Metabolic flux balance analysis and the in silicoanalysis of Escherichia coliK-12 gene deletions

Background

Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silicorepresentations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA). FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information.

Results

Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coliusing FBA and examined the optimal utilization of the E. colimetabolic pathways as a function of environmental variables. We have used an in silicoanalysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system) essential for aerobic growth of E. colion glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi ^-, zwf, and pta ^-mutant strains were examined in more detail by mapping the capabilities of these in silicoisogenic strains.

Conclusions

We found that computational models of E. colimetabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silicaresults lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: 10.1186/1471-2105-1-1     

Integrative network analysis reveals active microRNAs and their functions in gastric cancer

Background

Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO₂ and H₂ as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level.

Results

We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16.

Conclusion

The genome-scale metabolic model, RehMBEL1391, successfully represented metabolic characteristics of R. eutropha H16 at systems level. The reconstructed genome-scale metabolic model can be employed as an useful tool for understanding its metabolic capabilities, predicting its physiological consequences in response to various environmental and genetic changes, and developing strategies for systems metabolic engineering to improve its metabolic performance.     

In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production

Background

Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo.

Results

Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production.

Conclusions

Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to meet the required activities for an increased product formation in vivo.     

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

Background

Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO₂ due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood.

Results

In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysC^fbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose.

Conclusion

The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering.     

Genome and methylome of the oleaginous diatom <Emphasis Type="Italic">Cyclotella cryptica</Emphasis> reveal genetic flexibility toward a high lipid phenotype

Background

Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Results

We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches.

Conclusions

Functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.

     

Detecting positive selection from genome scans of linkage disequilibrium

Background

During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.

Results

Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.

Conclusions

Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.     

Modeling antibiotic and cytotoxic effects of the dimeric isoquinoline IQ-143 on metabolism and its regulation in Staphylococcus aureus, Staphylococcus epidermidis and human cells

Background

Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine.

Results

Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity.

Conclusions

The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics.     

The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum - two hookworms of animal health and zoonotic importance

Background

The anaerobic degradation of organic matter in natural environments, and the biotechnical use of anaerobes in energy production and remediation of subsurface environments, both require the cooperative activity of a diversity of microorganisms in different metabolic niches. The Geobacteraceae family contains members with three important anaerobic metabolisms: fermentation, syntrophic degradation of fermentation intermediates, and anaerobic respiration.

Results

In order to learn more about the evolution of anaerobic microbial communities, the genome sequences of six Geobacteraceae species were analyzed. The results indicate that the last common Geobacteraceae ancestor contained sufficient genes for anaerobic respiration, completely oxidizing organic compounds with the reduction of external electron acceptors, features that are still retained in modern Geobacter and Desulfuromonas species. Evolution of specialization for fermentative growth arose twice, via distinct lateral gene transfer events, in Pelobacter carbinolicus and Pelobacter propionicus. Furthermore, P. carbinolicus gained hydrogenase genes and genes for ferredoxin reduction that appear to permit syntrophic growth via hydrogen production. The gain of new physiological capabilities in the Pelobacter species were accompanied by the loss of several key genes necessary for the complete oxidation of organic compounds and the genes for the c-type cytochromes required for extracellular electron transfer.

Conclusion

The results suggest that Pelobacter species evolved parallel strategies to enhance their ability to compete in environments in which electron acceptors for anaerobic respiration were limiting. More generally, these results demonstrate how relatively few gene changes can dramatically transform metabolic capabilities and expand the range of environments in which microorganisms can compete.     

Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation

Background and aims

Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium isolated from sugarcane, rice, elephant grass, sweet potato, coffee, and pineapple. These plants have high level of asparagine, which promotes microbial growth and inhibits nitrogenase activity. The regulation of intracellular concentrations of this amino acid is essential for growth and biological nitrogen fixation (BNF) in this diazotroph; however its asparagine metabolic pathway has not yet been clearly established.

Methods

The work reported here is the first to demonstrate the use of an alternative route for asparaginyl-tRNA (Asn-tRNA) and asparagine formation in an endophytic nitrogen-fixing bacterium by using in silico and in vitro analysis.

Results

The indirect route involves transamidation of incorrectly charged tRNA via GatCAB transamidase. Nitrogenase activity was completely inhibited by 20?mM Asn in LGI-P medium, which in contrast promotes protein synthesis and microbial growth.

Conclusions

The analysis carried out in this work shows that intracellular levels of asparagine regulate the expression of nitrogenase nifD gene (GDI0437), suggesting that the presence of an alternative route to produce asparagine might give the G. diazotrophicus a tighter control over cell growth and BNF, and may be of importance in the regulation of the endophytic plant-microbe interaction.     

Genome-scale model-driven strain design for dicarboxylic acid production in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Background

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application.

