High Resolution Mapping of Mutations to Accelerate Pathway Engineering in E. coli

A fundamental goal of synthetic biology is to develop new paradigms for forward engineering of biological systems. Existing approaches are relatively straightforward to apply in monogenic traits, but the complexity underlying multigenic phenotypes requires innovative synthetic biology solutions. Traditionally, complex phenotypes have been manipulated via directed evolution strategies across thousands of generations, yielding adapted strains but poor knowledge on the genotype-phenotype relationship.

With recent advances in DNA reading and writing capabilities, novel approaches can be developed that allow us to more efficiently and rationally explore the genomic sequence space to engineer desirable traits. We recently developed a technology (CRISPR EnAbled Trackable genome Engineering or CREATE) that couples array-based DNA synthesis with CRISPR-Cas9 editing to generate 100,000s of designed edits in multiplex that can be tracked in parallel with single nucleotide resolution. Using CREATE, complex multigenic phenotypes can be interrogated to individually map the fitness contribution of thousands of mutations in parallel.

Here, we apply CREATE to demonstrate a new engineering paradigm on a pathway scale in E. coli. Specifically, the aspartate superpathway, containing genes for biosynthesis of the amino acids lysine, threonine and methionine, has been studied for decades for biotechnology applications as well as targets for development of specific antimicrobial drugs. To overcome the intricate feedback regulation of these genes, traditional approaches relied on random mutagenesis or adaptive evolution in the presence of end product analogs, such as S-2-aminoethyl-L-cysteine (AEC) for lysine. As such, the explored sequence space is relatively small and the learning step is limited to a few winners that dominate the selection.

We designed 16300 mutants across genes involved in lysine biosynthesis, degradation, transport and regulation, and challenged the library with the antimetabolite AEC. By mapping the fitness contribution of each of these mutants in parallel, CREATE allows us to explore the sequence space in this pathway at orders of magnitude more depth than previous approaches. We identified several mutants conferring high levels of resistance to AEC in mechanistically different ways, improving our knowledge on allosteric deregulation and antimicrobial resistance routes in this pathway. CREATE should substantially accelerate pathway engineering and improve our knowledge on pathway evolution and regulation.