Cross-evaluation of E. coli's operon structures via a whole-cell model suggests alternative cellular benefits for low- versus high-expressing operons.

Many bacteria use operons to coregulate genes, but it remains unclear how operons benefit bacteria. We integrated E. coli's 788 polycistronic operons and 1,231 transcription units into an existing whole-cell model and found inconsistencies between the proposed operon structures and the RNA-seq read counts that the model was parameterized from. We resolved these inconsistencies through iterative, model-guided corrections to both datasets, including the correction of RNA-seq counts of short genes that were misreported as zero by existing alignment algorithms. The resulting model suggested two main modes by which operons benefit bacteria. For 86% of low-expression operons, adding operons increased the co-expression probabilities of their constituent proteins, whereas for 92% of high-expression operons, adding operons resulted in more stable expression ratios between the proteins. These simulations underscored the need for further experimental work on how operons reduce noise and synchronize both the expression timing and the quantity of constituent genes. A record of this paper's transparent peer review process is included in the supplemental information.

SynRoute: A Retrosynthetic Planning Software.

Computer-assisted synthetic planning has seen major advancements that stem from the availability of large reaction databases and artificial intelligence methodologies. SynRoute is a new retrosynthetic planning software tool that uses a relatively small number of general reaction templates, currently 263, along with a literature-based reaction database to find short, practical synthetic routes for target compounds. For each reaction template, a machine learning classifier is trained using data from the Pistachio reaction database to predict whether new computer-generated reactions based on the template are likely to work experimentally in the laboratory. This reaction generation methodology is used together with a vectorized Dijkstra-like search of top-scoring routes organized by synthetic strategies for easy browsing by a synthetic chemist. SynRoute was able to find routes for an average of 83% of compounds based on selection of random subsets of drug-like compounds from the ChEMBL database. Laboratory evaluation of 12 routes produced by SynRoute, to synthesize compounds not from the previous random subsets, demonstrated the ability to produce feasible overall synthetic strategies for all compounds evaluated.

The EcoCyc Database (2023).

EcoCyc is a bioinformatics database available online at EcoCyc.org that describes the genome and the biochemical machinery of Escherichia coli K-12 MG1655. The long-term goal of the project is to describe the complete molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists and for biologists who work with related microorganisms. The database includes information pages on each E. coli gene product, metabolite, reaction, operon, and metabolic pathway. The database also includes information on the regulation of gene expression, E. coli gene essentiality, and nutrient conditions that do or do not support the growth of E. coli. The website and downloadable software contain tools for the analysis of high-throughput data sets. In addition, a steady-state metabolic flux model is generated from each new version of EcoCyc and can be executed online. The model can predict metabolic flux rates, nutrient uptake rates, and growth rates for different gene knockouts and nutrient conditions. Data generated from a whole-cell model that is parameterized from the latest data on EcoCyc are also available. This review outlines the data content of EcoCyc and of the procedures by which this content is generated.

Pathway Tools Management of Pathway/Genome Data for Microbial Communities.

The Pathway Tools (PTools) software provides a suite of capabilities for storing and analyzing integrated collections of genomic and metabolic information in the form of organism-specific Pathway/Genome Databases (PGDBs). A microbial community is represented in PTools by generating a PGDB from each metagenome-assembled genome (MAG). PTools computes a metabolic reconstruction for each organism, and predicts its operons. The properties of individual MAGs can be investigated using the many search and visualization operations within PTools. PTools also enables the user to investigate the properties of the microbial community by issuing searches across the full community, and by performing comparative operations across genome and pathway information. The software can generate a metabolic network diagram for the community, and it can overlay community omics datasets on that network diagram. PTools also provides a tool for searching for metabolic transformation routes across an organism community.

The EcoCyc Database in 2021.

The EcoCyc model-organism database collects and summarizes experimental data for Escherichia coli K-12. EcoCyc is regularly updated by the manual curation of individual database entries, such as genes, proteins, and metabolic pathways, and by the programmatic addition of results from select high-throughput analyses. Updates to the Pathway Tools software that supports EcoCyc and to the web interface that enables user access have continuously improved its usability and expanded its functionality. This article highlights recent improvements to the curated data in the areas of metabolism, transport, DNA repair, and regulation of gene expression. New and revised data analysis and visualization tools include an interactive metabolic network explorer, a circular genome viewer, and various improvements to the speed and usability of existing tools.

