Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

Abstract

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the ‘weak-link’ enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

Evolution of new enzymes by gene duplication and divergence

“…For example, the gene encoding E383A ProA (ProA*), which has a weak N ‐acetyl glutamyl phosphate reductase activity (Fig. 5), amplified rapidly during evolution of E. coli lacking N ‐acetyl glutamyl phosphate reductase (ArgC) on glucose + proline [79]. (Proline was added so that ProA* was free to evolve toward a neo‐ArgC in the absence of a requirement for its original function.)…”

“…Figure 20 shows amplification and later deamplification of proA* in one evolved lineage. The initial increase in growth rate was due to a mutation that corrects the known pyrimidine synthesis deficiency in K12 strains of E. coli [79‐81] . Following this mutation, a 41‐kb region surrounding proA* amplified to six copies.…”

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

“…Mutational target size is likely to explain why adaptive mutations often occur outside the gene whose product is being asked to perform a different function. When Escherichia coli is experimentally evolved to use an enzyme that normally participates in proline synthesis, proline mutant A (ProA), to catalyze a similar reaction in arginine synthesis, most of the adaptive mutations are in other genes of arginine synthesis pathway, not in ProA [63]. Mutations outside the focal gene are also found when proteins are asked to perform the same function in a novel cellular environment.…”

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

“…We amplified and cloned the target region with the SPM and sufficient unmutated end homology, which was used for recombination, into a plasmid and develop error‐prone PCR libraries (Fig ). We amplified and co‐transformed the linear donor error‐prone PCR library with the gRNA‐encoding plasmid in cells with active Cas9 and lambda Red recombination proteins, encoded by a single plasmid (preprint: Morgenthaler et al , ), to integrate the mutated donor onto the genome. The plasmid encodes cas9 expressed under the constitutive Pro1 promoter (Davis et al , ) and the lambda Red recombination genes exo , beta, and gam expressed using the heat‐inducible pL promoter, induced by heat shock at 42°C (Yu et al , ).…”

“…For our initial experiments, we used the pAM053 plasmids previously published (preprint: Morgenthaler et al , ), encoding cas9 expressed under the weak pro1 promoter (Davis et al , ) and lambda Red recombination proteins expressed under the lambda phage pL promoter, controlled by the temperature‐sensitive cI857 repressor. The cas9 +lambda Red recombination+ mutL‐E32K plasmid was constructed by cloning the mutL‐E32K gene in the lambda Red recombination operon of pSIM5 using the primers described previously (Nyerges et al , ).…”