Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype Isoprenoid Biosynthesis

Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for prototyping and designing biochemical synthesis pathways. To date, most CFME efforts use either purified enzymes or a crude extract from a source strain containing the entire enzymatic pathway. In this work, we used mixing of crude lysates, or extracts, from Escherichia coli to reconstitute a portion of the isoprenoid pathway; isoprenoids are a diverse class of compounds with applications as pharmaceuticals, fragrances, pesticides, biofuels, and chemical feedstocks. Specifically, we individually overexpressed three enzymes, mixed the resulting lysates, and utilized the native glycolytic enzyme cascade to convert glucose to mevalonate, an intermediate of isoprenoid synthesis. Avoiding the synthesis of cytotoxic HMG-CoA in the source strain, this plug-and-play approach was used to screen enzyme variants and optimize enzyme ratios which facilitated the construction of an all-in-one strain. The open nature of CFME allowed exploration of cofactor landscapes at varying ATP, NAD⁺, and CoA concentrations. Genomic modification to the source strain confirmed that metabolism of acetate salts via the ackA-pta pathway is critical for cofactor recycle and high titers (>10 g·L^-1). Our results highlight the high cofactor turnover of cell-free lysates and the ability to rapidly prototype and debug enzymatic pathways in vitro.