A Simple Recombinant E. coli Cell Lysate-Based Biocatalyst for ATP-Dependent Multi-step Reactions

The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps.