Development of a transposable elements free escherichia coli for the efficient production of recombinant therapeutic proteins

Abstract

Genome stability and integrity of a host strain are critical for the production of high quali-ty bioproducts from industrial microorganisms. However, the genomes of microorgan-isms are littered with transposable elements (TEs) called “jumping genes”, which are re-combinogenic factor causing genome instability and genetic variations. These elements cause many genetic rearrangements such as inversion, deletion, duplication and transposi-tion, thus causing adverse effects such as production of unwanted by-products and non-homogeneous products and masking expression of genes. To minimize these harmful ef-fects of TEs of Escherichia coli, we developed a TEs-free strain, in which all of TEs in-cluding insertion sequences (IS), transposons, phages and cryptic phages, were removed from E. coli MG1655 genome by a rapid markerless deletion system. Here, we compared the expression profiles of recombinant protein such as tumor necrosis factor-related apop-tosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and TEs-free strain. The hopping of ISs (IS1, IS3 or IS5) into the TRAIL and BMP2 genes oc-curred at the rate of $~10^{-8}/gene/h$ in MG1655 whereas no IS hopping was observed in TEs-free strain. Even though IS hopping occurred very rarely $(10^{-8} /gene/h)$, the IS-inserted TRAIL and BMP2 clones became dominant (~52 % of total population) in 28 h after fer-mentation because of its rapid growth, significantly reducing production of recombinant therapeutic proteins in batch fermentation. Our observation clearly indicates that TEs jumping is detrimental to the production of recombinant enzymes, strongly emphasizing the necessity of developing a TEs-free host strain. Thus, our TEs-free strain will be use-ful as a robust and suitable host for the production of recombinant therapeutic proteins.