Using Pathway Covering to Explore Connections among Metabolites

Interpreting changes in metabolite abundance in response to experimental treatments or disease states remains a major challenge in metabolomics. Pathway Covering is a new algorithm that takes a list of metabolites (compounds) and determines a minimum-cost set of metabolic pathways in an organism that includes (covers) all the metabolites in the list. We used five functions for assigning costs to pathways, including assigning a constant for all pathways, which yields a solution with the smallest pathway count; two methods that penalize large pathways; one that prefers pathways based on the pathway’s assigned function, and one that loosely corresponds to metabolic flux. The pathway covering set computed by the algorithm can be displayed as a multi-pathway diagram (“pathway collage”) that highlights the covered metabolites. We investigated the pathway covering algorithm by using several datasets from the Metabolomics Workbench. The algorithm is best applied to a list of metabolites with significant statistics and fold-changes with a specified direction of change for each metabolite. The pathway covering algorithm is now available within the Pathway Tools software and BioCyc website.

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Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis

PLoS ONE ◽

10.1371/journal.pone.0256802 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256802

Author(s):

Yuan Pan ◽

Xiao Zhao ◽

Yu Wang ◽

Jun Tan ◽

Da-xia Chen

Keyword(s):

Metabolic Pathways ◽

Metabolic Flux ◽

Developmental Stages ◽

Genomic Library ◽

Active Components ◽

Traditional Chinese Herbal Medicine ◽

Chinese Herbal ◽

Gardenia Jasminoides ◽

Natural Colorants ◽

Metabolite Abundance

Gardenia jasminoides Ellis (G. jasminoides) fruits are used as a resource for obtaining natural colorants and in traditional Chinese herbal medicine. However, G. jasminoides presents a relatively long flowering period and different ripening periods, so there are significant differences in the accumulation of metabolites in fruits of different colors. In addition, the complete metabolic pathways of iridoidsand crocins, which are used as medicinal composition of G. jasminoides, are poorly understood at present. In this research, we comprehensively compared the transcriptome and metabolites profiles of the developmental stages and locations of iridoid and crocin biosynthesis. A large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were detected in four groups of samples, and clear variation in the pattern of metabolite abundance and gene expression were observed among different fruit colors and parts. Geniposide and gardenoside mainly accumulated in the sarcocarp of green fruit (GFS) and the sarcocarp of red fruit (FS), respectively. Crocin mainly accumulated in the peel and sarcocarp of red fruits. In the iridoid pathway, we hypothesized that there was a transport mechanism from the sarcocarp to the peel of G. jasminoides because of the inconsistent expression of G8O, 10-HGO and IS associated with differences in fruit ripening. UGTs play an important role in the biosynthesis of the active components of G. jasminoides. Combined transcriptome and metabonomics analysis showed a negative correlation between the biosynthesis of geniposide and crocin. The redirection of the metabolic flux and the regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoid and crocin in G. jasminoides fruit. Our study expended valuable information for functional genomic library and provided new insights for metabolic engineering of secondary metabolite in G. Jasminoides.

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Metabolic Control of m6A RNA Modification

Metabolites ◽

10.3390/metabo11020080 ◽

2021 ◽

Vol 11 (2) ◽

pp. 80

Author(s):

Joohwan Kim ◽

Gina Lee

Keyword(s):

Cell Fate ◽

Metabolic Pathways ◽

Epigenetic Modification ◽

Genetic Material ◽

Rna Modification ◽

Cell Fate Decisions ◽

Disease States ◽

Evolutionarily Conserved ◽

Regulate Cell Growth ◽

Epigenetic Processes

Nutrients and metabolic pathways regulate cell growth and cell fate decisions via epigenetic modification of DNA and histones. Another key genetic material, RNA, also contains diverse chemical modifications. Among these, N6-methyladenosine (m6A) is the most prevalent and evolutionarily conserved RNA modification. It functions in various aspects of developmental and disease states, by controlling RNA metabolism, such as stability and translation. Similar to other epigenetic processes, m6A modification is regulated by specific enzymes, including writers (methyltransferases), erasers (demethylases), and readers (m6A-binding proteins). As this is a reversible enzymatic process, metabolites can directly influence the flux of this reaction by serving as substrates and/or allosteric regulators. In this review, we will discuss recent understanding of the regulation of m6A RNA modification by metabolites, nutrients, and cellular metabolic pathways.

