Rapid Cell-Free Combinatorial Mutagenesis Workflow Using Small Oligos Suitable for High-Iteration, Active Learning-Guided Protein Engineering

Active learning-guided protein engineering e2iciently navigates the challenging fitness landscape by screening designs iteratively in a model-guided design-build-test-learn cycle. However, while high iterations boost performance, current workflows reliance on tedious and costly cell-based cloning and expression steps limits the iterations they can practically implement. To address this problem, we present a novel combinatorial mutagenesis workflow that uses small (∼20-40 bp) mutagenic annealed-oligo fragments and cell-free expression to rapidly and conveniently screen protein variants in <9 hours. Using bulk-prepared mutagenic oligos eliminates the need for cloning, PCR-based mutagenesis, or ordering costly genes each screening round. Their >80% size reduction from current fragment-based shu2ling strategies also helps avoid including multiple mutations on the same fragment, reducing the number one must order to cover the design space. By screening 3-10 fragment assemblies for two di2erent proteins, we show our approach is a general, scalable, and cost-e2ective platform for high-iteration protein engineering.