Network-level architecture and the evolutionary potential of underground metabolism

Abstract

A central unresolved issue in evolutionary biology is how metabolic innovations emerge. Low-level enzymatic side activities are frequent and can potentially be recruited for new biochemical functions. However, the role of such underground reactions in adaptation toward novel environments has remained largely unknown and out of reach of computational predictions, not least because these issues demand analyses at the level of the entire metabolic network. Here, we provide a comprehensive computational model of the underground metabolism in Escherichia coli. Most underground reactions are not isolated and 45% of them can be fully wired into the existing network and form novel pathways that produce key precursors for cell growth. This observation allowed us to conduct an integrated genome-wide in silico and experimental survey to characterize the evolutionary potential of E. coli to adapt to hundreds of nutrient conditions. We revealed that underground reactions allow growth in new environments when their activity is increased. We estimate that at least ∼20% of the underground reactions that can be connected to the existing network confer a fitness advantage under specific environments. Moreover, our results demonstrate that the genetic basis of evolutionary adaptations via underground metabolism is computationally predictable. The approach used here has potential for various application areas from bioengineering to medical genetics.enzyme promiscuity | evolutionary innovation | molecular evolution | network evolution | phenotype microarray H ow do new molecular pathways evolve? In the best-studied molecular networks, small-molecule metabolism, the prevailing paradigm is that new pathways are patched together from preexisting enzymes borrowed from different parts of the network (1-3). Central to this "patchwork" model of pathway evolution is the notion that many enzymes have limited substrate specificities and can catalyze, albeit at low rates, reactions other than those for which they have evolved (also referred to as enzyme promiscuity) (4). These so-called underground (5) or side activities are prevalent (6-8) and were shown to serve as starting points for the evolution of novel functions both in directed evolution experiments (9) and in the diversification of gene families in the wild (7). However, how the underground catalytic repertoire encoded in the genome can generate novelties within the context of the existing metabolic network remains unknown. Do underground reactions remain isolated, or can they potentially be wired into the native network and allow the organism to survive in novel environments? Furthermore, would it be possible to computationally predict the genetic basis of phenotypic evolution based on a detailed knowledge of the organism's underground metabolism? Answering these questions requires both large-scale data on underground enzyme activities and systems-level approaches to analyze metabolic capabilities. Although systematic detection of underground activities by unbiased high-throughput ap...

Model-driven discovery of underground metabolic functions in Escherichia coli

“…5 (reaction abbreviations are provided in Table S5). The expanded reaction associations for the cases examined in this study support the hypothesis that functional overlap occurs for enzymes across metabolism, forming the basis of an underground metabolic network, and this concept has been supported by other recent works (6,8).…”

“…This likely expansion is evident in the appearance of complementary studies (8) that use computational modeling to isolate specific predicted functionalities through gene KO or overexpression and through determining media conditions that focus pressure on the predicted function(s). A comparison of the present study with the aforementioned study (8) shows some overlap between genes explored and thought to exhibit underground activity (ilvE and tyrB), although the suggested activities reported in each study for these genes are distinct. Workflows for discovering promiscuous and latent activities, such as the one presented here, will be critical for advancement of model-driven science.…”

“…In the present study, this task is approached from a different perspective by taking advantage of in vivo experimental techniques to gain insight into activities that are more physiologically relevant. In this way, as has been demonstrated in other in vivo studies, many of the challenges associated with removing enzymes from their native environment are circumvented (8). Specifically, the present study focuses on examination of the regulatory and evolutionary capacity of a cell in vivo.…”

“…The existence of promiscuous proteins further serves as a starting point for evolving new functions, allowing for novel adaptations. Thus, organisms may exhibit latent, underground metabolic pathways that form the basis of their capacity to adapt to changing environments (6)(7)(8). Substrate promiscuity, also referred to as "moonlighting activity" and "cross-reactivity," has thus been studied in terms of evolution, and ties have been made between enzymes and their superfamilies (9).…”

“…Thus, laboratory evolutions may provide insight into these evolutionary mechanisms involving enzyme promiscuity. Furthermore, exploration of an underground metabolic network that takes advantage of enzyme cross-reactivity through native regulatory adaptations is best examined in the context of a whole cell (6,8).…”