Machine-Learning-Assisted Pathway Optimization in Large Combinatorial Design Spaces: A p-Coumaric Acid Case Study.

Combinatorial pathway optimization is a powerful approach in metabolic engineering to improve strain performance. While machine learning (ML) has shown promise in guiding the Design-Build-Test-Learn (DBTL) cycle, most applications have been limited to small design spaces, thereby restricting the potential of predictive and exploration-exploitation strategies. In this work, we applied two DBTL cycles to optimize p-coumaric acid production in Saccharomyces cerevisiae. The first cycle involved constructing a large combinatorial library of 18 genes and 20 promoters (170 million possible designs). In the second cycle, we employed a gradient bandit-based machine learning recommendation strategy, tuned to balance exploration and exploitation. Our results show that this balanced strategy outperforms greedy, feature importance-based approaches, leading to greater diversity in strain performance and improved top-producer identification. Notably, applying the same strategy to an alternative parent strain yielded the highest p-coumaric acid titer (1.23 g/L), a 2.37-fold improvement over the original. These findings highlight the value of ML-guided exploration in large design spaces and demonstrate that balancing exploration and exploitation is critical for successful strain optimization.