were first reported by Patrick et al (17), who conducted a large-scale study to test whether chromosomal deletions of single genes in E. coli could be rescued by other E. coli genes overexpressed from a plasmid. In the case of the ΔserB auxotroph, Patrick et al (17) found that overexpression of HisB rescued the deletion.…”