(281e) Optimization of Pathway Gene Expression from the Escherichia coli Chromosome By High-Throughput Screening of Random Site Integration Libraries

Chromosomal integration of recombinant genes is desirable compared to expression from plasmids due to increased stability and elimination of the need for antibiotics for ensuring plasmid maintenance. The chromosomal location in which a gene is integrated has a significant impact on its expression level. Here, we present an approach for tuning pathway gene expression levels by integrating them into the genome in random locations by transposon integration, followed by high-throughput screening to identify those with optimal expression levels for production of a target molecule. For high-throughput screening we employ Syntrophic Co-culture Amplification of Production phenotype (SnoCAP) to convert the production phenotype into highly screenable growth phenotypes [1]. We demonstrate the approach by optimization of pathway gene expression for isobutanol production in Escherichia coli, generating strains that can stably produce isobutanol at high titers without addition of antibiotics. We compare the expression levels in various library members exhibiting differing production levels in order to elucidate factors that lead to superior production.

[1] Saleski, T. et al. “Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries.” In revision. bioRxiv: https://doi.org/10.1101/518639 (2019).