Lysate Proteome Engineering Enables High-Yield Metabolite Conversions in Crude Cell Extracts

Cell-free metabolic engineering (CFME) systems provide bioconversion opportunities for achieving near theoretical yields of high-value metabolites. High-yield biotransformations unattainable so far in live cells have been successfully demonstrated in purified enzyme systems. Recently, active endogenous metabolic pathways in cell extracts have been leveraged to convert simple sugars towards valuable metabolites. These lysate-based systems have several advantages over purified enzyme systems such as ease of preparation, cost, and scalability. However, due to the presence of unwanted enzymes in cell extracts, carbon flux from a supplemented substrate becomes distributed among competing pathways, potentially compromising yields of the desired end-product. To this end, we have developed a genome engineering enabled strategy to direct flux towards targeted metabolites in lysate-based cell-free systems. With the goal of concentrating carbon flux from a glucose substrate towards the central metabolic precursor pyruvate, we genome engineered E. coli BL21DE3 strains to express endogenous pyruvate-consuming enzymes with 6xHis-tags. These modified strains show no adverse growth phenotypes, and the proteomes of their derived extracts could be engineered. Specifically, 6xHis-tagged enzymes were removed from the derived lysates which enabled pooling of pyruvate at concentrations 40-fold higher than conventional CFME lysates. Therefore, our approach enables the reduction of flux through metabolic nodes that are difficult to engineer in vivo while bypassing the need to undergo multiple design-build-test cycles. This strategy was further demonstrated by an engineered lysate with an unconventional endogenous metabolic phenotype that generated 40-fold higher bioethanol than control E. coli lysates. Through optimization of our targeted depletion approach, as well as source strain cultivation conditions, we have further shown that this lysate can produce 0.52 g ethanol per g consumed glucose. Lysate proteome engineering can thus be used to focus flux towards targeted pathways in cell extracts, expanding the current CFME toolkit to improve bioproduction yields in lysate-based systems.