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Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13 C-metabolic flux analysis

The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13 C-Metabolic Flux Analysis ( 13 C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13 C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). In conclusion, using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.

13C-metabolic flux analysis↗

Recognition of partially occluded threat objects using the annealed Hopefield network

Recognition of partially occluded objects has been an important issue to airport security because occlusion causes significant problems in identifying and locating objects during baggage inspection. The neural network approach is suitable for the problems in the sense that the inherent parallelism of neural networks pursues many hypotheses in parallel resulting in high computation rates. Moreover, they provide a greater degree of robustness or fault tolerance than conventional computers. The annealed Hopfield network which is derived from the mean field annealing (MFA) has been developed to find global solutions of a nonlinear system. In the study, it has been proven that the system temperature of MFA is equivalent to the gain of the sigmoid function of a Hopfield network. In our early work, we developed the hybrid Hopfield network (HHN) for fast and reliable matching. However, HHN doesn't guarantee global solutions and yields false matching under heavily occluded conditions because HHN is dependent on initial states by its nature. In this paper, we present the annealed Hopfield network (AHN) for occluded object matching problems. In AHN, the mean field theory is applied to the hybird Hopfield network in order to improve computational complexity of the annealed Hopfield network and provide reliable matching under heavily occluded conditions. AHN is slower than HHN. However, AHN provides near global solutions without initial restrictions and provides less false matching than HHN. In conclusion, a new algorithm based upon a neural network approach was developed to demonstrate the feasibility of the automated inspection of threat objects from x-ray images. The robustness of the algorithm is proved by identifying occluded target objects with large tolerance of their features.

Kim, Jung H.↗

Satellite Communications Applications Research

This report describes work performed by SDL in the development of monolithically-integrated master oscillator power amplifiers under the NASA-SCAR research contract. The goals of the program were to demonstrate high power diffraction-limited operation from a semiconductor laser with the following characteristics: (1) Output power of 0.5 to 1.0 W continuous-wave (cw); (2) Single spectral mode with greater than 20 dB of side mode suppression; (3) Single spatial mode operation; (4) Modulation capability of 0.5 GHz; and (5) Operating wavelength between 830-860 nm. Cw diffraction-limited output powers as high as 2.2 W were achieved while maintaining a single longitudinal mode. Side mode suppression ratios greater than 20 dB are typically obtained. The single spatial mode output has a Strehl ratio estimated at 0.85, and a high-speed modulation capability was demonstrated with a small-signal 3-dB roll-off at 1.0 GHz. Finally, all of this performance was obtained in the 860 nm wavelength band, which corresponds to the peak sensitivity of Si detectors. During the first option period, three deliverables were fabricated and delivered that included 1 distributed Bragg reflector (DBR) laser operating at 970 nm, 1 DBR laser operating at 860 nm, and one monolithic flared amplifier master oscillator power amplifier (MFA-MOPA) providing 0.5 W cw diffraction-limited output. During the second option period, a total of 5 MFA-MOPA devices were delivered, each operating to better than 1 W cw diffraction-limited at 860 nm.

Lang, Robert J.↗

Tough Textiles Protect Payloads and Public Safety Officers

In order to create the Mars Pathfinder s mission-critical airbags in the 1990s, NASA s Jet Propulsion Laboratory collaborated with New Ipswich, New Hampshire s Warwick Mills Inc. to weave multi-layer textiles for the airbags for both Pathfinder and the Mars Exploration Rovers. Warwick Mills applied techniques from the collaboration to its puncture- and impact-resistant TurtleSkin product line. The company's metal flex armor (MFA) vests offer stab protection comparable with rigid steel plates and over 50,000 of the vests have sold. The SoftPlate body armor offers protection from handgun bullets, and like the MFA, is designed to be more comfortable than rigid vests. International public safety and military customers are now benefiting from the TurtleSkin products.

Source record↗

Model-free adaptive control of supercritical circulating fluidized-bed boilers

A novel 3-Input-3-Output (3.times.3) Fuel-Air Ratio Model-Free Adaptive (MFA) controller is introduced, which can effectively control key process variables including Bed Temperature, Excess O2, and Furnace Negative Pressure of combustion processes of advanced boilers. A novel 7-input-7-output (7.times.7) MFA control system is also described for controlling a combined 3-Input-3-Output (3.times.3) process of Boiler-Turbine-Generator (BTG) units and a 5.times.5 CFB combustion process of advanced boilers. Those boilers include Circulating Fluidized-Bed (CFB) Boilers and Once-Through Supercritical Circulating Fluidized-Bed (OTSC CFB) Boilers.

20 FOSSIL-FUELED POWER PLANTS↗

Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions

Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this paper, 13 C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37 °C in stage 1, and then the temperature was transitioned to 20 °C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5 mmol/L. On the contrary, ~0.02 mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed 13 C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Finally, interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).

