Reinforcing Synthetic Biology Against Evolutionary Failure

Unwanted or unintended evolution can rapidly degrade the performance of complex DNA-encoded devices and complicate large-scale strain engineering efforts. Acinetobacter baylyi ADP1 has been proposed as a new host organism for synthetic biology and metabolic engineering because it is naturally transformable. To understand the major causes of genetic instability in this "undomesticated" bacterium, we performed laboratory evolution experiments with ADP1 cultures under conditions where either adaptive mutations or genetic drift dominated. By analyzing whole-genome sequencing data from evolved strains using the breseq software program developed by our research group, we discovered that IS1236 transposable elements were largely responsible for the loss of several key traits during laboratory propagation, including competence for DNA uptake. To stabilize the ADP1 genome, we engineered an ISx strain in which all copies of IS1236 — the only native transposable element in ADP1 — were deleted from the chromosome. The ISx ADP1 strain exhibits a greater reduction in overall spontaneous mutation rates than predicted from the incidence of IS1236 insertions and an unexpected increase in transformation efficiency. These results may be due to the decreased accumulation of extracellular DNA from lysed cells in ISx compared to wild-type ADP1 cultures. Finally, we have demonstrated a generalizable directed evolution approach for discovering mutations that increase the evolutionary lifetime of a DNA device encoding a costly engineered function. Using this strategy, we isolated E. coli variants that are able to maintain fluorescent protein expression from a plasmid for more than twice as long as wild-type hosts. Genome sequencing of these variants found that they have mutations that likely result in lower mutation rates that would stabilize maintenance of any DNA sequence in these hosts. Together, these studies show that it is possible to engineer and evolve more genetically reliable host organisms to effectively prevent evolutionary failure in synthetic biology.