Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems. / text

Genome engineering, the deliberate alteration of an organism's genetic material, has revolutionized biotechnology and biomedical research, enabling precise modifications to DNA sequences. Among the tools developed for this purpose, site-specific recombinases (SSRs) stand out for their ability to catalyze targeted DNA rearrangements between defined target sites. The Cre/loxP system, in particular, has been widely used for conditional gene inactivation and recombinase-mediated cassette exchange, facilitating targeted DNA excision, inversion, or integration through the recognition and recombination of loxP target sites. While the inherent specificity of Cre towards the loxP target sequence has been invaluable, it also limits its application to other genomic loci of therapeutic interest. Understanding the factors that govern the enzyme’s DNA specificity opens the possibility to engineer and retarget the complex to non-native sequences, significantly broadening the range of targetable genomic loci. To address this challenge, I describe the development of a high-throughput method to quantify Cre recombination efficiency across a library of loxP-like spacer variants. This method systematically analyzes the impact of spacer sequence alterations to reveal DNA specificity determinants. Through comprehensive screening, the study identified spacer sequences that exhibit inefficient recombination by Cre, despite both full lox sites having matching spacer sequences. Directed evolution was used to enhance Cre activity on these previously 'inert' spacer sequences, generating variants with altered spacer specificity. Detailed molecular analyses, including mutational studies and molecular dynamics simulations, elucidated the structural basis for altered spacer selectivity in evolved Cre variants. The study provides mechanistic insights into the role of specific amino acid residues in determining spacer specificity and highlights the potential for the rational design of recombinases with tailored spacer preferences. Building upon this foundation, I describe the engineering of heterospecific Cre-type SSRs capable of recombining asymmetric DNA target sites. By combining two evolved Cre variants with unique half-site specificities, a functional heterotetrameric complex forms, capable of excising DNA fragments flanked by asymmetric target sequences naturally occurring in the human genome. This approach expands the applicability of SSRs and holds promise for correcting chromosomal inversions underlying genetic disorders, as demonstrated in the correction of the int1h inversion associated with hemophilia A. However, harnessing the full potential of heterospecific SSRs presents challenges, particularly concerning off-target effects resulting from the formation of undesired functional homotetrameric complexes. To mitigate these risks, I investigated strategies to render SSR monomers functionally active in heterotetrameric, but not homotetrameric complexes. Through substrate-linked directed evolution, I identified mutations that confer obligate heterospecificity, leading to safer and more precise genome engineering applications. Together, these studies highlight the transformative potential of engineered SSRs in genome editing and underscore the importance of ongoing research efforts to enhance their specificity, efficacy, and safety for therapeutic interventions and biotechnological applications. By manipulating the highly specific Cre/loxP complex to retarget different lox sequences and analyzing evolved or naturally occurring recombinase recombination specificity, we can better understand how these enzymes can be optimized for therapeutic applications. Furthermore, the ability to confer obligate heterospecificity increases the overall safety of these engineered SSRs, expanding their potential applications in genome engineering, particularly for therapeutic targets that require editing asymmetric (non-palindromic) target sites.