Random Chromosomal Integration and Screening Yields <i>E. coli</i> K-12 Derivatives Capable of Efficient Sucrose Utilization

Abstract

Chromosomal expression of heterologous genes offers stability and maintenance advantages over episomal expression, yet remains difficult to optimize through site-specific integration. The challenge has in large part been due to the variability of chromosomal gene expression, which has only recently been shown to be affected by multiple factors, including the local genomic context. In this work we utilize Tn5 transposase to randomly integrate a three-gene <i>csc</i> operon encoding nonphosphotransferase sucrose catabolism into the <i>E. coli</i> K-12 chromosome. Isolates from the transposon library yielded a range of growth rates on sucrose as the sole carbon source, including some that were comparable to that of <i>E. coli</i> K-12 on glucose (μ_max = 0.70 ± 0.03 h^-1). Narrowness of the growth rate distributions and faster growth compared to plasmids indicate that efficient <i>csc</i> expression is attainable. Furthermore, enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that sucrose catabolism is not limiting to cellular growth. We also show that transduction of a <i>csc</i> fast-growth locus into an isobutanol production strain yields high titer (7.56 ± 0.25 g/L) on sucrose as the sole carbon source. Our results demonstrate that random integration is an effective strategy for optimizing heterologous expression within the context of cellular metabolism for both fast growth and biochemical production phenotypes.