(288e) Chemically Inducible Chromosomal Evolution (CIChE): Increasing Genetic Stability by Avoiding the Pitfalls of Plasmids
Metabolic engineering offers the potential to produce robust microbes capable of efficient overproduction of fuels and chemicals for sustainable manufacturing using renewable resources. The great majority of metabolic engineering strategies presently rely on plasmids for heterologous pathway expression. These plasmids have fundamental genetic stability issues that, to date, have not been adequately addressed. We have developed a novel, plasmid-free, high copy expression system for the next generation of engineered microbes. The new method, Chemically Inducible Chromosomal Evolution (CIChE), can generate ~40-50 copies of a recombinant pathway integrated on the microbial genome. The chromosomally engineered pathways require no selection markers to maintain copy number, have no variation in copy number, and avoid plasmid instabilities by being physically linked in one strand of DNA, thereby improving genetic stability 10 fold. The method is demonstrated by a CIChE-engineered biopolymer production pathway that, compared to plasmids, had improved genetic stability exceeding >35 generations and increased (by 4x) growth-phase specific productivity. At the same time, it maintained equivalent product yields despite the high metabolic burden imposed and lack of any selection pressure. In another example, the yield of a small molecule nutraceutical, lycopene, was increased by 60%. CIChE is a generalizable, high-expression, genetically stable metabolic engineering tool that does not require selection pressure.