13C-MFA↗

Isotopically nonstationary 13 C flux analysis of cyanobacterial isobutyraldehyde production

Here, we applied isotopically nonstationary 13 C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway ( mdh and me ) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how 13 C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.

13C INST-MFA↗

Cyanobacterial carboxysome mutant analysis reveals the influence of enzyme compartmentalization on cellular metabolism and metabolic network rigidity

Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the ccm operon in Synechococcus sp. PCC 7002 ( ΔccmKLMN ) disrupts carboxysome formation and significantly affects cellular physiology. In this study, we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO 2 environments (1%), the ΔccmKLMN mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the ΔccmKLMN strain had significantly larger pool sizes of pyruvate (>18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the ΔccmKLMN mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO 2 was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses.

09 BIOMASS FUELS↗

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13 C-pathway tracing, 13 C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13 C-metabolic flux analysis ( 13 C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13 C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. In conclusion, three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.

09 BIOMASS FUELS↗

Probing Light-Dependent Regulation of the Calvin Cycle Using a Multi-Omics Approach

Photoautotrophic microorganisms are increasingly explored for the conversion of atmospheric carbon dioxide into biomass and valuable products. The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway for net CO 2 fixation within oxygenic photosynthetic organisms. The cyanobacteria, Synechocystissp. PCC 6803, is a model organism for the study of photosynthesis and a platform for many metabolic engineering efforts. The CBB cycle is regulated by complex mechanisms including enzymatic abundance, intracellular metabolite concentrations, energetic cofactors and post-translational enzymatic modifications that depend on the external conditions such as the intensity and quality of light. However, the extent to which each of these mechanisms play a role under different light intensities remains unclear. In this work, we conducted non-targeted proteomics in tandem with isotopically non-stationary metabolic flux analysis (INST-MFA) at four different light intensities to determine the extent to which fluxes within the CBB cycle are controlled by enzymatic abundance. The correlation between specific enzyme abundances and their corresponding reaction fluxes is examined, revealing several enzymes with uncorrelated enzyme abundance and their corresponding flux, suggesting flux regulation by mechanisms other than enzyme abundance. Additionally, the kinetics of 13 C labeling of CBB cycle intermediates and estimated inactive pool sizes varied significantly as a function of light intensity suggesting the presence of metabolite channeling, an additional method of flux regulation. These results highlight the importance of the diverse methods of regulation of CBB enzyme activity as a function of light intensity, and highlights the importance of considering these effects in future kinetic models.

54 ENVIRONMENTAL SCIENCES↗

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13 C‐metabolite fingerprinting

ABSTRACT For rapid analysis of microbial metabolisms, 13 C‐fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous 13 C‐metabolic flux analysis ( 13 C‐MFA), this method aims to provide metabolic insights without expensive flux measurements. Using 13 C‐fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value‐added chemicals. Specifically, seven metabolic insights were gathered as follows: (1) glucose metabolism mainly via the Entner–Doudoroff (ED) pathway; (2) lack of glucose catabolite repression during phenol co‐utilization; (3) simultaneous operation of gluconeogenesis and the ED pathway for the co‐metabolism of glucose and phenol; (4) an active glyoxylate shunt in acetate‐fed culture; (5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase); (6) presence of alternative glycine synthesis pathway via glycine dehydrogenase; and (7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a 13 C‐fingerprinting kit was developed for studying the central metabolism of non‐model microbial species. This low‐cost kit can be used to characterize microbial metabolisms and facilitate the design‐build‐test‐learn cycle during the development of microbial cell factories. Biotechnol. Bioeng. 2016;113: 91–100. © 2015 Wiley Periodicals, Inc.

Hollinshead, Whitney D.↗

Two-Scale 13 C Metabolic Flux Analysis for Metabolic Engineering

Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13 C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13 C Metabolic Flux Analysis, and it introduces the capability to use 13 C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13 C Metabolic Flux Analysis (2S- 13 C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is illustrated through a step-by-step demonstration, which is also contained in a digital Jupyter Notebook format that enhances reproducibility and provides the capability to be adopted to the user's specific needs. As an open-source software project, users can modify and extend the code base and make improvements at will, providing a base for future modeling efforts.

