An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing. Cell-free systems enable the design of biosynthetic pathways for sustainable chemical synthesis. Here the authors create an integrated framework for accelerating design using extracts from genetically rewired strains.

Automated summary

Cell-free systems using genetically rewired cell extracts enable faster and more efficient design of biosynthetic pathways for sustainable chemical synthesis. This integrated framework allows for significant increases in product titers and volumetric productivities through systematic optimization of the cell-free reaction environment. The approach is generalizable to other biosynthesis pathways, showcasing the potential for synthetic biology prototyping and cell-free biomanufacturing.