A. Barghout, R.; Chinas Serrano, L.; Sanchez-Lengeling, B.; Mahadevan, R.

The construction of accurate enzyme-constrained genome-scale models (ecGEMs) remains a critical challenge in systems biology, limited by sparse kinetic data and the need for biologically meaningful representations. This work presents an integrated framework combining CPI-Pred, a deep learning model to predict kinetic parameters (kcat) from sequence and compound embeddings, with kinGEMs, a pipeline to incorporate these uncertainty-aware parameters into ecGEMs for metabolic optimization. By leveraging representations at multiple scales, the approach captures sequence, structure, and kinetic data to enhance model generalizability and accuracy. Rigorous benchmarking demonstrates the framework's capability to predict growth rates and fluxes that are consistent with experimental observations and measured proteome fractions, reduce median flux variability by several folds, and yield better-constrained metabolic models by explicitly accounting for the uncertainty inherent in ML-predicted kinetic parameters. kinGEMs successfully generated ecGEMs for 93 models spanning a phylogenetically diverse set of organisms including Gram-negative and Gram-positive bacteria, mycobacteria, parasitic protists, fungi, and mammalian cell lines. These innovations breakdown the barrier to application of ecGEMs to non-model organisms and open new avenues for metabolic engineering and synthetic biology in more industrially relevant hosts.