Chemically Inducible Chromosomal Evolution (CIChE) for Multicopy Metabolic Pathway Engineering.

Chemically inducible chromosomal evolution (CIChE) was developed for stable multicopy chromosomal integration of heterologous genes. In this technique, flanking an antibiotic selection marker and a gene of interest with identical regions of homology permits gene duplication via recA mediated homologous recombination. A strong selective pressure for gene duplication can be applied by increasing antibiotic concentration, and in a week's time one can create a set of strains with a wide range of cassette copy numbers (upward of 20×), which can be made stable by deletion of recA. Herein, we describe a generalized workflow for this methodology.