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Metabolomics in nutrition research–a powerful window into nutritional metabolism

Essays in Biochemistry ◽

10.1042/ebc20160029 ◽

2016 ◽

Vol 60 (5) ◽

pp. 451-458 ◽

Cited By ~ 13

Author(s):

Lorraine Brennan

Keyword(s):

Mass Spectrometry ◽

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Data Acquisition ◽

Small Molecules ◽

Biological Samples ◽

Metabolic Pathways ◽

Recent Trend ◽

Disease States ◽

Nutrition Research

Metabolomics is the study of small molecules present in biological samples. In recent years it has become evident that such small molecules, called metabolites, play a key role in the development of disease states. Furthermore, metabolomic applications can reveal information about alterations in certain metabolic pathways under different conditions. Data acquisition in metabolomics is usually performed using nuclear magnetic resonance (NMR)-based approaches or mass spectrometry (MS)-based approaches with a more recent trend including the application of multiple platforms in order to maximise the coverage in terms of metabolites measured. The application of metabolomics is rapidly increasing and the present review will highlight applications in nutrition research.

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Effects of microcompartmentation on flux distribution and metabolic pools in Chlamydomonas reinhardtii chloroplasts

eLife ◽

10.7554/elife.37960 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 8

Author(s):

Anika Küken ◽

Frederik Sommer ◽

Liliya Yaneva-Roder ◽

Luke CM Mackinder ◽

Melanie Höhne ◽

...

Keyword(s):

Experimental Data ◽

Chlamydomonas Reinhardtii ◽

Metabolic Pathways ◽

Metabolic Flux ◽

Flux Distribution ◽

Photosynthetic Performance ◽

Computational Approach ◽

Cellular Processes ◽

Diffusional Barrier ◽

Computational Strategy

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.

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Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

Nucleic Acids Research ◽

10.1093/nar/gkz1123 ◽

2019 ◽

Vol 48 (2) ◽

pp. 996-1009 ◽

Cited By ~ 19

Author(s):

Yaokang Wu ◽

Taichi Chen ◽

Yanfeng Liu ◽

Rongzhen Tian ◽

Xueqin Lv ◽

...

Keyword(s):

Bacillus Subtilis ◽

Metabolic Pathways ◽

Metabolic Flux ◽

Target Genes ◽

Fine Tuning ◽

Dual Control ◽

Genetic Circuits ◽

Dynamic Regulation ◽

Batch Bioreactor ◽

Acetoin Production

Abstract Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

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Towards Decoding the Metabolic Plasticity in Cancer: Coupling of Gene Regulation and Metabolic Pathways

10.1101/428367 ◽

2018 ◽

Author(s):

Dongya Jia ◽

Mingyang Lu ◽

Kwang Hwa Jung ◽

Jun Hyoung Park ◽

Linglin Yu ◽

...

