Literature mining supports a next-generation modeling approach to predict cellular byproduct secretion

The metabolic byproducts secreted by growing cells can be easily measured and provide a window into the state of a cell; they have been essential to the development of microbiology, cancer biology, and biotechnology. Progress in computational modeling of cells has made it possible to predict metabolic byproduct secretion with bottom-up reconstructions of metabolic networks. However, owing to a lack of data, it has not been possible to validate these predictions across a wide range of strains and conditions. Through literature mining, we were able to generate a database of Escherichia coli strains and their experimentally measured byproduct secretions. We simulated these strains in six historical genome-scale models of E. coli, and we report that the predictive power of the models has increased as they have expanded in size and scope. The latest genome-scale model of metabolism correctly predicts byproduct secretion for 35/89 (39%) of designs. The next-generation genome-scale model of metabolism and gene expression (ME-model) correctly predicts byproduct secretion for 40/89 (45%) of designs, and we show that ME-model predictions could be further improved through kinetic parameterization. We analyze the failure modes of these simulations and discuss opportunities to improve prediction of byproduct secretion.     

Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity “swaps” for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast—including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine—and non-native products in E. coli—including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.     

Integration of Biomass Formulations of Genome-Scale Metabolic Models with Experimental Data Reveals Universally Essential Cofactors in Prokaryotes

The composition of a cell in terms of macromolecular building blocks and other organic molecules underlies the metabolic needs and capabilities of a species. Although some core biomass components such as nucleic acids and proteins are evident for most species, the essentiality of the pool of other organic molecules, especially cofactors and prosthetic groups, is yet unclear. Here we integrate biomass compositions from 71 manually curated genome-scale models, 33 large-scale gene essentiality datasets, enzyme-cofactor association data and a vast array of publications, revealing universally essential cofactors for prokaryotic metabolism and also others that are specific for phylogenetic branches or metabolic modes. Our results revise predictions of essential genes in Klebsiella pneumoniae and identify missing biosynthetic pathways in models of Mycobacterium tuberculosis. This work provides fundamental insights into the essentiality of organic cofactors and has implications for minimal cell studies as well as for modeling genotype-phenotype relations in prokaryotic metabolic networks.     

Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

In this study, the intracellular proteome of Escherichia coli O157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-¹³C₆]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 µM 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.     

Characterization of E. coli MG1655 and frdA and sdhC mutants at various aerobiosis levels

Depending on the availability of oxygen, Escherichia coli is able to switch between aerobic respiratory metabolism and anaerobic mixed acid fermentation. An important, yet understudied, metabolic mode is the micro-aerobic metabolism at intermediate oxygen availabilities. The relationship between oxygen input, physiology and gene expression of E. coli MG1655 and two isogenic mutants lacking succinate dehydrogenase (SDH) and fumarate reductase (FRD) activities was analyzed at different aerobiosis levels. Growth rate and cell yield were very similar to the parent strain. By-product formation was altered in the sdhC mutant to higher acetic acid and glutamate production in batch cultures. In continuous cultures with defined oxygen input gene expression analysis revealed a dependency of many catabolic genes to aerobiosis. Acetate excretion was still detectable under aerobic conditions in the sdhC mutant; the frdA mutant lacked anaerobic succinate excretion. Anaerobic repression of the sdh operon was diminished in the frdA strain, possibly to allow SDH to partially replace FRD. The experiments illustrate the remarkable adaptability of E. coli physiology—to compensate for the absence of important metabolic genes by altering carbon flux and/or gene expression such that there are only minor changes in growth capability across the aerobiosis range.     

Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD⁺/NADH or, to a lesser extent, NADP⁺/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation.     

An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions.     

From coarse to fine: the absolute Escherichia coli proteome under diverse growth conditions

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.     

An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli

Background  

Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis.     

Improved computational performance of MFA using elementary metabolite units and flux coupling

Extending the scope of isotope mapping models becomes increasingly important in order to analyze strains and drive improved product yields as more complex pathways are engineered into strains and as secondary metabolites are used as starting points for new products. Here we present how the elementary metabolite unit (EMU) framework and flux coupling significantly decrease the computational burden of metabolic flux analysis (MFA) when applied to large-scale metabolic models. We applied these techniques to a previously published isotope mapping model of Escherichia coli accounting for 238 reactions. We find that the combined use of EMU and flux coupling analysis leads to a ten-fold decrease in the number of variables in comparison to the original isotope distribution vector (IDV) version of the model. In addition, using OptMeas the task of identifying additional measurement choices to fully specify the flows in the metabolic network required only 2% of the computation time of the one using IDVs. The observed computational savings reveal the rapid progress in performing MFA with increasingly larger isotope models with the ultimate goal of handling genome-scale models of metabolism.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6′-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.     

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.     

An allosteric model for control of pore opening by substrate binding in the EutL microcompartment shell protein

The ethanolamine utilization (Eut) microcompartment is a protein-based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of this elaborate protein complex is composed from a few thousand copies of BMC-domain shell proteins, which form a semi-permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear. Previous work has revealed two conformations of the EutL shell protein, providing substantial evidence for a gated pore that might allow the passage of large cofactors. Here we report structural and biophysical evidence to show that ethanolamine, the substrate of the Eut microcompartment, acts as a negative allosteric regulator of EutL pore opening. Specifically, a series of X-ray crystal structures of EutL from Clostridium perfringens, along with equilibrium binding studies, reveal that ethanolamine binds to EutL at a site that exists in the closed-pore conformation and which is incompatible with opening of the large pore for cofactor transport. The allosteric mechanism we propose is consistent with the cofactor requirements of the Eut microcompartment, leading to a new model for EutL function. Furthermore, our results suggest the possibility of redox modulation of the allosteric mechanism, opening potentially new lines of investigation.     

The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

Background

In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.

Methodology/Principal Findings

Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP⁺ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.

Conclusions/Significance

Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP⁺, whereas in contrast the human enzyme utilises NAD⁺. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.     

Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.