Recoding of synonymous genes to expand evolutionary landscapes requires control of secondary structure affecting translation

Abstract

Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability.Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles. Although synonymous, both alleles yielded less IntI1 protein and were less active in recombination assays than intI1. We spliced the three alleles and mapped the activity decrease to the beginning of alternative sequences. Mfold predicted the presence of more stable secondary structures in the alternative genes.Using synonymous mutations, we decreased their stability and recovered full activity.Following a design-build-test approach, we have now updated ELP to consider such structures and provide streamlined alternative sequences. Our results support the possibility of modulating gene activity through the ad hoc design of 5′ secondary structures in synthetic genes.DNA design, integrons, mRNA secondary structure, optimized gene, synonymous genes, synthetic biology

Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions

“…Here, the prevalence of 5' UTR mutations is high enough to suspect that this region is non-optimal. We assessed if the 5' region of the transcript could be interfering with transcription of the alt2 8mut gene (as we found in 42 ), but saw no differences in folding energies between WT and mutated 5'-UTRs (-6.1 kcal mol -1 in all cases). This, combined with the linkage of UTR mutations to substitutions that appear alone in other codes, led us to consider UTR mutations in alt2 8mut as hitchhiking events.…”

“…and alt2 l.e. , showed recombination activities comparable to those of intI1 and were used as a starting point for directed evolution experiments 42 .…”

“…Using the adaptive landscape metaphor -in which genotype space is plotted on the x and y axis and fitness on the z vertical axis-our data suggest that that the peaks representing the ancestral and modern functions are connected by some form of ridge that may facilitate the transition from one to the other, fostering innovation. ELP has been recently updated to avoid the formation of secondary structures in the 5' region of the gene that interfere with transcription 42 .…”

“…Notably, in the second round of enrichment cycles, the number of alt2 8mut alleles bearing mutations in the 5'UTR of the gene is high enough to be potentially interpreted as if this region was non-optimal. We hence explored the possibility that the 5' folding of the transcript was interfering with transcription of the alt2 8mut gene (as we saw in 42 ), but folding energies were identical between WT and mutated 5' UTRs (-6.1 kcal mol -1 in all cases). Here, using the evolutionary rationale, and on the basis of frequency of appearance,…”

“…To explore this, in this work we have evolved the modern integrase IntI1 towards its ancestral function, the recombination of ds-substrates. With a succession of in vitro evolution experiments, using synonymous genes to extend the range of explored mutations 41,42 , we have identified mutations increasing the ancestral function ( Fig. 1d).…”