Gene sharing during enzyme recruitment reveals distinct adaptive strategies including massive, sustained duplication without divergence

Abstract Organisms expand their metabolism by repurposing enzymes to perform new reactions. To be repurposed, an enzyme must balance its original and new functions if both contribute to fitness. If an enzyme cannot balance its functions, another candidate may take its place. Here, we used adaptive evolution on a glucokinase-deficient Escherichia coli containing four promiscuous surrogates to investigate enzyme recruitment when the preferred candidate, N -acetyl-D-mannosamine kinase (NanK), is under selective pressure to maintain its original function. We find that NanK is still recruited to restore glycolysis under conditions requiring both functions via two distinct mechanisms that leave native activity largely unaltered. In one mechanism, small-scale gene amplification precedes the appearance of two non-synonymous mutations in nanK that increase glucokinase activity but have little or no effect on N -acetyl-D-mannosamine kinase activity. In another mechanism, recruitment occurs via amplification of a ∼1000 base pair fragment that narrowly encompasses nanK and reaches copy numbers as high as 127. Despite maintenance of amplification for hundreds of generations, we observe no persistent mutations in any nanK duplicate at the level of resolution provided by 75X whole genome sequencing coverage. Our results demonstrate that gene sharing can alter the trajectory but not necessarily prevent the recruitment of a preferred promiscuous candidate during adaptive evolution when other, seemingly equal candidates are available. Our findings also reveal that evolution by Innovation-Amplification-Divergence may only be facilitated at moderate levels of gene amplification, and hindered by massive amplification, as increased gene copy number diminishes returns of individual adaptive point mutations. Classification: Evolution, Biochemistry Significance The Innovation-Amplification-Divergence model posits that single multifunctional enzymes evolve into multiple monofunctional enzymes through two events: First, selective pressure on a multifunctional enzyme leads to a duplication of the gene encoding that enzyme in an organism’s genome. Second, the duplicate gene copy can freely accumulate mutations that enhance one of the encoded enzyme’s functions while the original copy can accumulate mutations that enhance the enzyme’s other encoded function. Intriguingly, our results reveal sequence divergence only in cells that experience mild amplification, and no divergence in cells that experience massive amplification. This suggests that sequence divergence may be suppressed above a certain number of gene copies, providing a new perspective on a widely accepted theory of evolution. Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.