Two approaches for metabolic pathway analysis?

Metabolic pathway analysis is becoming increasingly important for assessing inherent network properties in (reconstructed) biochemical reaction networks. Of the two most promising concepts for pathway analysis, one relies on elementary flux modes and the other on extreme pathways. These concepts are closely related because extreme pathways are a subset of elementary modes. Here, the common features, differences and applicability of these concepts are discussed. Assessing metabolic systems by the set of extreme pathways can, in general, give misleading results owing to the exclusion of possibly important routes. However, in certain network topologies, the sets of elementary modes and extreme pathways coincide. This is quite often the case in realistic applications. In our opinion, the unification of both approaches into one common framework for metabolic pathway analysis is necessary and achievable.     

基于图论和约束优化的异源代谢途径设计方法及应用

构建高产高附加值产品的微生物细胞工厂是代谢工程的研究目标之一,设计高效的产品合成途径是实现这一目标的重要方式.不同微生物底盘因其代谢能力有所差异,故而可以利用的底物和生产的产品范围有限.为了扩大其生产能力,需要进行代谢途径从无到有的设计.传统代谢工程基于经验进行异源途径设计的方式低效且无法确保结果的全面性,而系统生物学...     

Experimental and mathematical approaches to modeling plant metabolic networks

To support their sessile and autotrophic lifestyle higher plants have evolved elaborate networks of metabolic pathways. Dynamic changes in these metabolic networks are among the developmental forces underlying the functional differentiation of organs, tissues and specialized cell types. They are also important in the various interactions of a plant with its environment. Further complexity is added by the extensive compartmentation of the various interconnected metabolic pathways in plants. Thus, although being used widely for assessing the control of metabolic flux in microbes, mathematical modeling approaches that require steady-state approximations are of limited utility for understanding complex plant metabolic networks. However, considerable progress has been made when manageable metabolic subsystems were studied. In this article, we will explain in general terms and using simple examples the concepts underlying stoichiometric modeling (metabolic flux analysis and metabolic pathway analysis) and kinetic approaches to modeling (including metabolic control analysis as a special case). Selected studies demonstrating the prospects of these approaches, or combinations of them, for understanding the control of flux through particular plant pathways are discussed. We argue that iterative cycles of (dry) mathematical modeling and (wet) laboratory testing will become increasingly important for simulating the distribution of flux in plant metabolic networks and deriving rational experimental designs for metabolic engineering efforts.     

浓香型白酒发酵过程微生物合成正丙醇途径解析

【目的】揭示浓香型白酒窖内发酵过程与正丙醇合成相关的微生物和代谢途径。【方法】通过对浓香型白酒窖内发酵过程酒醅中微生物的宏转录组进行分析,解析与正丙醇合成相关的微生物和代谢途径,并验证相关微生物合成正丙醇的能力。【结果】浓香型白酒窖内发酵过程中有3条可能的酒醅微生物合成正丙醇的途径。真菌主要通过2-甲基苹果酸代谢途径和苏氨酸代谢途径合成正丙醇,细菌则主要通过丙酸代谢途径合成并参与苏氨酸代谢途径。宏转录组测序分析表明,这3条途径对白酒窖内发酵过程正丙醇的合成与积累均有贡献,并且微生物通过这3条途径合成正丙醇的时期和能力存在较大差异。此外,对分离自酒醅的酵母和乳酸菌合成正丙醇能力分析发现,它们均与浓香型白酒窖内发酵过程正丙醇的合成有关。【结论】本研究揭示了浓香型白酒窖内发酵过程中正丙醇合成相关的微生物和代谢途径,为阐明白酒发酵过程中正丙醇的形成机制奠定了理论基础。     

Metabolic systems cost-benefit analysis for interpreting network structure and regulation

