Genome-scale mapping models and algorithms for stationary and instationary MFA-based metabolic flux elucidation

Metabolic models used in 13C metabolic flux analysis (13C-MFA) generally include a limited number of reactions primarily from central metabolism, neglecting degradation pathways and atom transition contributions for reactions outside central metabolism. This study addresses the impact on prediction fidelity of scaling-up core bacterial and cyanobacterial mapping models to a genome-scale carbon mapping (GSCM) models, imEco726 (668 reaction and 566 metabolites) and imSyn711 (731 reactions, 679 metabolites) for E. coli and Synechocystis PCC 6803, respectively, representing a ten-fold increase in model size. The GSCM models are constructed using the CLCA algorithm following reduction of the corresponding metabolic models, iAF1260 and iSyn731, using experimentally measured biomass and product yield during growth on glucose and CO2, respectively. The mapping models are then deployed for flux elucidation using isotopic steady-state MFA for E. coli to recapitulate experimentally observed labeling distributions of 12 measured amino acids, and isotopic instationary MFA for Synechocystis, to recapitulate labeling dynamics of 15 central metabolites. In both models, 80% of all fluxes varies less than onetenth of the basis carbon substrate uptake rate primarily due to the flux coupling with biomass production. Overall, we find that both the topology and estimated values of the metabolic fluxes remain largely consistent between the core and GSMM models for E. coli. Stepping up to a genome-scale mapping model leads to wider flux inference ranges for 20 key reactions present in the core model. The glycolysis flux range doubles due to the possibility of active gluconeogenesis, the TCA flux range expanded by 80% due to the availability of a bypass through arginine consistent with labeling data, and the transhydrogenase reaction flux was essentially unresolved due to the presence of as many as five routes for the inter-conversion of NADPH to NADH afforded by the genome-scale model. By globally accounting for ATP demands in the GSMM model the unused ATP decreased drastically with the lower bound matching the maintenance ATP requirement. A non-zero flux for the arginine degradation pathway was identified to meet biomass precursor demands as detailed in the iAF1260 model. Significant flux range shifts were observed using a GSCM model compared to a core model in Synechocystis arising from the inclusion of 18 novel carbon paths in the GSCM model. In particular, no flux is channeled through the oxidative pentose phosphate pathway, resulting in a reduced carbon fixation flux. In addition, a higher flux is seen through the Transaldolase reaction, serving as a bypass route to Fructose bisphosphatase. Serine and glycine are found to be synthesized from 3-phosphoglycerate and the photorespiratory pathway, respectively. Pyruvate is synthesized exclusively via the malate bypass with trace contributions from pyruvate kinase. Furthermore, trace flux is predicted through the lower TCA cycle indicating either pathway incompleteness or dispensability during photoautotrophic growth. Despite these differences, 80% of all reactions in both genome-scale models are resolved to within 10% of the respective substrate uptake rate due to the presence of 411 and 407 growth-coupled reactions in E. coli and Synechocystis, respectively. Flux ranges obtained with GSCM models are compared with those obtained upon projecting core model ranges on to a genome-scale metabolic model to elucidate the loss of information and erroneous biological inferences about pathway usage arising from assumptions contained within core models, reaffirming the importance of using mapping models with global carbon path coverage in 13C metabolic flux analysis

MetRxn: a knowledgebase of metabolites and reactions spanning metabolic models and databases

<p>Abstract</p> <p>Background</p> <p>Increasingly, metabolite and reaction information is organized in the form of genome-scale metabolic reconstructions that describe the reaction stoichiometry, directionality, and gene to protein to reaction associations. A key bottleneck in the pace of reconstruction of new, high-quality metabolic models is the inability to directly make use of metabolite/reaction information from biological databases or other models due to incompatibilities in content representation (i.e., metabolites with multiple names across databases and models), stoichiometric errors such as elemental or charge imbalances, and incomplete atomistic detail (e.g., use of generic R-group or non-explicit specification of stereo-specificity).</p> <p>Description</p> <p>MetRxn is a knowledgebase that includes standardized metabolite and reaction descriptions by integrating information from BRENDA, KEGG, MetaCyc, Reactome.org and 44 metabolic models into a single unified data set. All metabolite entries have matched synonyms, resolved protonation states, and are linked to unique structures. All reaction entries are elementally and charge balanced. This is accomplished through the use of a workflow of lexicographic, phonetic, and structural comparison algorithms. MetRxn allows for the download of standardized versions of existing genome-scale metabolic models and the use of metabolic information for the rapid reconstruction of new ones.</p> <p>Conclusions</p> <p>The standardization in description allows for the direct comparison of the metabolite and reaction content between metabolic models and databases and the exhaustive prospecting of pathways for biotechnological production. This ever-growing dataset currently consists of over 76,000 metabolites participating in more than 72,000 reactions (including unresolved entries). MetRxn is hosted on a web-based platform that uses relational database models (MySQL).</p

