Research And Application Of Multiplex Iterative Plasmid Engineering (MIPE)

A major challenge in synthetic biology is to engineer complex biological systemswith novel functions. Due to the inherent complexity of biological systems, it is oftendifficult to rationally design every component in a synthetic gene network to arrive atoptimal performance. Engineering complex biological systems typically requirescombinatorial optimization to achieve the desired functionality. Combinatorialengineering is an important solution to this problem and can greatly facilitate theconstruction of novel biological functions. Here, we present Multiplex IterativePlasmid Engineering (MIPE), which is a highly efficient and customized method forcombinatorial diversification of plasmid sequences. MIPE exploits ssDNA mediated λRed recombineering for the introduction of mutations, allowing it to target severalsites simultaneously. We also describe “Restriction Digestion mediated Co-Selection(RD CoS)”, which enables MIPE to produce enhanced recombineering efficiencieswith greatly simplified co-selection procedures. To demonstrate this approach, weapplied MIPE to fine-tune gene expression level in the5-gene riboflavin biosyntheticpathway and successfully isolated a clone with2.67-fold improved production in lessthan a week. We further demonstrated the ability of MIPE for highly multiplexeddiversification of protein coding sequence by simultaneously targeting23codonsscattered along the750bp sequence. We anticipate this method to benefit theoptimization of diverse biological systems in synthetic biology and metabolicengineering.