Leveraging Curation Among Escherichia coli Pathway/Genome Databases Using Ortholog-Based Annotation Propagation.

Updating genome databases to reflect newly published molecular findings for an organism was hard enough when only a single strain of a given organism had been sequenced. With multiple sequenced strains now available for many organisms, the challenge has grown significantly because of the still-limited resources available for the manual curation that corrects errors and captures new knowledge. We have developed a method to automatically propagate multiple types of curated knowledge from genes and proteins in one genome database to their orthologs in uncurated databases for related strains, imposing several quality-control filters to reduce the chances of introducing errors. We have applied this method to propagate information from the highly curated EcoCyc database for Escherichia coli K-12 to databases for 480 other Escherichia coli strains in the BioCyc database collection. The increase in value and utility of the target databases after propagation is considerable. Target databases received updates for an average of 2,535 proteins each. In addition to widespread addition and regularization of gene and protein names, 97% of the target databases were improved by the addition of at least 200 new protein complexes, at least 800 new or updated reaction assignments, and at least 2,400 sets of GO annotations.

Pathway Tools Visualization of Organism-Scale Metabolic Networks.

Metabolomics, synthetic biology, and microbiome research demand information about organism-scale metabolic networks. The convergence of genome sequencing and computational inference of metabolic networks has enabled great progress toward satisfying that demand by generating metabolic reconstructions from the genomes of thousands of sequenced organisms. Visualization of whole metabolic networks is critical for aiding researchers in understanding, analyzing, and exploiting those reconstructions. We have developed bioinformatics software tools that automatically generate a full metabolic-network diagram for an organism, and that enable searching and analyses of the network. The software generates metabolic-network diagrams for unicellular organisms, for multi-cellular organisms, and for pan-genomes and organism communities. Search tools enable users to find genes, metabolites, enzymes, reactions, and pathways within a diagram. The diagrams are zoomable to enable researchers to study local neighborhoods in detail and to see the big picture. The diagrams also serve as tools for comparison of metabolic networks and for interpreting high-throughput datasets, including transcriptomics, metabolomics, and reaction fluxes computed by metabolic models. These data can be overlaid on the metabolic charts to produce animated zoomable displays of metabolic flux and metabolite abundance. The BioCyc.org website contains whole-network diagrams for more than 18,000 sequenced organisms. The ready availability of organism-specific metabolic network diagrams and associated tools for almost any sequenced organism are useful for researchers working to better understand the metabolism of their organism and to interpret high-throughput datasets in a metabolic context.

Pathway Tools version 23.0 update: software for pathway/genome informatics and systems biology.

MOTIVATION: Biological systems function through dynamic interactions among genes and their products, regulatory circuits and metabolic networks. Our development of the Pathway Tools software was motivated by the need to construct biological knowledge resources that combine these many types of data, and that enable users to find and comprehend data of interest as quickly as possible through query and visualization tools. Further, we sought to support the development of metabolic flux models from pathway databases, and to use pathway information to leverage the interpretation of high-throughput data sets. RESULTS: In the past 4 years we have enhanced the already extensive Pathway Tools software in several respects. It can now support metabolic-model execution through the Web, it provides a more accurate gap filler for metabolic models; it supports development of models for organism communities distributed across a spatial grid; and model results may be visualized graphically. Pathway Tools supports several new omics-data analysis tools including the Omics Dashboard, multi-pathway diagrams called pathway collages, a pathway-covering algorithm for metabolomics data analysis and an algorithm for generating mechanistic explanations of multi-omics data. We have also improved the core pathway/genome databases management capabilities of the software, providing new multi-organism search tools for organism communities, improved graphics rendering, faster performance and re-designed gene and metabolite pages. AVAILABILITY: The software is free for academic use; a fee is required for commercial use. See http://pathwaytools.com. CONTACT: pkarp@ai.sri.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Briefings in Bioinformatics online.

The MetaCyc database of metabolic pathways and enzymes - a 2019 update.

