Combining Machine Learning and Directed Evolution for Optimization of a Monooxygenase
L-3,4-dihydroxyphenylalanine (L-Dopa) is an important pharmaceutical for the treatment of Parkinsons disease and a precursor to numerous catechol-containing compounds. The flavin-dependent monooxygenase HpaBC is a promising biocatalyst for microbial L-Dopa production but exhibits limited native activity toward L-tyrosine. Although structure-based machine learning (ML) models have become increasingly popular for protein engineering, relatively few studies have systematically compared their performance or evaluated their integration into iterative engineering workflows. Here, we benchmarked multiple ML models for their ability to predict activity enhancing mutations in HpaBC. Experimentally validated single mutants were used to seed combinatorial design with EVOLVEpro, generating progressively improved higher-order variants. We next evaluated how expanding the EVOLVEpro training set with directed evolution derived variants influenced combinatorial predictions and finally explored an expanded sequence space by allowing combinations of both machine learning derived and directed evolution derived mutations. This workflow produced HpaBC variants with substantially improved activity. Although incorporating directed evolution data substantially altered EVOLVEpros predicted mutational trajectories, both training strategies converged on variants with comparable activities, demonstrating that distinct regions of sequence space can yield similarly optimized enzymes. Together, these results provide a systematic comparison of zero-shot ML models and establish an iterative framework for integrating machine learning with directed evolution to accelerate enzyme engineering.