Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systemslevel adaptations are poorly understood. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set ('underground metabolism') can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in E. coli K-12 MG1655. After as few as 20 generations, the evolving populations repeatedly acquired the capacity to grow on five predicted novel substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/310946 doi: bioRxiv preprint first posted online May. 3, 2018; and monomethyl succinate-none of which could support growth in wild-type cells. Promiscuous enzyme activities played key roles in multiple phases of adaptation. Altered promiscuous activities not only established novel highefficiency pathways, but also suppressed undesirable metabolic routes. Further, structural mutations shifted enzyme substrate turnover rates towards the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity. Computational approaches will be essential to synthesize the complex role of promiscuous activities in applied biotechnology and in models of evolutionary adaptation.