Agent Clustering Strategy Based on Metabolic Flux Distribution and Transcriptome Expression for Novel Drug Development

The network module-based method has been used for drug repositioning. The traditional drug repositioning method only uses the gene characteristics of the drug but ignores the drug-triggered metabolic changes. The metabolic network systematically characterizes the connection between genes, proteins, and metabolic reactions. The differential metabolic flux distribution, as drug metabolism characteristics, was employed to cluster the agents with similar MoAs (mechanism of action). In this study, agents with the same pharmacology were clustered into one group, and a total of 1309 agents from the CMap database were clustered into 98 groups based on differential metabolic flux distribution. Transcription factor (TF) enrichment analysis revealed the agents in the same group (such as group 7 and group 26) were confirmed to have similar MoAs. Through this agent clustering strategy, the candidate drugs which can inhibit (Japanese encephalitis virus) JEV infection were identified. This study provides new insights into drug repositioning and their MoAs.

A rice QTL GS3.1 regulates grain size through metabolic-flux distribution between flavonoid and lignin metabolons without affecting stress tolerance

Grain size is a key component trait of grain weight and yield. Numbers of quantitative trait loci (QTLs) have been identified in various bioprocesses, but there is still little known about how metabolism-related QTLs influence grain size and yield. The current study report GS3.1, a QTL that regulates rice grain size via metabolic flux allocation between two branches of phenylpropanoid metabolism. GS3.1 encodes a MATE (multidrug and toxic compounds extrusion) transporter that regulates grain size by directing the transport of p-coumaric acid from the p-coumaric acid biosynthetic metabolon to the flavonoid biosynthetic metabolon. A natural allele of GS3.1 was identified from an African rice with enlarged grains, reduced flavonoid content and increased lignin content in the panicles. Notably, the natural allele of GS3.1 caused no alterations in other tissues and did not affect stress tolerance, revealing an ideal candidate for breeding efforts. This study uncovers insights into the regulation of grain size though metabolic-flux distribution. In this way, it supports a strategy of enhancing crop yield without introducing deleterious side effects on stress tolerance mechanisms.

Efficient Production of Propionic Acid in the Fed-Batch Fermentation of Propionibacterium acidipropionici and Its Metabolic Flux Analysis

To improve the fermentation efficiency of Propionibacterium acidipropionici, a simplified metabolic network was established to provide theoretical guidance for medium optimization and process regulation. The effect of glucose and glycerol on propionic acid production and metabolic flux distribution was investigated and the combination of glucose and glycerol was optimized. The results showed that the productivity of propionic acid could be improved by enhancing the synthesis of pyruvate and its flux distribution to the oxaloacetate branch. Finally, the scaled-up fed-batch fermentation of P. acidipropionici was conducted. The concentration of propionic acid reached 51.75 ± 3.62g/L with a glucose/glycerol ratio of 4:1, an improvement of 79.25% relative to the use of glucose alone, and the corresponding productivity and yield were 0.39g/(L· h) and 0.52g/g, respectively. Therefore, the combination of glucose and glycerol significantly improved the productivity of propionic acid and provides a new strategy for industrial production.

Comparing Metabolites and Functional Properties of Various Tomatoes Using Mass Spectrometry-Based Metabolomics Approach

Tomato is one of the world's most consumed vegetables, and thus, various cultivars have been developed. Therefore, metabolic differences and nutrient contents of various tomatoes need to be discovered. To do so, we performed metabolite profiling along with evaluation of morphological and physicochemical properties of five representative tomato types. Common tomato cultivars, bigger and heavier than other tomatoes, contained higher levels of amino acids, organic acids, and lipids. On the contrary, cherry tomato cultivars contained a higher proportion of phenylpropanoids, lycopene, β-carotene, and α-carotene than the other tomatoes. Also, the highest antioxidant activity and total phenolic and flavonoid contents were observed in cherry tomato cultivars. Furthermore, to understand metabolic distributions in various tomato cultivars, we constructed a metabolic pathway map. The higher metabolic flux distribution of most primary metabolite synthetic pathways was observed in common tomatoes, while cherry tomato cultivars showed a significantly elevated flux in secondary metabolite synthetic pathways. Accordingly, these results provide valuable information of different characteristics in various tomatoes, which can be considered while purchasing and improving tomato cultivars.

