Transcriptome-guided parsimonious flux analysis improves predictions with metabolic networks in complex environments

The metabolic responses of bacteria to dynamic extracellular conditions drives not only the behavior of single species, but also entire communities of microbes. Over the last decade, genome-scale metabolic network reconstructions have assisted in our appreciation of important metabolic determinants of bacterial physiology. These network models have been a powerful force in understanding the metabolic capacity that species may utilize in order to succeed in an environment. Increasingly, an understanding of context-specific metabolism is critical for elucidating metabolic drivers of larger phenotypes and disease. However, previous approaches to use network models in concert with omics data to better characterize experimental systems have met challenges due to assumptions necessary by the various integration platforms or due to large input data requirements. With these challenges in mind, we developed RIPTiDe (Reaction Inclusion by Parsimony and Transcript Distribution) which uses both transcriptomic abundances and parsimony of overall flux to identify the most cost-effective usage of metabolism that also best reflects the cell’s investments into transcription. Additionally, in biological samples where it is difficult to quantify specific growth conditions, it becomes critical to develop methods that require lower amounts of user intervention in order to generate accurate metabolic predictions. Utilizing a metabolic network reconstruction for the model organism Escherichia coli str. K-12 substr. MG1655 (iJO1366), we found that RIPTiDe correctly identifies context-specific metabolic pathway activity without supervision or knowledge of specific media conditions. We also assessed the application of RIPTiDe to in vivo metatranscriptomic data where E. coli was present at high abundances, and found that our approach also effectively predicts metabolic behaviors of host-associated bacteria. In the setting of human health, understanding metabolic changes within bacteria in environments where growth substrate availability is difficult to quantify can have large downstream impacts on our ability to elucidate molecular drivers of disease-associated dysbiosis across the microbiota. Our results indicate that RIPTiDe may have potential to provide understanding of context-specific metabolism of bacteria within complex communities.Author SummaryTranscriptomic analyses of bacteria have become instrumental to our understanding of their responses to changes in their environment. While traditional analyses have been informative, leveraging these datasets within genome-scale metabolic network reconstructions (GENREs) can provide greatly improved context for shifts in pathway utilization and downstream/upstream ramifications for changes in metabolic regulation. Many previous techniques for GENRE transcript integration have focused on creating maximum consensus with input datasets, but these approaches were recently shown to generate less accurate metabolic predictions than a transcript-agnostic method of flux minimization (pFBA), which identifies the most efficient/economic patterns of metabolism given certain growth constraints. Despite this success, growth conditions are not always easily quantifiable and highlights the need for novel platforms that build from these findings. Our new method, RIPTiDe, combines these concepts and utilizes overall minimization of flux weighted by transcriptomic analysis to identify the most energy efficient pathways to achieve growth that include more highly transcribed enzymes, without previous insight into extracellular conditions. Utilizing a well-studied GENRE from Escherichia coli, we demonstrate that this new approach correctly predicts patterns of metabolism utilizing a variety of both in vitro and in vivo transcriptomes. This platform could be important for revealing context-specific bacterial phenotypes in line with governing principles of adaptive evolution, that drive disease manifestation or interactions between microbes.