Keyword(s):

Gene Regulation ◽

Cancer Cells ◽

Metabolic Pathway ◽

Metabolic Pathways ◽

Breast Cancer Cell Lines ◽

Acid Oxidation ◽

Master Regulators ◽

Metabolic Plasticity ◽

Metabolite Abundance

AbstractMetabolic plasticity enables cancer cells to switch their metabolism phenotypes between glycolysis and oxidative phosphorylation (OXPHOS) during tumorigenesis and metastasis. However, it is still largely unknown how cancer cells orchestrate gene regulation to balance their glycolysis and OXPHOS activities for better survival. Here, we establish a theoretical framework to model the coupling of gene regulation and metabolic pathways in cancer. Our modeling results demonstrate a direct association between the activities of AMPK and HIF-1, master regulators of OXPHOS and glycolysis respectively, with the activities of three metabolic pathways: glucose oxidation, glycolysis and fatty acid oxidation (FAO). Guided by the model, we develop metabolic pathway signatures to quantify the activities of glycolysis, FAO and the citric acid cycle of tumor samples by evaluating the expression levels of enzymes involved in corresponding processes. The association of AMPK/HIF-1 activity with metabolic pathway activity, predicted by the model and verified by analyzing the gene expression and metabolite abundance data of patient samples, is further validated by in vitro studies of aggressive triple negative breast cancer cell lines.

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Omics-based label-free metabolic flux inference reveals dysregulation of glucose metabolism in liver associated with obesity

10.1101/2021.06.21.449220 ◽

2021 ◽

Author(s):

Saori Uematsu ◽

Satoshi Ohno ◽

Kaori Y. Tanaka ◽

Atsushi Hatano ◽

Toshiya Kokaji ◽

...

Keyword(s):

Glucose Metabolism ◽

Glucose Homeostasis ◽

Metabolic Pathways ◽

Metabolic Flux ◽

Label Free ◽

Transcriptomic Data ◽

Metabolic Fluxes ◽

Flux Estimation

Glucose homeostasis is maintained by modulating metabolic flux through various metabolic pathways in metabolic organs such as liver, and a change in metabolic flux is regulated by enzymes and metabolites. Obesity represents dysregulation of the glucose homeostasis, but changes in metabolic fluxes and their regulations associated with obesity have not been fully understood. Here we introduce Omics-based Metabolic flux Estimation without Labeling for Extended Trans-omic analysis (OMELET), an approach that uses metabolomic, proteomic and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes and transcripts to the changes in metabolic flux. We identified obesity-associated increases in metabolic fluxes through gluconeogenesis and pyruvate cycle in fasting ob/ob mice. The increased metabolic flux through gluconeogenesis was contributed by increased transcripts, while that through pyruvate cycle by increased transcripts and substrates. OMELET provided quantitative insights into alteration and dysregulation of metabolic flux in liver associated with obesity.

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Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

Applied and Environmental Microbiology ◽

10.1128/aem.01175-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7849-7855 ◽

Cited By ~ 42

Author(s):

Howard H. Chou ◽

Jay D. Keasling

Keyword(s):

Renewable Resources ◽

Metabolic Pathways ◽

Metabolic Flux ◽

Biological Pathways ◽

Enzymatic Reactions ◽

Isopentenyl Diphosphate ◽

New Approach ◽

Synthetic Pathway ◽

Bifunctional Enzymes ◽

Evolution Of Metabolic Pathways

ABSTRACTSynthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell.

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Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

Journal of Bacteriology ◽

10.1128/jb.00490-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4452-4461 ◽

Cited By ~ 102

Author(s):

Daniel Amador-Noguez ◽

Xiao-Jiang Feng ◽

Jing Fan ◽

Nathaniel Roquet ◽

Herschel Rabitz ◽

...

Keyword(s):

Metabolic Pathways ◽

Genome Annotation ◽

Clostridium Acetobutylicum ◽

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Anaerobic Bacteria ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Fermentation Products

ABSTRACT Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/α-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

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Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux

Communications Biology ◽

10.1038/s42003-021-02541-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Lin Lin ◽

Junyu Chen ◽

Ruchira Mitra ◽

Quanxiu Gao ◽

Feiyue Cheng ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Pathways ◽

Carbon Flux ◽

Metabolic Flux ◽

Citrate Synthase ◽

Fine Tuning ◽

Type I ◽

Haloferax Mediterranei ◽

Target Region ◽

Fermentation Period

AbstractThe haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

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