Cell-Free Biofuel Production using an In Vitro Synthetic Biochemistry Platform and Quantification of Isobutanol Tolerance of Synthesis Enzymes

In recent years, the growing threat of anthropogenic climate change has raised interest in and incentivized the development of low-carbon fuel and energy technologies. Liquid biofuels, derived from harvested plant biomass, have the potential to provide substantial greenhouse gas (GHG) emission reductions to the transport sector and serve as alternatives to traditional petroleum-based fuels, like gasoline and diesel. The production of these fossil fuel substitutes often times requires the use of microbial organisms, genetically manipulated to mass produce these fuels from fermentable sugars. However, in general, cell-based synthesis tends to be somewhat inefficient, due to, among other factors, the metabolic constraints of biological life and issues related to intermediate and product toxicity. Recently, a new method, termed synthetic biochemistry or in vitro metabolic engineering, has emerged as a solution to some of these challenges. In this approach, the cell is essentially tossed out, and only the desired biochemical pathway is reconstituted in vitro, facilitating rapid bio-commodity production at high yields and high titers. Using this approach and fourteen enzymes from the Embden-Meyerhof-Parnas pathway, the valine biosynthesis pathway and the Ehrlich pathway, we have been able to demonstrate for the first time the complete conversion (100% yield) of the fermentable sugar, D-glucose, to the advanced biofuel molecule, isobutanol, and achieve titers of nearly 15 g/L without the use of any living cells. Though several enzymes were found to be particularly susceptible to product inhibition at high concentrations of isobutanol and may need to be engineered for greater organic solvent tolerance to achieve higher titers in the future, the results demonstrated here lend credibility to the concept of cell-free synthetic biochemistry as an efficient method for high-yield biofuel production