(585ar) Long-Term Adaptive Evolution of Amberless Escherichia coli strains Reveals Selective Mutations in Translation Machinery | AIChE

(585ar) Long-Term Adaptive Evolution of Amberless Escherichia coli strains Reveals Selective Mutations in Translation Machinery

Authors 

Kunjapur, A. M. - Presenter, Harvard Medical School
Wannier, T. M., Harvard Medical School
Desai, M. M., Department of Organismic and Evolutionary Biology
Church, G. M., Harvard University
Advances in genome engineering and DNA synthesis technologies have facilitated large-scale recoding of bacterial genomes for objectives that include non-standard amino acid incorporation, multi-virus resistance, and biocontainment. An example is the recoded Escherichia coli strain C321.∆A, in which all instances of UAG (“amber”) stop codons were substituted for UAA and release factor 1 (RF1) was deleted. Unfortunately, C321.∆A exhibits a significant growth defect, which is particularly severe in industrially relevant glucose minimal media (Glu-MM), where its growth rate is nearly half that of its parental strain (ECNR2). To improve strain fitness and concurrently shed light on recoded genome evolution, we performed adaptive evolution of C321.∆A strains for more than 1,000 generations each in 14 independent replicate populations in Glu-MM. Average growth rates of evolved recoded populations significantly exceed the growth rates of the ancestral C321.∆A and ECNR2 strains in Glu-MM and in some lineages approach the growth rate of ECNR2 in LB media. We used whole genome sequencing of two clones each from population to determine statistically significant mutations and subsequently reconstructed them in ancestral strains using multiplex automatable genome engineering (MAGE). Overall, we observe that recoding introduced a burden to E. coli cellular translation machinery under a wide range of industrially relevant defined media, but that point mutations in release factor 2 (RF2) known to provide increased activity on UAA codons are selected for and recover much of the fitness. Furthermore, we observe that natural selection exhibits a variety of mechanisms to correct the most detrimental off-target mutations introduced during engineering of the recoded strain, including the use of premature termination codons in essential genes. Our findings demonstrate that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional rounds of genome engineering that may introduce further unintended mutations.