Metabolic fluxes and metabolic engineering

Metabolic engineering is the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology. As such, metabolic engineering emphasizes metabolic pathway integration and relies on metabolic fluxes as determinants of cell physiology and measures of metabolic control. The combination of analytical methods to quantify fluxes and their control with molecular biological techniques to implement genetic modifications is the essence of metabolic engineering. Strategies for metabolic flux determination are reviewed in this paper and it is shown how metabolic fluxes can be used in the systematic elucidation of metabolic control in the framework of reaction grouping and top-down metabolic control analysis.     

PathAligner

MOTIVATION: Analysis of metabolic pathways is a central topic in understanding the relationship between genotype and phenotype. The rapid accumulation of biological data provides the possibility of studying metabolic pathways at both the genomic and the metabolic levels. Retrieving metabolic pathways from current biological data sources, reconstructing metabolic pathways from rudimentary pathway components, and aligning metabolic pathways with each other are major tasks. Our motivation was to develop a conceptual framework and computational system that allows the retrieval of metabolic pathway information and the processing of alignments to reveal the similarities between metabolic pathways. RESULTS: PathAligner extracts metabolic information from biological databases via the Internet and builds metabolic pathways with data sources of genes, sequences, enzymes, metabolites etc. It provides an easy-to-use interface to retrieve, display and manipulate metabolic information. PathAligner also provides an alignment method to compare the similarity between metabolic pathways. AVAILABILITY: PathAligner is available at http://bibiserv.techfak.uni-bielefeld.de/pathaligner.     

代谢工程的发展及其应用

本首先论述了代谢工程的代谢网络理论、代谢分析、节点分析和中心代谢物作用机理等代谢工程理论基础。然后,分析了代谢工程的各种具体设计思路,并以实际例子作了详细说明。另外还对代谢工程的新兴研究方向－逆代谢工程进行了简单说明。     

An algorithm for efficient identification of branched metabolic pathways

This article presents a new graph-based algorithm for identifying branched metabolic pathways in multi-genome scale metabolic data. The term branched is used to refer to metabolic pathways between compounds that consist of multiple pathways that interact biochemically. A branched pathway may produce a target compound through a combination of linear pathways that split compounds into smaller ones, work in parallel with many compounds, and join compounds into larger ones. While branched metabolic pathways predominate in metabolic networks, most previous work has focused on identifying linear metabolic pathways. The ability to automatically identify branched pathways is important in applications that require a deeper understanding of metabolism, such as metabolic engineering and drug target identification. The algorithm presented in this article utilizes explicit atom tracking to identify linear metabolic pathways and then merges them together into branched metabolic pathways. We provide results on several well-characterized metabolic pathways that demonstrate that the new merging approach can efficiently find biologically relevant branched metabolic pathways.     

Filament formation by metabolic enzymes—A new twist on regulation

Compartmentalization of metabolic enzymes through protein–protein interactions is an emerging mechanism for localizing and regulating metabolic activity. Self-assembly into linear filaments is a common strategy for cellular compartmentalization of enzymes. Polymerization is often driven by changes in the metabolic state of the cell, suggesting that it is a strategy for shifting metabolic flux in response to cellular demand. Although polymerization of metabolic enzymes is widespread, observed from bacteria to humans, we are just beginning to appreciate their role in regulating cellular metabolism. In most cases, one functional role of metabolic enzyme filaments is allosteric control of enzyme activity. Here, we highlight recent findings, providing insight into the structural and functional significance of filamentation of metabolic enzymes in cells.     

Inbreeding depression in the effects of body mass on energy use

Large organisms have higher metabolic rates than small organisms but, if we compare their relative metabolic rates (i.e. per gram of tissue), this relationship is very often reversed. The pervasiveness of this phenomenon, called metabolic scaling, has attracted several theoretical explanations, and also produced lingering debate over whether metabolic scaling is a physically constrained and universally constant phenomenon or a more variable and evolutionarily malleable trait. To bring novel insights to this debate, we manipulated male Gryllodes sigillatus crickets' coefficients of inbreeding to determine whether metabolic scaling is sensitive to the manipulation of genetic quality. Because inbreeding depression is inversely related to past selection, our results indicate that selection has favoured an overall lower metabolic rate and a less steep slope of metabolic scaling. Altered metabolic scaling as a result of inbreeding was found to be caused by increased variation in metabolic rate, suggesting the existence of balancing selection towards intermediate metabolic rates. Although we found effects of inbreeding on metabolic scaling, much of the relationship between body mass and metabolic rate remained unexplained, leaving plenty of room for speculation concerning the fixed constraints that might affect evolutionary trajectories. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 309–317.     

Physiology of microbial cell and metabolic engineering

This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering. The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes, and prospects of physiological and evolutionary approaches in metabolic engineering.     