Cyanobacterial Alkanes Modulate Photosynthetic Cyclic Electron Flow to Assist Growth under Cold Stress

All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons at concentrations similar to chlorophyll. Recently, two universal but mutually exclusive hydrocarbon production pathways in cyanobacteria were discovered. We engineered a mutant of Synechocystis sp. PCC 6803 that produces no alkanes, which grew poorly at low temperatures. We analyzed this defect by assessing the redox kinetics of PSI. The mutant exhibited enhanced cyclic electron flow (CEF), especially at low temperature. CEF raises the ATP:NADPH ratio from photosynthesis and balances reductant requirements of biosynthesis with maintaining the redox poise of the electron transport chain. We conducted in silico flux balance analysis and showed that growth rate reaches a distinct maximum for an intermediate value of CEF equivalent to recycling 1 electron in 4 from PSI to the plastoquinone pool. Based on this analysis, we conclude that the lack of membrane alkanes causes higher CEF, perhaps for maintenance of redox poise. In turn, increased CEF reduces growth by forcing the cell to use less energy-efficient pathways, lowering the quantum efficiency of photosynthesis. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacterial thylakoid membranes: they regulate redox balance and reductant partitioning in these oxygenic photosynthetic cells under stress

Cyanobacterial Alkanes Modulate Photosynthetic Cyclic Electron Flow to Assist Growth under Cold Stress

All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons at concentrations similar to chlorophyll. Recently, two universal but mutually exclusive hydrocarbon production pathways in cyanobacteria were discovered. We engineered a mutant of Synechocystis sp. PCC 6803 that produces no alkanes, which grew poorly at low temperatures. We analyzed this defect by assessing the redox kinetics of PSI. The mutant exhibited enhanced cyclic electron flow (CEF), especially at low temperature. CEF raises the ATP:NADPH ratio from photosynthesis and balances reductant requirements of biosynthesis with maintaining the redox poise of the electron transport chain. We conducted in silico flux balance analysis and showed that growth rate reaches a distinct maximum for an intermediate value of CEF equivalent to recycling 1 electron in 4 from PSI to the plastoquinone pool. Based on this analysis, we conclude that the lack of membrane alkanes causes higher CEF, perhaps for maintenance of redox poise. In turn, increased CEF reduces growth by forcing the cell to use less energy-efficient pathways, lowering the quantum efficiency of photosynthesis. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacterial thylakoid membranes: they regulate redox balance and reductant partitioning in these oxygenic photosynthetic cells under stress

IPRO+/-: a computational protein design tool allowing not only for amino acid changes but also insertions and deletions

The need for enzymes with new or improved catalytic properties and specificities underpins many challenges in both biotechnology and pharmaceutical industry. This is typically carried out by changing the native amino acid composition through single or multiple mutations or recombination. Many computational strategies have been developed for suggesting amino acid changes (i.e., mutations) likely to usher an altered substrate or cofactor specificity, improved thermostability or higher turnover. However, by perusing protein family alignments one can immediately notice the ubiquitous presence of gaps. These gaps imply that not all active enzyme variants have the same backbone length with insertions and deletions (indels) contributing significantly to the possibilities of altering enzyme activity by drastically affecting protein repacking. Currently, no algorithms exist which can systemically position multiple insertions or deletions during in silico protein redesign. In this contribution we introduce IPRO+/-, a first of its kind integrated environment for protein redesign with respect to a single or multiple binding imperatives by not only predicting amino acid changes, but also insertions and deletions (see Figure 1). IPRO+/- allows the user to run standalone programs for (a) predicting energy minimized structural models of an enzyme with a desired indels and/or mutations, (b) computing binding free energies between proteins and small molecules, and (c) performing energy minimization on any protein or protein complex. The contribution will provide an overview of the tasks involved in IPRO+/-, input language terminology, algorithmic details, software implementation specifics and application highlights. IPRO+/- will be made freely downloadable from http://www.maranasgroup.com/software.htm upon publication. Please click Additional Files below to see the full abstract

