Abstract
Chromosomal integration of recombinant genes is desirable compared to expression from plasmids due to increased stability, reduced cell-to-cell variability, and the elimination of antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integrations followed by high-throughput screening. We demonstrate multiplexed pathway gene integration and optimization of expression levels for isobutanol production in Escherichia coli. The integrated strains could, with significantly lower expression levels than observed from multicopy plasmid-based expression, produce high titers (up to 10.0 ± 0.9 g/L isobutanol in 48 h) and yields (up to 69 % of the theoretical maximum). Close examination of pathway gene 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. This new method for multiplexed pathway gene integration and expression optimization could be readily extended and applied to a wide range of pathways and chassis to create robust and efficient production strains.