Accelerating Genome Engineering in E. coli: From Single Step Pathway Integration to Trackable Genomic Diversity

Engineering the E. coli genome for a broad range of biotechnology applications commonly require two tasks: the ability to import heterologous functions stably into the host genome and to search the genome space efficiently for beneficial mutations affecting the phenotype of interest. We facilitated the process of importing new functions by developing a highly efficient CRISPR-based strategy that allows integration of entire metabolic pathways. This strategy accomplishes integration in a single step without requirement of selectable markers, maintaining high efficiency in distinct genomic positions. Using this strategy, we integrated a 10 kb construct to implement isobutanol production in a single day in E. coli with 50% efficiency. Such capability will increase the throughputs of testing metabolic pathways for interesting biological functions. To efficiently search the genome for beneficial mutations, we previously developed a technology (CREATE) that allows generation of thousands of rationally designed genomic mutations using multiplexed oligonucleotide synthesis and cloning. The fitness of each mutant can then be tracked in parallel using the CREATE plasmid as a trans-acting barcode. We applied this technology to introduce more then 50,000 mutations across the genome and identified enriched mutants in libraries spanning from a single protein to whole genome scale. We are now improving the tracking strategy by introducing a barcode recording element into the CREATE plasmid. As a result, each designed genomic edit is accompanied by the integration of a small barcode into a neutral site in the genome, maintaining a log of previously introduced mutations in the cell. We demonstrated high correlation between genomic edits and barcode recording. This technology allows trackable generation of combinatorial mutants as well as generation of portable libraries that can be shuttled to other organisms. Together, both of these technologies will facilitate genome engineering in E. coli and accelerate the process of generating interesting strains from both a scientific and biotechnology perspective.