The landscape of metabolic pathway dependencies in cancer cell lines

The metabolic reprogramming of cancer cells creates metabolic vulnerabilities that can be therapeutically targeted. However, our understanding of metabolic dependencies and the pathway crosstalk that creates these vulnerabilities in cancer cells remains incomplete. Here, by integrating gene expression data with genetic loss-of-function and pharmacological screening data from hundreds of cancer cell lines, we identified metabolic vulnerabilities at the level of pathways rather than individual genes. This approach revealed that metabolic pathway dependencies are highly context-specific such that cancer cells are vulnerable to inhibition of one metabolic pathway only when activity of another metabolic pathway is altered. Notably, we also found that the no single metabolic pathway was universally essential, suggesting that cancer cells are not invariably dependent on any metabolic pathway. In addition, we confirmed that cell culture medium is a major confounding factor for the analysis of metabolic pathway vulnerabilities. Nevertheless, we found robust associations between metabolic pathway activity and sensitivity to clinically approved drugs that were independent of cell culture medium. Lastly, we used parallel integration of pharmacological and genetic dependency data to confidently identify metabolic pathway vulnerabilities. Taken together, this study serves as a comprehensive characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines.     

Physiology of microbial cells and metabolic engineering

This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering. The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes, and prospects of physiological and evolutionary approaches in metabolic engineering.     

代谢工程——生物工程学科的新兴研究领域

代谢工程包括推理性代谢工程及逆代谢工程。针对限制生物活性的因素 ,从不同的途径设计代谢改变策略 ,采用重组DNA技术解除或削弱该影响 ,提高目的产物的产率 ,是生化工程学科提高生物活性的新兴研究领域。讨论了推理性代谢工程及逆代谢工程的设计及应用。     

Probing metabolic pathways with isotopic tracers: insights from mammalian metabolic physiology

Metabolic Engineering offers an opportunity to forge a link between metabolic physiologists, working with mammalian systems and metabolic engineers. Many parallels may be drawn between the specific modification of metabolic networks to improve cellular properties and the analysis of metabolic networks in search of causes of disease. At the core of both fields is the measurement of fluxes. This issue of Metabolic Engineering highlights important topics: mammalian metabolic physiology where estimating fluxes is challenging. The challenges come from compartmentation of metabolites, from dilution of tracer by endogenous pools, and from the difficulty of sampling relevant pools. The common theme across these investigations is the use of isotopic tracers. The wide variety of tracers and tracer analysis techniques in use in mammalian metabolic physiology reflects the complexity of the systems under study. In presenting these examples from the field of mammalian metabolic physiology, our goal is to strengthen the linkages between physiologists and engineers as we develop our knowledge and appreciation of the complexity of metabolic networks.     

Active and resting metabolism in birds: allometry, phylogeny and ecology

Variation in resting metabolic rate is strongly correlated with differences in body weight among birds. The lowest taxonomic level at which most of the variance in resting metabolic rate and body weight is evident for the sample is among families within orders. The allometric exponent across family points is 0.67. This exponent accords with the surface area interpretation of metabolic scaling based on considerations of heat loss. Deviations of family points from this allometric line are used to examine how resting metabolic rates differ among taxa, and whether variation in resting metabolic rate is correlated with broad differences in ecology and behaviour. Despite the strong correlation between resting metabolic rate and body weight, there is evidence for adaptive departures from the allometric line, and possible selective forces are discussed.
The allometric scaling of active metabolic rate is compared with that of resting metabolic rate. The allometric exponents for the two levels of energy expenditure differ, demonstrating that active small-bodied birds require proportionately more energy per unit time above resting levels than do active large-bodied birds. No consistent evidence was found to indicate that the different methods used to estimate active metabolic rate result in systematic bias. Birds require more energy relative to body size when undertaking breeding activities than at other stages of the annual cycle.     

Immunometabolism features of metabolic deregulation and cancer

Immunometabolism is a branch dealing at the interface of immune functionalities and metabolic regulations. Considered as a bidirectional trafficking, metabolic contents and their precursors bring a considerable change in immune cells signal transductions which as a result affect the metabolic organs and states as an implication. Lipid metabolic ingredients form a major chunk of daily diet and have a proven contribution in immune cells induction, which then undergo metabolic pathway shuffling inside their ownself. Lipid metabolic states activate relevant metabolic pathways inside immune cells that in turn prime appropriate responses to outside environment in various states including lipid metabolic disorders itself and cancers as an extension. Although data on Immunometabolism are still growing, but scientific community need to adjust and readjust according to recent data on given subject. This review attempts to provide current important data on Immunometabolism and consequently its metabolic ramifications. Incumbent data on various lipid metabolic deregulations like obesity, metabolic syndrome, obese asthma and atherosclerosis are analysed. Further, metabolic repercussions on cancers and its immune modalities are also analysed.     

