A Cell-Free Protein Evolution Platform
Authors:
Grant Landwehr1* ([email protected]), Bastian Vögeli1, and Michael Jewett1,2
Institutions:
1Northwestern University; and 2Stanford University
Abstract
The biosynthesis of high-value sustainable chemicals is a major goal of synthetic biology and securing the energy future. For many important molecular biotransformations, efficient enzymes have yet to be discovered or engineered. High-throughput methods to prepare enzyme mutants and measure their activity, as well as evaluate their engineering potential and transform them into industrial tools are important to overcoming this bottleneck. Researchers addressed this challenge by developing a cell-free DNA assembly and protein synthesis platform, allowing researchers to rapidly screen 1000’s of sequence defined enzyme mutants in iterative design-build-test-learn cycles. These rich datasets are also amenable to machine learning algorithms that try to capture the sequence-fitness landscape. As a model, researchers demonstrate the utility of the platform by engineering a formyl-CoA synthetase for the activation of formate, a transformation that does not readily occur in nature and has implications in building synthetic carbon-fixing metabolic pathways. To design, researchers used molecular structure and sequence homology to guide selection of amino acid residues for creating new-to-nature enzymes that catalyze the desired chemistry. To build, researchers used site saturation mutagenesis to create a DNA library of enzyme variants containing mutations at the identified residues. To test, researchers developed a visual detection scheme to bypass the need for traditional chromatography-based analytics. The team anticipates the work engineering a formyl-CoA synthetase will serve as a blueprint to enable future efforts aimed at the maturation of a diverse array of biocatalysts.
Funding Information
This work was supported by DOE Office of Science, Office of Biological and Environmental Research (BER), grant DE-SC0023278, and an NSF Graduate Research Fellowship to G.M.L. (DGE-1842165).