MOTIVATION: Interpretation of bioinformatics data in terms of cellular function is a major challenge facing systems biology. This question is complicated by robust metabolic networks filled with structural features like parallel pathways and isozymes. Under conditions of nutrient sufficiency, metabolic networks are well known to be regulated for thermodynamic efficiency however; efficient biochemical pathways are anabolically expensive to construct. While parameters like thermodynamic efficiency have been extensively studied, a systems-based analysis of anabolic proteome synthesis 'costs' and the cellular function implications of these costs has not been reported. RESULTS: A cost-benefit analysis of an in silico Escherichia coli network revealed the relationship between metabolic pathway proteome synthesis requirements, DNA-coding sequence length, thermodynamic efficiency and substrate affinity. The results highlight basic metabolic network design principles. Pathway proteome synthesis requirements appear to have shaped biochemical network structure and regulation. Under conditions of nutrient scarcity and other general stresses, E. coli expresses pathways with relatively inexpensive proteome synthesis requirements instead of more efficient but also anabolically more expensive pathways. This evolutionary strategy provides a cellular function-based explanation for common network motifs like isozymes and parallel pathways and possibly explains 'overflow' metabolisms observed during nutrient scarcity. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

FluxAnalyzer: exploring structure,pathways, and flux distributions in metabolic networks on interactive flux maps

MOTIVATION: The analysis of structure, pathways and flux distributions in metabolic networks has become an important approach for understanding the functionality of metabolic systems. The need of a user-friendly platform for stoichiometric modeling of metabolic networks in silico is evident. RESULTS: The FluxAnalyzer is a package for MATLAB and facilitates integrated pathway and flux analysis for metabolic networks within a graphical user interface. Arbitrary metabolic network models can be composed by instances of four types of network elements. The abstract network model is linked with network graphics leading to interactive flux maps which allow for user input and display of calculation results within a network visualization. Therein, a large and powerful collection of tools and algorithms can be applied interactively including metabolic flux analysis, flux optimization, detection of topological features and pathway analysis by elementary flux modes or extreme pathways. The FluxAnalyzer has been applied and tested for complex networks with more than 500,000 elementary modes. Some aspects of the combinatorial complexity of pathway analysis in metabolic networks are discussed. AVAILABILITY: Upon request from the corresponding author. Free for academic users (license agreement). Special contracts are available for industrial corporations. SUPPLEMENTARY INFORMATION: http://www.mpi-magdeburg.mpg.de/projects/fluxanalyzer.     

Exploring biochemical pathways for mono-ethylene glycol (MEG) synthesis from synthesis gas

Mono-ethylene glycol (MEG) is an important petrochemical with widespread use in numerous consumer products. The current industrial MEG-production process relies on non-renewable fossil fuel-based feedstocks, such as petroleum, natural gas, and naphtha; hence, it is useful to explore alternative routes of MEG-synthesis from gases as they might provide a greener and more sustainable alternative to the current production methods. Technologies of synthetic biology and metabolic engineering of microorganisms can be deployed for the expression of new biochemical pathways for MEG-synthesis from gases, provided that such promising alternative routes are first identified. We used the BNICE.ch algorithm to develop novel and previously unknown biological pathways to MEG from synthesis gas by leveraging the Wood-Ljungdahl pathway of carbon fixation of acetogenic bacteria. We developed a set of useful pathway pruning and analysis criteria to systematically assess thousands of pathways generated by BNICE.ch. Published genome-scale models of Moorella thermoacetica and Clostridium ljungdahlii were used to perform the pathway yield calculations and in-depth analyses of seven (7) newly developed biological MEG-producing pathways from gases, including CO₂, CO, and H₂. These analyses helped identify not only better candidate pathways, but also superior chassis organisms that can be used for metabolic engineering of the candidate pathways. The pathway generation, pruning, and detailed analysis procedures described in this study can also be used to develop biochemical pathways for other commodity chemicals from gaseous substrates.     

On-line metabolic pathway analysis based on metabolic signal flow diagram

In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose. This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth. The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture. Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy. The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity. The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop. All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process.     

Metabolic flux analysis of Arthrobacter sp. CGMCC 3584 for cAMP production based on C tracer experiments and gas chromatography–mass spectrometry

Arthrobacter sp. CGMCC 3584 are able to produce cAMP from glucose by the purine synthesis pathway via de novo or salvage biosynthesis. In order to gain an improved understanding of its metabolism, ¹³C-labeling experiment and gas chromatography–mass spectrometry (GC–MS) analysis were employed to determine the metabolic network structure and estimate the intracellular fluxes. GC–MS analysis helps to reflect the activity of the intracellular pathways and reactions. The metabolic network mainly contains glycolytic and pentose phosphate pathways, the tricarboxylic acid cycle, and the inactive glyoxylate shunt. Hypoxanthine as a precursor of cAMP and sodium fluoride as an inhibitor of glycolysis were found to increase the cAMP production, as well as the flux through the PP pathway. The effects of adding hypoxanthine and sodium fluoride are discussed based on the enzyme assays and metabolic flux analysis. In conclusion, our results provide quantitative insights into how cells manipulate the metabolic network under different culture conditions and this may be of value in metabolic regulation for desirable production.     

