(500g) Development of Non-Model Microbes As Chassis Organisms for Bioconversion

Many non-model microbes have native phenotypic advantages over model organisms like E. coli and S. cerevisiae, such as the ability to catabolize polymeric biomass feedstocks, extreme tolerance to various stressors, or high flux through complex metabolic pathways. Unfortunately, these non-model microbes typically lack the genetic tools that would enable further modifications and metabolic engineering. We routinely improve transformation efficiency or demonstrate first-of-kind transformation in phylogenetically diverse bacteria by evading restriction systems and then use in-house libraries of genetic parts to enable gene deletion and heterologous expression. Beyond initial transformation technologies, we have also developed a high efficiency DNA integration tool called SAGE (Serine-recombinase Assisted Genome Engineering) using site-specific DNA recombinases. This system uses a suite of orthogonal site-specific DNA recombinases to enable rapid, stable, and iterative insertion of heterologous DNA into the chromosome of the target host. This allows rapid screening of both genetic parts (e.g., promoters, terminators, insulators, reporter genes) and metabolic pathways in diverse organisms. Because of the high efficiency, these recombinases also enable combinatorial libraries to be constructed in vivo for pathway optimization. Together, these approaches allow the rapid development of non-model microbes into bioengineering platforms for the conversion of plant biomass and waste feedstocks like plastics into the next generation of fuels and chemicals.