Engineering gene overlaps to sustain genetic constructs in vivo.

Arnaud Gutierrez,Edward L Braun,Aude Bernheim,Chantal Lotton,Antoine Frénoy,Ariel B Lindner,Antoine L Decrulle,Thomas A Meiller-Legrand

doi:10.1371/journal.pcbi.1009475

Abstract

Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.

Highlights

Synthetic biology attempts to use engineering principles to manipulate and reprogram living organisms [1]
This permits the existence of gene overlaps, often observed in microbial genomes, where two different proteins are encoded on the same piece of DNA, but in different reading frames
Gene overlaps are classically considered an obstacle for both evolution and genetic engineering, as mutations in overlapping regions likely have pleitrotropic effects on several genes

Summary

Introduction

Synthetic biology attempts to use engineering principles to manipulate and reprogram living organisms [1]. This could hold promise for many of the world’s challenges, for example with microbes engineered for bioremediation [2,3,4] and drugs or fuel biosynthesis [5,6,7]). Unforeseen evolution of genetic circuits released in the wild raises strong concerns. Containment and control of engineered organisms outside of the laboratory relies on sophisticated gene circuits that should be made as evolutionary-proof as possible [17,18,19]

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Discussion

Conclusion