Conversion of Glucose to 2,3-Butanediol in Escherichia coli Cell-Free Lysates: A Model System

In recent decades we are seeing more and more examples of whole pathways being activated in crude lysates to generate a desired product in vitro. For instance, inexpensive substrates glucose or maltose have been used to regenerate ATP to power cell-free protein synthesis. Buffers and reaction conditions designed to mimic the cytoplasm have allowed a remarkable number of enzymes to be coordinately activated and to remain stable for up to about a week. It begs the question, of whether cytoplasmic mimicry will benefit other endeavors such as rapid enzyme screening or in vitro multi-enzyme catalysis.    

     Here, as a model study, we sought to determine if the conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications, could be achieved in an in vitro system designed to have high cofactor regeneration. 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 have 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 have found the system to be robust to working concentrations of antibiotics and antifoam, and other compounds that are toxic to cell growth but do not denature or inhibit relevant enzymes. Current work includes tuning enzyme ratios to try to understand what is necessary and limiting in the system, and testing potential enzyme screening protocols. 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 bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation.