Abstract: Inverse metabolic engineering is a kind of new genetic engineering, which can get complex phenotypes regulated by multiple genes by constructing a library of cells with genetic diversity, following with efficient high throughput screen. Currently, it has become a powerful tool for researching biological metabolic pathways and modifying the biological properties of strains in industrial biotechnology. One of the bottleneck problems in inverse metabolic engineering is how to obtain a variety of cell phenotype mutant library. This paper briefly introduced several classic and recently developed ways to solve the bottleneck problem, and listed the latest research progresses of these methods.