Mobile Translation Systems Generate Genomically Engineered Escherichia coli Cells with Improved Growth Phenotypes

Abstract

Cellular translation is responsible for the synthesis of proteins, a highly diverse class of macromolecules that form the basis of biological function. In Escherichia coli, harnessing and engineering of the biomolecular components of translation, such as ribosomes, transfer RNAs (tRNAs), and aminoacyl-tRNA synthetases, has led to both biotechnology products and an expanded genetic code. However, the engineering potential of molecular translation is hampered by the limited capabilities for rapidly sampling the large genomic space necessary to evolve well-coordinated synthetic translation networks inside cells. To address this limitation, we developed a genome engineering method inspired by the action of mobile genetic elements termed mobilization. Mobilization utilizes the stochastic action of the recombinase flippase (FLP) to generate up to ∼400 million genomic insertions, deletions, or rearrangements at flippase recognition target sites per milliliter of culture per OD in living E. coli cells. As a model, we applied our approach to evolve faster-growing E. coli strains living exclusively off genomically expressed tethered ribosomes. In an iterative "pulse-passaging scheme," we generated genomic libraries of cells via induction of FLP recombinase (pulse) followed by passaging the population without induction of FLP to enrich the resulting population for cells with higher fitness. We observed large structural genomic diversity across these cells, with the fastest growing strains exhibiting a 71% increase in growth rate compared to the ancestral strain. We anticipate that both these strains and the mobilization method will be useful tools for synthetic biology efforts to engineer translation systems.

A self‐propagating, barcoded transposon system for the dynamic rewiring of genomic networks

“…We anticipate our genome evolution platform playing an important role in screening efforts to improve strains for industrial and therapeutic applications. As an example, fitness‐conferring mutations that improve growth on diverse feedstocks (Mundhada et al , 2017 ; Gowland & Jewett, 2022 ) could be identified through the transposon‐mediated rewiring of GRNs. Similar mutations could then be rationally introduced without the transposon components to establish a final, stable strain.…”

“…A major challenge in directed genome evolution is our limited ability to dynamically track the mutations across a genome and mechanistically link these changes to the emergent phenotype (Luo et al , 2018 ). As a result, many of the processes generating genome‐wide structural heterogeneity in cells—for example, the duplication, translocation, transposition, or recombination of DNA segments (Kirchberger et al , 2020 )—have yet to be adapted into engineering approaches for fitness landscape exploration during laboratory evolution (Ma et al , 2019 ; Gowland & Jewett, 2022 ).…”

Biomimetic Vesicles with Designer Phospholipids Can Sense Environmental Redox Cues

“…C ell-like supramolecular assemblies, protocell models, and semisynthetic organisms can be employed for studying origins of cellular life, 1 membrane biogenesis and regulation, 2 cell development, 3 differentiation, 4 and signaling, 5 for producing biofuel, 6,7 or hormones 8 , for detecting metabolites, 9 pathogens, 10 or agricultural pollutants, 11 and for building cytomimetic tissues in regenerative medicine, 12 and targeted drug delivery. 13,14 A central goal in building cell-like materials is to configure the minimal requirements for cellular processes, which include, among others, sensing "extracellular" environment.…”

“…Cell-like supramolecular assemblies, protocell models, and semisynthetic organisms can be employed for studying origins of cellular life, membrane biogenesis and regulation, cell development, differentiation, and signaling, for producing biofuel, , or hormones, for detecting metabolites, pathogens, or agricultural pollutants, and for building cytomimetic tissues in regenerative medicine, and targeted drug delivery. , …”