MetaCyc (MetaCyc.org) is a comprehensive reference database of metabolic pathways and enzymes from all domains of life. It contains 2749 pathways derived from more than 60 000 publications, making it the largest curated collection of metabolic pathways. The data in MetaCyc are evidence-based and richly curated, resulting in an encyclopedic reference tool for metabolism. MetaCyc is also used as a knowledge base for generating thousands of organism-specific Pathway/Genome Databases (PGDBs), which are available in BioCyc.org and other genomic portals. This article provides an update on the developments in MetaCyc during September 2017 to August 2019, up to version 23.1. Some of the topics that received intensive curation during this period include cobamides biosynthesis, sterol metabolism, fatty acid biosynthesis, lipid metabolism, carotenoid metabolism, protein glycosylation, antibiotics and cytotoxins biosynthesis, siderophore biosynthesis, bioluminescence, vitamin K metabolism, brominated compound metabolism, plant secondary metabolism and human metabolism. Other additions include modifications to the GlycanBuilder software that enable displaying glycans using symbolic representation, improved graphics and fonts for web displays, improvements in the PathoLogic component of Pathway Tools, and the optional addition of regulatory information to pathway diagrams.

Metabolic route computation in organism communities.

BACKGROUND: Microbiomes are complex aggregates of organisms, each of which has its own extensive metabolic network. A variety of metabolites are exchanged between the microbes. The challenge we address is understanding the overall metabolic capabilities of a microbiome: through what series of metabolic transformations can a microbiome convert a starting compound to an ending compound? RESULTS: We developed an efficient software tool to search for metabolic routes that include metabolic reactions from multiple organisms. The metabolic network for each organism is obtained from BioCyc, where the network was inferred from the annotated genome. The tool searches for optimal metabolic routes that minimize the number of reactions in each route, maximize the number of atoms conserved between the starting and ending compounds, and minimize the number of organism switches. The tool pre-computes the reaction sets found in each organism from BioCyc to facilitate fast computation of the reactions defined in a researcher-specified organism set. The generated routes are depicted graphically, and for each reaction in a route, the tool lists the organisms that can catalyze that reaction. We present solutions for three route-finding problems in the human gut microbiome: (1) production of indoxyl sulfate, (2) production of trimethylamine N-oxide (TMAO), and (3) synthesis and degradation of autoinducers. The optimal routes computed by our multi-organism route-search (MORS) tool for indoxyl sulfate and TMAO were the same as routes reported in the literature. CONCLUSIONS: Our tool quickly found plausible routes for the discussed multi-organism route-finding problems. The routes shed light on how diverse organisms cooperate to perform multi-step metabolic transformations. Our tool enables scientists to consider multiple alternative routes and identifies the organisms responsible for each reaction.

A Comparison of Microbial Genome Web Portals.

Microbial genome web portals have a broad range of capabilities that address a number of information-finding and analysis needs for scientists. This article compares the capabilities of the major microbial genome web portals to aid researchers in determining which portal(s) are best suited to their needs. We assessed both the bioinformatics tools and the data content of BioCyc, KEGG, Ensembl Bacteria, KBase, IMG, and PATRIC. For each portal, our assessment compared and tallied the available capabilities. The strengths of BioCyc include its genomic and metabolic tools, multi-search capabilities, table-based analysis tools, regulatory network tools and data, omics data analysis tools, breadth of data content, and large amount of curated data. The strengths of KEGG include its genomic and metabolic tools. The strengths of Ensembl Bacteria include its genomic tools and large number of genomes. The strengths of KBase include its genomic tools and metabolic models. The strengths of IMG include its genomic tools, multi-search capabilities, large number of genomes, table-based analysis tools, and breadth of data content. The strengths of PATRIC include its large number of genomes, table-based analysis tools, metabolic models, and breadth of data content.

The BioCyc collection of microbial genomes and metabolic pathways.

