Establishment of a system for small molecule synthesis by Crude Lysates of Escherichia Coli

In recent decades we have seen several examples of central metabolism being activated in crude lysates to generate a desired product in vitro. For instance, inexpensive substrates glucose, maltose, and starch have been used to regenerate ATP to power cell-free protein synthesis. We attribute this success to identification of appropriate lysate preparation methods and a reaction composition designed to mimic the cytoplasm. In this work, we sought to use similar reaction conditions to activate native metabolism in crude lysates to supply energy and carbon to a heterologous pathway; in other words, in vitro multi-enzyme catalysis.

As a proof-of-principle study, we selected the conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Extracts of Escherichia coli expressing three heterologous enzymes are able to convert glucose to m2,3-BD at surprisingly high rates and concentrations. With no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD / g glucose) and concentrations of 82 ± 8 g/L m2,3-BD in batch reactions. We found the system to be robust to working concentrations of antibiotics and other compounds that are toxic to cell growth but do not denature or inhibit relevant enzymes. These results highlight the ability for high-level co-factor regeneration in cell-free lysates and suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when cellular toxicity, transport characteristics, bioconversion yields, and/or productivities limit commercial feasibility of whole-cell fermentation.