Abstract
Synthetic biology enables the reprogramming of cellular functions for various applications. However, challenges in scalability and predictability persist due to context-dependent performance and complex circuit-host interactions. This study introduces an iModulon-based engineering approach, utilizing machine learning-defined co-regulated gene groups (iModulons) as design parts containing essential genes for specific functions. This approach identifies the necessary components for genetic circuits across different contexts, enhancing genome engineering by improving target selection and predicting module behavior. We demonstrate several distinct uses of iModulons: (i) discovery of unknown iModulons to increase protein productivity, heat tolerance and fructose utilization; (ii) an iModulon boosting approach, which amplifies the activity of specific iModulons, improved cell growth under osmotic stress with minimal host regulation disruption; (iii) an iModulon rebalancing strategy, which adjusts the activity levels of iModulons to balance cellular functions, significantly increased oxidative stress tolerance while minimizing trade-offs and (iv) iModulon-based gene annotation enabled natural competence activation by predictably rewiring iModulons. Comparative experiments with traditional methods showed our approach offers advantages in efficiency and predictability of strain engineering. This study demonstrates the potential of iModulon-based strategies to systematically and predictably reprogram cellular functions, offering refined and adaptable control over complex regulatory networks.