Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals

•Identification of gene targets is a central problem of metabolic engineering.•Tractable and transferable genetic libraries are employed to identify target genes.•A hybrid approach using various libraries will confer versatility on microbes.

Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed.