Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

Abstract

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

Intestinal Engineered Probiotics as Living Therapeutics: Chassis Selection, Colonization Enhancement, Gene Circuit Design, and Biocontainment

“…For example, researchers have inserted bipA ( l -4,4′-biphenylalanine) into the sites of various essential proteins to generate an escape probability of less than 2 × 10 –12 within 2 weeks. They demonstrated that low BipA concentrations could support the stability of engineered microorganisms within 100 days of evolution . In addition, ligand-dependent essential gene-based auxotrophies require essential genes to function only in the presence of synthetic ligands.…”

“…218 An orthogonalized genetic central dogma strategy can overcome the deficiencies of auxotrophy. Utilizing artificial elements (XNA, 219 nonstandard amino acids (nsAA), 220 synthetic ligands, 221 etc.) and coding principles (orthogonal DNA replication, 222 transcription, 223 and translation processes) to achieve orthogonalization of the central genetic dogma, the survival or specific gene expression of engineered probiotics depends on non-natural synthetic substances to achieve biocontainment (Figure 6c).…”

“…They demonstrated that low BipA concentrations could support the stability of engineered microorganisms within 100 days of evolution. 220 In addition, ligand-dependent essential genebased auxotrophies require essential genes to function only in the presence of synthetic ligands. This method can achieve an escape rate similar to that of the nsAA strategy and has been developed in a short time at a very low cost.…”

Swapped genetic code blocks viral infections and gene transfer

“…Finally, we also sought to develop a tightly biocontained version of Ec_Syn61∆3-SL because a virus-resistant strain might have a competitive advantage in natural ecosystems due to the lack of predating bacteriophages. Synthetic auxotrophy based on the engineered reliance of essential proteins on human-provided nonstandard amino acids (nsAAs), e.g., L-4,4'biphenylalanine (bipA), offers tight, likely escape-free biocontainment that remains stable under long-term evolution [30][31][32] . Therefore, we generated a recombination deficient (i.e., ΔrecA), biocontained version of Ec_Syn61∆3-SL bearing a bipA-dependent essential adk gene and the bipA aminoacyl-tRNA synthetase/tRNA-bipA CUA system, by first performing adaptive laboratory evolution on a recA knock-out Syn61∆3, and then replacing the genomic adk copy with its bipAdependent variant 33,34 (Methods).…”

In vivo, in vitro and in silico: an open space for the development of microbe‐based applications of synthetic biology

“…We should expect to understand before long the functions that have been altered in this organism, allowing it to grow on 5‐chlorouracil while being inhibited by thymine. This entails understanding how these chassis will behave in the long run, but we can be confident that there is some hope for stability as demonstrated in stable evolution after 100 days of an E. coli strain after continuous co‐culture with mammalian cells (Kunjapur et al ., 2021).…”