Abstract
Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.