Protein-constrained models pinpoints the role of underground metabolism in robustness of metabolic phenotypes.

Integrating enzyme parameters into constraint-based models have significantly improved the prediction of physiological and molecular traits. To further improve these models, we integrated promiscuous enzyme activities that jointly comprise the so-called underground metabolism by developing the CORAL toolbox, which increases the resolution of modeled enzyme resource allocation. Applying CORAL to a protein-constrained model of revealed that underground metabolism resulted in larger flexibility of metabolic fluxes and enzyme usage. Simulating metabolic defects where the main activity of a promiscuous enzyme was blocked but promiscuous activities remained functional showed a small enzyme redistribution to the side activities. Further, blocking pairs of main activities showed that non-promiscuous enzymes exhibited larger impact on growth than promiscuous enzymes. These simulations showed that promiscuous enzymes can compensate for these defects, in line with experimental evidence. Together, our results indicated that promiscuous enzyme activities are vital to maintain robust metabolic function and growth.