13C Metabolic flux analysis↗

Strong Correlation Effect in Ferrimagnetic Half-Metallic V{sub 2}CoAl and V{sub 2}CoGa Heusler Compounds

We present first-principles calculations based on the full-potential linearized augmented plane wave (FP-LAPW) method to investigate the structural, elastic, electronic, and magnetic properties of the Heusler compounds V{sub 2}CoAl and V{sub 2}CoGa. We explore different magnetic configurations in both regular and inverse Heusler structures: the Cu{sub 2}MnAl-type and Hg{sub 2}CuTi-type structures respectively. Our compounds are found to be ferrimagnets in the inverse Heusler structure. To take into account the effects of the strong correlation among the localized d-states, we perform a calculation using the GGA+U method (the general gradient approximation with the Hubbard on-site Coulomb correction U). Then we show that both compounds exhibit a half-metallic character and have a magnetic moment of 2.00 μ{sub B} per formula unit. This latter statement is coherent with the generalized Slater-Pauling rule (SP). In terms of the band structure calculations, we explain the formation of the indirect energy band gaps in the spin-majority channels. We also use the mean-field approximation (MFA) to discuss the magnetic exchange couplings and predict the Curie temperature for both compounds.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses

Abstract Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana , Nicotiana tabacum , and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

59 BASIC BIOLOGICAL SCIENCES↗

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ~40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

59 BASIC BIOLOGICAL SCIENCES↗

Adapting isotopic tracer and metabolic flux analysis approaches to study C1 metabolism

Single-carbon (C1, or one-carbon) substrates are promising feedstocks for sustainable biofuel and biochemical production. Crucial to the goal of engineering C1-utilizing strains for improved production is a quantitative understanding of the organization, regulation and rates of the reactions that underpin C1 metabolism. 13 C Metabolic flux analysis (MFA) is a well-established platform for interrogating these questions with multi-carbon substrates, and uses the differential labeling of metabolites that results from feeding a substrate with position-specific incorporation of 13 C in order to infer quantitative fluxes and pathway topology. Furthermore, adapting isotopic tracer approaches to C1 metabolism, where position-specific substrate labeling is impossible, requires additional experimental considerations. Here we review recent studies that have developed isotopic tracer approaches to overcome the challenge of uniform metabolite labeling and provide quantitative insight into C1 metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

Primary and secondary battery consumption trends in Sweden 1996–2013: Method development and detailed accounting by battery type

Highlights: • Developed MFA method was validated by the national statistics. • Exponential increase of EEE sales leads to increase in integrated battery consumption. • Digital convergence is likely to be a cause for primary batteries consumption decline. • Factors for estimation of integrated batteries in EE are provided. • Sweden reached the collection rates defined by European Union. - Abstract: In this article, a new method based on Material Flow Accounting is proposed to study detailed material flows in battery consumption that can be replicated for other countries. The method uses regularly available statistics on import, industrial production and export of batteries and battery-containing electric and electronic equipment (EEE). To promote method use by other scholars with no access to such data, several empirically results and their trends over time, for different types of batteries occurrence among the EEE types are provided. The information provided by the method can be used to: identify drivers of battery consumption; study the dynamic behavior of battery flows – due to technology development, policies, consumers behavior and infrastructures. The method is exemplified by the study of battery flows in Sweden for years 1996–2013. The batteries were accounted, both in units and weight, as primary and secondary batteries; loose and integrated; by electrochemical composition and share of battery use between different types of EEE. Results show that, despite a fivefold increase in the consumption of rechargeable batteries, they account for only about 14% of total use of portable batteries. Recent increase in digital convergence has resulted in a sharp decline in the consumption of primary batteries, which has now stabilized at a fairly low level. Conversely, the consumption of integrated batteries has increased sharply. In 2013, 61% of the total weight of batteries sold in Sweden was collected, and for the particular case of alkaline manganese dioxide batteries, the value achieved 74%.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

A High-Quality Genome-Scale Model for Rhodococcus opacus Metabolism

Rhodococcus opacus is a bacterium that has a high tolerance to aromatic compounds and can produce significant amounts of triacylglycerol (TAG). Here, we present iGR1773, the first genome-scale model (GSM) of R. opacus PD630 metabolism based on its genomic sequence and associated data. The model includes 1773 genes, 3025 reactions, and 1956 metabolites, was developed in a reproducible manner using CarveMe, and was evaluated through Metabolic Model tests (MEMOTE). We combine the model with two Constraint-Based Reconstruction and Analysis (COBRA) methods that use transcriptomics data to predict growth rates and fluxes: E-Flux2 and SPOT (Simplified Pearson Correlation with Transcriptomic data). Growth rates are best predicted by E-Flux2. Flux profiles are more accurately predicted by E-Flux2 than flux balance analysis (FBA) and parsimonious FBA (pFBA), when compared to 44 central carbon fluxes measured by 13C-Metabolic Flux Analysis (13C-MFA). Under glucose-fed conditions, E-Flux2 presents an R2 value of 0.54, while predictions based on pFBA had an inferior R2 of 0.28. We attribute this improved performance to the extra activity information provided by the transcriptomics data. For phenol-fed metabolism, in which the substrate first enters the TCA cycle, E-Flux2’s flux predictions display a high R2 of 0.96 while pFBA showed an R2 of 0.93. We also show that glucose metabolism and phenol metabolism function with similar relative ATP maintenance costs. These findings demonstrate that iGR1773 can help the metabolic engineering community predict aromatic substrate utilization patterns and perform computational strain design.

Roell, Garrett W.↗