Piace: Parallel Integration and Chromosomal Expansion of Biofuels Pathways in E. coli

Robust fermentation of biomass-derived sugars into biofuels demands the reliable microbial expression of underlying metabolic pathways. Previous studies have shown that plasmid-based expression systems, which may suffer from instability issues (despite selective pressure), can result in inconsistent and highly variable biofuel production rates and titers. An established mitigation strategy, Chemical Induced Chromosomal Expansion (CIChE; Tyo et al. 2009), exploits recA-mediated copy number expansion of a pathway post integration at a single chromosomal locus, and offers the gene copy numbers of plasmids while maintaining pathway stability even without the addition of antibiotics (a significant process cost-savings). Innovating beyond CIChE, we are developing PIACE (Parallel Integration and Chromosomal Expansion) to facilitate the simultaneous and independently-adjustable expansions of multiple pathways at separate chromosomal loci. PIACE offers a set of BglBrick-compatible suicide vectors, each of which site-specifically integrates into a distinct chromosomal locus via a single homologous cross-over and has high efficiency for transformation and chromosomal integration of long (> 3kb) pathways that reside in super-coiled sequence-validated plasmids. As a proof of principle for PIACE, we have integrated rfp and gfp at two separate chromosomal loci, expanded rfp and gfp independently in the presence of heat-curable plasmid pRedi2RecA, and demonstrated that both expansions are stable over 50 cellular generations in the absence of antibiotic selection pressure. We are now applying PIACE to the chromosomal stabilization and optimization of the isopentenol fuel pathway in E. coli DH1. PIACE affords an efficient, tunable method for improving the robustness, productivity, and titers of engineered metabolic pathways.