Synthetic overlapping genes stabilize genetic systems.

Overlapping genes-wherein two different proteins are translated from alternative reading frames of the same DNA sequence-provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapping gene. However, creating overlapping gene pairs is challenging, as it requires redesigning both protein products to accommodate overlap constraints. Here, we present a new "overlapping, alternate-frame insertion" (OAFI) method for creating synthetic overlapping genes by inserting an "inner" gene, encoded in an alternate frame, into a flexible region of an "outer" gene. Using OAFI, we create new overlapping gene pairs of genetic reporters and bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain function despite redesign, with translation of the inner gene influenced by its overlap position in the outer gene. Importantly, we show that, despite these inner gene sequences not contributing to outer gene function, selection for the outer gene alters the permitted inactivating mutations in the inner gene, and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment. IMPORTANCE: Genetically engineered microbes promise to improve human health and help solve global climate crises. However, the widespread adoption of these microbes is often hindered by genetic instability caused by mutations and by the unpredictable spread of synthetic genes in the environment. We present a simple but effective method for creating synthetic overlapping genes to stabilize genes against mutations and prevent their spread in the environment. This method is broadly useful for constructing stable genetically engineered microbes and studying how they evolve in the environment.