Multiplexed <i>in Vivo</i> His-Tagging of Enzyme Pathways for <i>in Vitro</i> Single-Pot Multienzyme Catalysis

Abstract

Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced <i>in vitro</i> reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism <i>Escherichia coli</i> to reconstitute functional synthetic proteomes <i>in vitro</i>. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes <i>in vivo</i> that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis <i>in vitro</i>. Our approach can be applied to a growing area of applications in <i>in vitro</i> one-pot multienzyme catalysis (MEC) to manipulate or enhance <i>in vitro</i> pathways such as natural product or carbohydrate biosynthesis