BioCyc.org is a microbial genome Web portal that combines thousands of genomes with additional information inferred by computer programs, imported from other databases and curated from the biomedical literature by biologist curators. BioCyc also provides an extensive range of query tools, visualization services and analysis software. Recent advances in BioCyc include an expansion in the content of BioCyc in terms of both the number of genomes and the types of information available for each genome; an expansion in the amount of curated content within BioCyc; and new developments in the BioCyc software tools including redesigned gene/protein pages and metabolite pages; new search tools; a new sequence-alignment tool; a new tool for visualizing groups of related metabolic pathways; and a facility called SmartTables, which enables biologists to perform analyses that previously would have required a programmer's assistance.

The EcoCyc Database.

EcoCyc is a bioinformatics database available at EcoCyc.org that describes the genome and the biochemical machinery of Escherichia coli K-12 MG1655. The long-term goal of the project is to describe the complete molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists and for biologists who work with related microorganisms. The database includes information pages on each E. coli gene product, metabolite, reaction, operon, and metabolic pathway. The database also includes information on E. coli gene essentiality and on nutrient conditions that do or do not support the growth of E. coli. The website and downloadable software contain tools for analysis of high-throughput data sets. In addition, a steady-state metabolic flux model is generated from each new version of EcoCyc and can be executed via EcoCyc.org. The model can predict metabolic flux rates, nutrient uptake rates, and growth rates for different gene knockouts and nutrient conditions. This review outlines the data content of EcoCyc and of the procedures by which this content is generated.

The MetaCyc database of metabolic pathways and enzymes.

MetaCyc (https://MetaCyc.org) is a comprehensive reference database of metabolic pathways and enzymes from all domains of life. It contains more than 2570 pathways derived from >54 000 publications, making it the largest curated collection of metabolic pathways. The data in MetaCyc is strictly evidence-based and richly curated, resulting in an encyclopedic reference tool for metabolism. MetaCyc is also used as a knowledge base for generating thousands of organism-specific Pathway/Genome Databases (PGDBs), which are available in the BioCyc (https://BioCyc.org) and other PGDB collections. This article provides an update on the developments in MetaCyc during the past two years, including the expansion of data and addition of new features.

The EcoCyc database: reflecting new knowledge about Escherichia coli K-12.

EcoCyc (EcoCyc.org) is a freely accessible, comprehensive database that collects and summarizes experimental data for Escherichia coli K-12, the best-studied bacterial model organism. New experimental discoveries about gene products, their function and regulation, new metabolic pathways, enzymes and cofactors are regularly added to EcoCyc. New SmartTable tools allow users to browse collections of related EcoCyc content. SmartTables can also serve as repositories for user- or curator-generated lists. EcoCyc now supports running and modifying E. coli metabolic models directly on the EcoCyc website.

Pathway Tools version 19.0 update: software for pathway/genome informatics and systems biology.

Pathway Tools is a bioinformatics software environment with a broad set of capabilities. The software provides genome-informatics tools such as a genome browser, sequence alignments, a genome-variant analyzer and comparative-genomics operations. It offers metabolic-informatics tools, such as metabolic reconstruction, quantitative metabolic modeling, prediction of reaction atom mappings and metabolic route search. Pathway Tools also provides regulatory-informatics tools, such as the ability to represent and visualize a wide range of regulatory interactions. This article outlines the advances in Pathway Tools in the past 5 years. Major additions include components for metabolic modeling, metabolic route search, computation of atom mappings and estimation of compound Gibbs free energies of formation; addition of editors for signaling pathways, for genome sequences and for cellular architecture; storage of gene essentiality data and phenotype data; display of multiple alignments, and of signaling and electron-transport pathways; and development of Python and web-services application programming interfaces. Scientists around the world have created more than 9800 Pathway/Genome Databases by using Pathway Tools, many of which are curated databases for important model organisms.

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.

The MetaCyc database (MetaCyc.org) is a freely accessible comprehensive database describing metabolic pathways and enzymes from all domains of life. The majority of MetaCyc pathways are small-molecule metabolic pathways that have been experimentally determined. MetaCyc contains more than 2400 pathways derived from >46,000 publications, and is the largest curated collection of metabolic pathways. BioCyc (BioCyc.org) is a collection of 5700 organism-specific Pathway/Genome Databases (PGDBs), each containing the full genome and predicted metabolic network of one organism, including metabolites, enzymes, reactions, metabolic pathways, predicted operons, transport systems, and pathway-hole fillers. The BioCyc website offers a variety of tools for querying and analyzing PGDBs, including Omics Viewers and tools for comparative analysis. This article provides an update of new developments in MetaCyc and BioCyc during the last two years, including addition of Gibbs free energy values for compounds and reactions; redesign of the primary gene/protein page; addition of a tool for creating diagrams containing multiple linked pathways; several new search capabilities, including searching for genes based on sequence patterns, searching for databases based on an organism's phenotypes, and a cross-organism search; and a metabolite identifier translation service.

