Establishing Cell-Free Metabolic Engineering for Pathway Debugging and the Production of Sustainable Chemicals

Rapid population growth, a rise in global living standards, and climate change concerns have intensified the need for sustainable, low-cost production of bioenergy, commodity chemicals, and natural products. Industrial biotechnology is one of the most attractive approaches for addressing this need, particularly when large-scale chemical synthesis is untenable. While the number of microbial metabolic engineering success stories is rapidly growing, the fraction of biochemicals amenable to economical production is still limited because engineering whole-cell microorganisms with biosynthetic pathways remains costly and slow. Common problems afflicting the current state-of-the-art include low volumetric productivities (g/L/h), build-up of toxic intermediates or products, byproduct losses via competing pathways, and constraints arising from the fact that microbial growth and adaptation objectives are often diametrically opposed to the overproduction and release of a single product. To overcome these limitations, we are expanding the scope of the traditional bioengineering model by using cell-free systems to harness ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells for the production of target products. In this presentation, I will discuss our efforts to demonstrate ultra-high productivities and titers for metabolic conversion and also perform design-build-test (DBT) iterations without the need to re-engineer organisms. This experimental approach holds great promise to increase our ability to debug and optimize modular construction of pathways in cellular lysates through the use of simple, well-defined experimental conditions. We anticipate that cell-free systems will open new frontiers for biomanufacturing when cellular toxicity limits commercial feasibility of whole-cell fermentation.