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/.     

Identification of distributed metabolic objectives in the hypermetabolic liver by flux and energy balance analysis

A methodology for inferring distributed metabolic objectives from time series flux data is developed by combining metabolic flux analysis, pathway identification, free energy balances, and nested optimization. This methodology is used to investigate the metabolic response of the rat liver to burn injury-induced whole body inflammation. Gibbs free energy changes were computed for stoichiometrically balanced sequences of reactions, or pathways, rather than individual reactions, to account for energetic coupling between reactions. Systematic enumeration of pathways proceeded by elementary flux mode (EFM) analysis. Together with stoichiometric balances and external metabolite flux measurements, the DeltaG(PATH)(o) criterion provided sufficient constraints to solve a series of nested optimization problems on the metabolic goal functions and associated flux distributions of fasted livers during the first-week time course of burn injury. The optimization results suggest that there is a consistent metabolic goal function for the liver that is insensitive to the changing metabolic burdens experienced by the liver during the first-week time course. As defined by the goal function coefficients, the global metabolic objective was to distribute the metabolic resources between amino acid metabolism and ketone body synthesis. These findings point to a role for the time-invariant structure of the metabolic reaction network, expressed as stoichiometric and thermodynamic constraints, in shaping the cellular metabolic objective.     

Flux analysis and metabolomics for systematic metabolic engineering of microorganisms

Rational engineering of metabolism is important for bio-production using microorganisms. Metabolic design based on in silico simulations and experimental validation of the metabolic state in the engineered strain helps in accomplishing systematic metabolic engineering. Flux balance analysis (FBA) is a method for the prediction of metabolic phenotype, and many applications have been developed using FBA to design metabolic networks. Elementary mode analysis (EMA) and ensemble modeling techniques are also useful tools for in silico strain design. The metabolome and flux distribution of the metabolic pathways enable us to evaluate the metabolic state and provide useful clues to improve target productivity. Here, we reviewed several computational applications for metabolic engineering by using genome-scale metabolic models of microorganisms. We also discussed the recent progress made in the field of metabolomics and ¹³C-metabolic flux analysis techniques, and reviewed these applications pertaining to bio-production development. Because these in silico or experimental approaches have their respective advantages and disadvantages, the combined usage of these methods is complementary and effective for metabolic engineering.     

Interpreting metabolic complexity via isotope-assisted metabolic flux analysis

Isotope-assisted metabolic flux analysis (iMFA) is a powerful method to mathematically determine the metabolic fluxome from experimental isotope labeling data and a metabolic network model. While iMFA was originally developed for industrial biotechnological applications, it is increasingly used to analyze eukaryotic cell metabolism in physiological and pathological states. In this review, we explain how iMFA estimates the intracellular fluxome, including data and network model (inputs), the optimization-based data fitting (process), and the flux map (output). We then describe how iMFA enables analysis of metabolic complexities and discovery of metabolic pathways. Our goal is to expand the use of iMFA in metabolism research, which is essential to maximizing the impact of metabolic experiments and continuing to advance iMFA and biocomputational techniques.     

Evolutionary models of metabolism, behaviour and personality

I explore the relationship between metabolism and personality by establishing how selection acts on metabolic rate and risk-taking in the context of a trade-off between energy and predation. Using a simple time budget model, I show that a high resting metabolic rate is not necessarily associated with a high daily energy expenditure. The metabolic rate that minimizes the time spent foraging does not maximize the net gain rate while foraging, and it is not always advantageous for animals to have a higher metabolic rate when food availability is high. A model based on minimizing the ratio of mortality rate to net gain rate is used to determine how a willingness to take risks should be correlated with metabolic rate. My results establish that it is not always advantageous for animals to take greater risks when metabolic rate is high. When foraging intensity and metabolic rate coevolve, I show that in a particular case different combinations of foraging intensity and metabolic rate can have equal fitness.     

MetaFluxNet: the management of metabolic reaction information and quantitative metabolic flux analysis

SUMMARY: MetaFluxNet is a program package for managing information on the metabolic reaction network and for quantitatively analyzing metabolic fluxes in an interactive and customized way. It allows users to interpret and examine metabolic behavior in response to genetic and/or environmental modifications. As a result, quantitative in silico simulations of metabolic pathways can be carried out to understand the metabolic status and to design the metabolic engineering strategies. The main features of the program include a well-developed model construction environment, user-friendly interface for metabolic flux analysis (MFA), comparative MFA of strains having different genotypes under various environmental conditions, and automated pathway layout creation. AVAILABILITY: http://mbel.kaist.ac.kr/ Supplementary information: A manual for MetaFluxNet is available as PDF file.