Representation and inference of cellular architecture for metabolic reconstruction and modeling.

MOTIVATION: Metabolic modeling depends on accurately representing the cellular locations of enzyme-catalyzed and transport reactions. We sought to develop a representation of cellular compartmentation that would accurately capture cellular location information. We further sought a representation that would support automated inference of the cellular compartments present in newly sequenced organisms to speed model development, and that would enable representing the cellular compartments present in multiple cell types within a multicellular organism. RESULTS: We define the cellular architecture of a unicellular organism, or of a cell type from a multicellular organism, as the collection of cellular components it contains plus the topological relationships among those components. We developed a tool for inferring cellular architectures across many domains of life and extended our Cell Component Ontology to enable representation of the inferred architectures. We provide software for visualizing cellular architectures to verify their correctness and software for editing cellular architectures to modify or correct them. We also developed a representation that records the cellular compartment assignments of reactions with minimal duplication of information. AVAILABILITY AND IMPLEMENTATION: The Cell Component Ontology is freely available. The Pathway Tools software is freely available for academic research and is available for a fee for commercial use. CONTACT: pkarp@ai.sri.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational Metabolomics Operations at BioCyc.org.

BioCyc.org is a genome and metabolic pathway web portal covering 5500 organisms, including Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae and Escherichia coli. These organism-specific databases have undergone variable degrees of curation. The EcoCyc (Escherichia coli Encyclopedia) database is the most highly curated; its contents have been derived from 27,000 publications. The MetaCyc (Metabolic Encyclopedia) database within BioCyc is a "universal" metabolic database that describes pathways, reactions, enzymes and metabolites from all domains of life. Metabolic pathways provide an organizing framework for analyzing metabolomics data, and the BioCyc website provides computational operations for metabolomics data that include metabolite search and translation of metabolite identifiers across multiple metabolite databases. The site allows researchers to store and manipulate metabolite lists using a facility called SmartTables, which supports metabolite enrichment analysis. That analysis operation identifies metabolite sets that are statistically over-represented for the substrates of specific metabolic pathways. BioCyc also enables visualization of metabolomics data on individual pathway diagrams and on the organism-specific metabolic map diagrams that are available for every BioCyc organism. Most of these operations are available both interactively and as programmatic web services.

Optimal metabolic route search based on atom mappings.

MOTIVATION: A key computational problem in metabolic engineering is finding efficient metabolic routes from a source to a target compound in genome-scale reaction networks, potentially considering the addition of new reactions. Efficiency can be based on many factors, such as route lengths, atoms conserved and the number of new reactions, and the new enzymes to catalyze them, added to the route. Fast algorithms are needed to systematically search these large genome-scale reaction networks. RESULTS: We present the algorithm used in the new RouteSearch tool within the Pathway Tools software. This algorithm is based on a general Branch-and-Bound search and involves constructing a network of atom mappings to facilitate efficient searching. As far as we know, it is the first published algorithm that finds guaranteed optimal routes where atom conservation is part of the optimality criteria. RouteSearch includes a graphical user interface that speeds user understanding of its search results. We evaluated the algorithm on five example metabolic-engineering problems from the literature; for one problem the published solution was equivalent to the optimal route found by RouteSearch; for the remaining four problems, RouteSearch found the published solution as one of its best-scored solutions. These problems were each solved in less than 5 s of computational time. AVAILABILITY AND IMPLEMENTATION: RouteSearch is accessible at BioCyc.org by using the menu command Metabolism --> Metabolic RouteSearch and by downloading Pathway Tools. Pathway Tools software is freely available to academic users, and for a fee to commercial users. Download from: http://biocyc.org/download.shtml.