Results

In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica.

Conclusion

In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.

     

Metabolic modeling and response surface analysis of an Escherichia coli strain engineered for shikimic acid production

Background

Classic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media.

Results

The obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process.

Conclusions

The combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.

     

Effects of introducing heterologous pathways on microbial metabolism with respect to metabolic optimality

Although optimality of microbial metabolism under genetic and environmental perturbations is well studied, the effects of introducing heterologous reactions on the overall metabolism are not well understood. This point is important in the field of metabolic engineering because heterologous reactions are more frequently introduced into various microbial hosts. The genome-scale metabolic simulations of Escherichia coli strains engineered to produce 1,4-butanediol, 1,3-propanediol, and amorphadiene suggest that microbial metabolism shows much different responses to the introduced heterologous reactions in a strain-specific manner than typical gene knockouts in terms of the energetic status (e.g., ATP and biomass generation) and chemical production capacity. The 1,4-butanediol and 1,3-propanediol producers showed greater metabolic optimality than the wild-type strains and gene knockout mutants for the energetic status, while the amorphadiene producer was metabolically less optimal. For the optimal chemical production capacity, additional gene knockouts were most effective for the strain producing 1,3-propanediol, but not for the one producing 1,4-butanediol. These observations suggest that strains having heterologous metabolic reactions have metabolic characteristics significantly different from those of the wild-type strain and single gene knockout mutants. Finally, comparison of the theoretically predicted and ¹³C-based flux values pinpoints pathways with non-optimal flux values, which can be considered as engineering targets in systems metabolic engineering strategies. To our knowledge, this study is the first attempt to quantitatively characterize microbial metabolisms with different heterologous reactions. The suggested potential reasons behind each strain’s different metabolic responses to the introduced heterologous reactions should be carefully considered in strain designs.     

Literacy and recent history of diarrhoea are predictive of Plasmodium falciparum parasitaemia in Kenyan adults

Background

Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked.

Methods

The^ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses.

Results

Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa.

Conclusion

The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.     

Mesothelioma mortality in Europe: impact of asbestos consumption and simian virus 40

Background

Hypophosphatasia (HP) is an inborn error of bone metabolism characterized by a genetic defect in the gene encoding the tissue-nonspecific alkaline phosphatase (TNSALP). There is a lack of knowledge as to how the variability and clinical severity of the HP phenotype (especially pain and walking impairment) are related to metabolic disturbances or impairments, subsequent to the molecular defect.

Methods

We analyzed the changes in clinical symptoms and the prostaglandin (PG) metabolism in response to treatment with non-steroidal anti-inflammatory drugs (NSAIDs) in six children affected by childhood HP. In addition, by exposing HP fibroblasts to pyridoxal phosphate and/or calcium pyrophosphate in vitro, we analyzed whether the alterations in PG levels are sequelae related to the metabolic defect.

Results

Childhood HP patients, who often complain about pain in the lower limbs without evident fractures, have systemic hyperprostaglandinism. Symptomatic anti-inflammatory treatment with NSAIDs significantly improved pain-associated physical impairment. Calcium pyrophosphate, but not pyridoxal phosphate, induced cyclooxygenase-2 (COX-2) gene expression and PG production in HP and normal fibroblasts in vitro.

Conclusion

Clinical features of childhood HP related to pain in the lower legs may be, at least in part, sequelae related to elevated PG levels, secondary to the primary metabolic defect. Consequently, NSAID treatment does improve the clinical features of childhood HP.     

Metabolic reprogramming by viruses in the sunlit and dark ocean

Background

Marine ecosystem function is largely determined by matter and energy transformations mediated by microbial community interaction networks. Viral infection modulates network properties through mortality, gene transfer and metabolic reprogramming.

Results

Here we explore the nature and extent of viral metabolic reprogramming throughout the Pacific Ocean depth continuum. We describe 35 marine viral gene families with potential to reprogram metabolic flux through central metabolic pathways recovered from Pacific Ocean waters. Four of these families have been previously reported but 31 are novel. These known and new carbon pathway auxiliary metabolic genes were recovered from a total of 22 viral metagenomes in which viral auxiliary metabolic genes were differentiated from low-level cellular DNA inputs based on small subunit ribosomal RNA gene content, taxonomy, fragment recruitment and genomic context information. Auxiliary metabolic gene distribution patterns reveal that marine viruses target overlapping, but relatively distinct pathways in sunlit and dark ocean waters to redirect host carbon flux towards energy production and viral genome replication under low nutrient, niche-differentiated conditions throughout the depth continuum.

Conclusions

Given half of ocean microbes are infected by viruses at any given time, these findings of broad viral metabolic reprogramming suggest the need for renewed consideration of viruses in global ocean carbon models.