Theory for the systemic definition of metabolic pathways and their use in interpreting metabolic function from a pathway-oriented perspective

Cellular metabolism is most often described and interpreted in terms of the biochemical reactions that make up the metabolic network. Genomics is providing near complete information regarding the genes/gene products participating in cellular metabolism for a growing number of organisms. As the true functional units of metabolic systems are its pathways, the time has arrived to define metabolic pathways in the context of whole-cell metabolism for the analysis of the structural design and capabilities of the metabolic network. In this study, we present the theoretical foundations for the identification of the unique set of systemically independent biochemical pathways, termed extreme pathways, based on system stochiometry and limited thermodynamics. These pathways represent the edges of the steady-state flux cone derived from convex analysis, and they can be used to represent any flux distribution achievable by the metabolic network. An algorithm is presented to determine the set of extreme pathways for a system of any complexity and a classification scheme is introduced for the characterization of these pathways. The property of systemic independence is discussed along with its implications for issues related to metabolic regulation and the evolution of cellular metabolic networks. The underlying pathway structure that is determined from the set of extreme pathways now provides us with the ability to analyse, interpret, and perhaps predict metabolic function from a pathway-based perspective in addition to the traditional reaction-based perspective. The algorithm and classification scheme developed can be used to describe the pathway structure in annotated genomes to explore the capabilities of an organism.     

Cell free biosynthesis of isoprenoids from isopentenol

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d -erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.     

An algorithm for linear metabolic pathway alignment

Metabolic pathway alignment represents one of the most powerful tools for comparative analysis of metabolism. It involves recognition of metabolites common to a set of functionally-related metabolic pathways, interpretation of biological evolution processes and determination of alternative metabolic pathways. Moreover, it is of assistance in function prediction and metabolism modeling. Although research on genomic sequence alignment is extensive, the problem of aligning metabolic pathways has received less attention. We are motivated to develop an algorithm of metabolic pathway alignment to reveal the similarities between metabolic pathways. A new definition of the metabolic pathway is introduced. The algorithm has been implemented into the PathAligner system; its web-based interface is available at http://bibiserv.techfak.uni-bielefeld.de/pathaligner/.     

Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins

This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol–methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:28–37, 2014     

Entamoeba histolytica: observations on metabolism based on the genome sequence

The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E. histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.     

Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering

Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acid-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.     

In silico assessment of the metabolic capabilities of an engineered functional reversal of the β-oxidation cycle for the synthesis of longer-chain (C≥4) products

The modularity and versatility of an engineered functional reversal of the β-oxidation cycle make it a promising platform for the synthesis of longer-chain (C≥4) products. While the pathway has recently been exploited for the production of n-alcohols and carboxylic acids, fully capitalizing on its potential for the synthesis of a diverse set of product families requires a system-level assessment of its biosynthetic capabilities. To this end, we utilized a genome scale model of Escherichia coli, in combination with Flux Balance Analysis and Flux Variability Analysis, to determine the key characteristics and constraints of this pathway for the production of a variety of product families under fermentative conditions. This analysis revealed that the production of n-alcohols, alkanes, and fatty acids of lengths C3–C18 could be coupled to cell growth in a strain lacking native fermentative pathways, a characteristic enabling product synthesis at maximum rates, titers, and yields. While energetic and redox constraints limit the production of target compounds from alternative platforms such as the fatty acid biosynthesis and α-ketoacid pathways, the metabolic efficiency of a β-oxidation reversal allows the production of a wide range of products of varying length and functionality. The versatility of this platform was investigated through the simulation of various termination pathways for product synthesis along with the use of different priming molecules, demonstrating its potential for the efficient synthesis of a wide variety of functionalized compounds. Overall, specific metabolic manipulations suggested by this systems-level analysis include deletion of native fermentation pathways, the choice of priming molecules and specific routes for their synthesis, proper choice of termination enzymes, control of flux partitioning at the pyruvate node and the pentose phosphate pathway, and the use of an NADH-dependent trans-enoyl-CoA reductase instead of a ferredoxin-dependent enzyme.