Genome-scale metabolic representation of Amycolatopsis balhimycina.

Infection caused by methicillin-resistant Staphylococcus aureus (MRSA) is an increasing societal problem. Typically, glycopeptide antibiotics are used in the treatment of these infections. The most comprehensively studied glycopeptide antibiotic biosynthetic pathway is that of balhimycin biosynthesis in Amycolatopsis balhimycina. The balhimycin yield obtained by A. balhimycina is, however, low and there is therefore a need to improve balhimycin production. In this study, we performed genome sequencing, assembly and annotation analysis of A. balhimycina and further used these annotated data to reconstruct a genome-scale metabolic model for the organism. Here we generated an almost complete A. balhimycina genome sequence comprising 10,562,587 base pairs assembled into 2,153 contigs. The high GC-genome (∼ 69%) includes 8,585 open reading frames (ORFs). We used our integrative toolbox called SEQTOR for functional annotation and then integrated annotated data with biochemical and physiological information available for this organism to reconstruct a genome-scale metabolic model of A. balhimycina. The resulting metabolic model contains 583 ORFs as protein encoding genes (7% of the predicted 8,585 ORFs), 407 EC numbers, 647 metabolites and 1,363 metabolic reactions. During the analysis of the metabolic model, linear, quadratic and evolutionary programming algorithms using flux balance analysis (FBA), minimization of metabolic adjustment (MOMA), and OptGene, respectively were applied as well as phenotypic behavior and improved balhimycin production were simulated. The A. balhimycina model shows a good agreement between in silico data and experimental data and also identifies key reactions associated with increased balhimycin production. The reconstruction of the genome-scale metabolic model of A. balhimycina serves as a basis for physiological characterization. The model allows a rational design of engineering strategies for increasing balhimycin production in A. balhimycina and glycopeptide production in general.

Detecting structural invariants in biological reaction networks.

The detection and analysis of structural invariants in cellular reaction networks is of central importance to achieve a more comprehensive understanding of metabolism. In this work, we review different kinds of structural invariants in reaction networks and their Petri net-based representation. In particular, we discuss invariants that can be obtained from the left and right null spaces of the stoichiometric matrix which correspond to conserved moieties (P-invariants) and elementary flux modes (EFMs, minimal T-invariants). While conserved moieties can be used to detect stoichiometric inconsistencies in reaction networks, EFMs correspond to a mathematically rigorous definition of the concept of a biochemical pathway. As outlined here, EFMs allow to devise strategies for strain improvement, to assess the robustness of metabolic networks subject to perturbations, and to analyze the information flow in regulatory and signaling networks. Another important aspect addressed by this review is the limitation of metabolic pathway analysis using EFMs to small or medium-scale reaction networks. We discuss two recently introduced approaches to circumvent these limitations. The first is an algorithm to enumerate a subset of EFMs in genome-scale metabolic networks starting from the EFM with the least number of reactions. The second approach, elementary flux pattern analysis, allows to analyze pathways through specific subsystems of genome-scale metabolic networks. In contrast to EFMs, elementary flux patterns much more accurately reflect the metabolic capabilities of a subsystem of metabolism as well as its integration into the entire system.

Can the whole be less than the sum of its parts? Pathway analysis in genome-scale metabolic networks using elementary flux patterns.

Elementary modes represent a valuable concept in the analysis of metabolic reaction networks. However, they can only be computed in medium-size systems, preventing application to genome-scale metabolic models. In consequence, the analysis is usually constrained to a specific part of the known metabolism, and the remaining system is modeled using abstractions like exchange fluxes and external species. As we show by the analysis of a model of the central metabolism of Escherichia coli that has been previously analyzed using elementary modes, the choice of these abstractions heavily impacts the pathways that are detected, and the results are biased by the knowledge of the metabolic capabilities of the network by the user. In order to circumvent these problems, we introduce the concept of elementary flux patterns, which explicitly takes into account possible steady-state fluxes through a genome-scale metabolic network when analyzing pathways through a subsystem. By being similar to elementary mode analysis, our concept now allows for the application of many elementary-mode-based tools to genome-scale metabolic networks. We present an algorithm to compute elementary flux patterns and analyze a model of the tricarboxylic acid cycle and adjacent reactions in E. coli. Thus, we detect several pathways that can be used as alternative routes to some central metabolic pathways. Finally, we give an outlook on further applications like the computation of minimal media, the development of knockout strategies, and the analysis of combined genome-scale networks.