1 Department of Systems Biology, Technical University of Denmark2 Eucaryotic Molecular Cell Biology, Department of Systems Biology, Technical University of Denmark
Biological systems are characterized by a high degree of complexity wherein the individual components (e.g. proteins) are inter-linked in a way that leads to emergent behaviors that are difficult to decipher. Uncovering system complexity requires, at least, answers to the following three questions: what are the components of the systems, how are the different components interconnected and how do these networks perform the functions that make the resulting system behavior? Modern analytical technologies allow us to unravel the constituents and interactions happening in a given system; however, the third question is the ultimate challenge for systems biology. The work of this thesis systematically addresses this question in the context of metabolic networks, which are arguably the most well characterized cellular networks in terms of their constituting components and interactions among them. Furthermore, there is large interest in understanding and manipulating cellular metabolism from health as well as biotechnological perspectives. Fundamentally different biological questions are investigated in different core chapters of the thesis, though all are linked by the common thread of the functioning of cellular metabolism. The three main topics addressed are: i) transcriptional regulation of metabolite concentration, ii) transcriptional dys-regulation of skeletal muscle metabolism in type 2 diabetes, and iii) metabolic interactions in microbial ecosystems. The overall objective is to obtain novel understanding underlying the operating principles of metabolic networks. Cellular responses to environmental perturbations and genetic/epigenetic modifications are to a large extent controlled through transcription, which is one of the fundamental mechanism/means of cellular regulation. An important question is to what extent gene expression can explain metabolic phenotype, in other words, how well changes in metabolite concentrations can be explained by the changes in related enzyme-coding transcripts? Attempts to predict changes in the metabolome from gene expression data have so far remained unsolved. Here, I challenge this question by proposing a mechanistic explanation of the interplay between metabolite concentrations, transcripts and fluxes based on Michaelis-Menten kinetics at the network-scale. The work demonstrates that in steadystate systems, changes of intracellular metabolites concentrations are linked with the changes in gene expression of both reactions that produce and reactions that consume a given metabolite. Analysis of a large compendium of gene expression data further suggested that, contrary to previous thinking, transcriptional regulation at metabolic branch points is highly plastic and, in several cases, the objective of the regulation appears to be metabolite-oriented as opposed to pathway-oriented. The study thus provides a fundamental and novel view of metabolic network regulation in Saccharomyces cerevisiae. Metabolism is a conserved system across all domains of life. Nowadays, metabolism has become a focal point in diagnosing and treating diseases such as diabetes and cancer. Type 2 diabetes mellitus is a complex metabolic disease which is recognized as one of the largest threats to human health in the 21st century. Recent studies of gene expression levels in human tissue samples have indicated that multiple metabolic pathways are dys-regulated in diabetes and in individuals at risk for diabetes; which of these are primary, or central to disease pathogenesis, remains a key question. Cellular metabolic networks are highly interconnected and often tightly regulated; any perturbations at a single node can thus rapidly diffuse to the rest of the network. Such complexity presents a considerable challenge in pinpointing key molecular mechanisms and signatures associated with insulin resistance and type 2 diabetes. The present work addresses this problem by using a methodology that integrates gene expression data with the human cellular metabolic network. The approach is demonstrated by analysis of two skeletal muscle gene expression datasets. The proposed methodology identified transcription factors and metabolites that represent potential targets for therapeutic agents and future clinical diagnostics for type 2 diabetes and impaired glucose metabolism. In a broader context, the study provides a framework for analysis of gene expression datasets from complex heterogeneous diseases, genetic, and environmental perturbations that are reflected in and/or mediated through changes in metabolism. In nature, microorganisms do not exist as pure cultures, but evolve and co-exist with other species. Microbial communities have a variety of potential applications, including metabolic disease therapies and biotechnology. For example, microbial consortia consisting of various bacteria and fungi are known to exhibit a biodegradation performance superior to pure cultures, making them attractive research targets. It is believed that nutrition plays a crucial role in shaping microbial communities. Interspecies metabolite cross-feeding can confer several advantages to the community as a whole. For example, more efficient and complete use of available nutrients, or increased ability to survive under diverse/changing nutrition availability potentially induces fitness of individuals. The third topic of this thesis investigates the role of metabolic interaction in co-occurring microbial communities. The study aims to identify metabolic properties that shape the community structures. The analysis based on a global metagenomic dataset and genome-scale metabolic models suggested that species within coexisting communities have higher potential of metabolic cooperation compared to random controls. This work yielded a novel methodology (termed species metabolic coupling analysis) for studying metabolic interaction and interdependencies within microbial communities. Species metabolic coupling analysis has a spectrum of applications to real-world problems, including investigation of metabolic interactions within the human microbiome, host -pathogen interactions and development of stable microbial communities. Overall, this work contributes with novel insights, tools and methodologies to study the operation of cellular metabolism.