Optimization based automated curation of metabolic reconstructions

<p>Abstract</p> <p>Background</p> <p>Currently, there exists tens of different microbial and eukaryotic metabolic reconstructions (e.g., <it>Escherichia coli, Saccharomyces cerevisiae</it>, <it>Bacillus subtilis</it>) with many more under development. All of these reconstructions are inherently incomplete with some functionalities missing due to the lack of experimental and/or homology information. A key challenge in the automated generation of genome-scale reconstructions is the elucidation of these gaps and the subsequent generation of hypotheses to bridge them.</p> <p>Results</p> <p>In this work, an optimization based procedure is proposed to identify and eliminate network gaps in these reconstructions. First we identify the metabolites in the metabolic network reconstruction which cannot be produced under any uptake conditions and subsequently we identify the reactions from a customized multi-organism database that restores the connectivity of these metabolites to the parent network using four mechanisms. This connectivity restoration is hypothesized to take place through four mechanisms: a) reversing the directionality of one or more reactions in the existing model, b) adding reaction from another organism to provide functionality absent in the existing model, c) adding external transport mechanisms to allow for importation of metabolites in the existing model and d) restore flow by adding intracellular transport reactions in multi-compartment models. We demonstrate this procedure for the genome- scale reconstruction of <it>Escherichia coli </it>and also <it>Saccharomyces cerevisiae </it>wherein compartmentalization of intra-cellular reactions results in a more complex topology of the metabolic network. We determine that about 10% of metabolites in <it>E. coli </it>and 30% of metabolites in <it>S. cerevisiae </it>cannot carry any flux. Interestingly, the dominant flow restoration mechanism is directionality reversals of existing reactions in the respective models.</p> <p>Conclusion</p> <p>We have proposed systematic methods to identify and fill gaps in genome-scale metabolic reconstructions. The identified gaps can be filled both by making modifications in the existing model and by adding missing reactions by reconciling multi-organism databases of reactions with existing genome-scale models. Computational results provide a list of hypotheses to be queried further and tested experimentally.</p

Computational redesign of acyl-ACP thioesterase with improved selectivity towards medium chain fatty acids at high production levels

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Our computational procedure was demonstrated by tuning substrate binding of the highly-active ‘TesA thioesterase in Escherichia coli in favor of medium-chain (C6-C12) lengths. Specifically, the Iterative Protein Redesign & Optimization procedure (IPRO) was used to design ‘TesA variants with enhanced C12- or C8specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8), the fatty acid products of thioesterase-mediated catalysis. The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A similar sized library created through random mutagenesis failed to identify medium-chain specific, highly-active variants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. In this work, we not only successfully modified ‘TesA substrate preference but in doing so, we identified the third most C12-specific and tenth most C8-specific thioesterase characterized to date. These results are significant because medium-chain fatty acids are limited in natural abundance relative to long-chain fatty acids. This limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. We expect that the new thioesterase variants will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals. Furthermore, we anticipate that the lessons learned from both successful and failed computational designs can guide algorithmic advancements aiding with future enzyme engineering endeavors

\u3ci\u3eZea mays i\u3c/i\u3eRS1563: A Comprehensive Genome-Scale Metabolic Reconstruction of Maize Metabolism

The scope and breadth of genome-scale metabolic reconstructions have continued to expand over the last decade. Herein, we introduce a genome-scale model for a plant with direct applications to food and bioenergy production (i.e., maize). Maize annotation is still underway, which introduces significant challenges in the association of metabolic functions to genes. The developed model is designed to meet rigorous standards on gene-protein-reaction (GPR) associations, elementally and charged balanced reactions and a biomass reaction abstracting the relative contribution of all biomass constituents. The metabolic network contains 1,563 genes and 1,825 metabolites involved in 1,985 reactions from primary and secondary maize metabolism. For approximately 42% of the reactions direct literature evidence for the participation of the reaction in maize was found. As many as 445 reactions and 369 metabolites are unique to the maize model compared to the AraGEM model for A. thaliana. 674 metabolites and 893 reactions are present in Zea mays iRS1563 that are not accounted for in maize C4GEM. All reactions are elementally and charged balanced and localized into six different compartments (i.e., cytoplasm, mitochondrion, plastid, peroxisome, vacuole and extracellular). GPR associations are also established based on the functional annotation information and homology prediction accounting for monofunctional, multifunctional and multimeric proteins, isozymes and protein complexes. We describe results from performing flux balance analysis under different physiological conditions, (i.e., photosynthesis, photorespiration and respiration) of a C4 plant and also explore model predictions against experimental observations for two naturally occurring mutants (i.e., bm1 and bm3). The developed model corresponds to the largest and more complete to-date effort at cataloguing metabolism for a plant species