Metabolic flux analysis of simultaneous production of vitamin B12 and propionic acid in a coupled fermentation process by Propionibacterium freudenreichii

The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanisms of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.

ΔFBA: Predicting metabolic flux alterations using genome-scale metabolic models and differential transcriptomic data

Genome-scale metabolic models (GEMs) provide a powerful framework for simulating the entire set of biochemical reactions occurring in a cell. Constraint-based modeling tools like flux balance analysis (FBA) developed for the purposes of predicting metabolic flux distribution using GEMs face considerable difficulties in estimating metabolic flux alterations between experimental conditions. Particularly, the most appropriate metabolic objective for FBA is not always obvious, likely context-specific, and not necessarily the same between conditions. Here, we propose a new method, called ΔFBA (deltaFBA), that employs constraint-based modeling, in combination with differential gene expression data, to evaluate changes in the intracellular flux distribution between two conditions. Notably, ΔFBA does not require specifying the cellular objective to produce the flux change predictions. We showcased the performance of ΔFBA through several case studies involving the prediction of metabolic alterations caused by genetic and environmental perturbations in Escherichia coli and caused by Type-2 diabetes in human muscle.

Metabolic mechanism of Saccharomyces cerevisiae under different physiological conditions based on non-stationary 13C metabolic flux analysis

Crabtree effect is well known for Saccharomyces cerevisiae, and is defined as glucose-induced repression of respiratory flux. Even though a number of hypotheses have been formulated, its triggering mechanisms are still unknown. At present, the information about intracellular metabolic flux can be obtained by the 13C isotope labeling experiments. 13C metabolic flux analysis(13C-MFA) is a traditional method for calculating metabolic flux based on isotopic steady state. Another new method (INST-13C-MFA: Isotopically nonstationary metabolic flux analysis) based on isotope non-steady state is being used by researchers. In this review, we have chemostatized S. cerevisiae at three different dilution rates (D=0.12, 0.22, 0.32 h-1) and obtained the metabolic flux distribution of the intracellular central carbon metabolic of S. cerevisiae using INST-13C-MFA. Combined with the metabolome and metabolic fluxome data, we found obvious metabolic flux shift under the three different physiological states. In this process, pyruvate decarboxylase, ethanol dehydrogenase and acetyl-CoA synthase(AcCoA) catalyzed reactions were key points. Negative correlation between relative flux of embden meyerh of pathway(EMP) and tricarboxylic acid cycle(TCA) and biomass yield, while positive correlation for pentose phosphate pathway(PPP) were observed. Yield of acetate and glycerol did not change significantly, while that of ethanol increased sharply. In the central carbon metabolism (CCM), most of the carbon flux (70%) was directed to the EMP. At the same time, the energy charge increased with dilution rate, and the cell's energy supply mode gradually shifted from oxidative respiration to substrate level phosphorylation mode.

Synthesis, Molecular Docking Studies and Antibacterial Activities of Novel Monocationic Indole-benzimidazole Derivatives

Background: Finding efficient therapy against hospital-acquired MRSA infections has become rather important in the last decade. To this end, inhibition of the enzyme pyruvate kinase (PK) is being investigated for antibacterial activity, since this enzyme controls energy generation and metabolic flux distribution. Our main scaffold consists of benzimidazole and indole rings fused together. Both rings are famous for antibacterial properties and promising anti-MRSA compounds include indole ring. Methods: Several 1-substituted-2-(1H-indol-3-yl)-N-substituted-1H-benzimidazole-5-carboxamidine analogues were developed, synthesized and their antibacterial activities were evaluated against Staphylococcus aureus (ATCC 25923), Methicillin resistant Staphylococcus aureus (MRSA) (ATCC 43300), and Staphylococcus epidermidis (ATCC 12228) by using tube dilution method. Molecular docking analysis with a characteristic protein called MRSA- Pyruvate Kinase has been conducted for the assessment of the activities of our compounds against Methicillin-resistant S.aureus (MRSA). Results: Among all the tested compounds, the most potent compound 36 had MIC values as 3.12, 3.12 and 6.25 µg/mL against S. aureus, Methicillin-resistant S. aureus (MRSA), and S. epidermidis, respectively. This compound had much better docking energy value than standard ampicillin and also created the link between two residues in different monomers of PK. Discussion: . This approach of using indol-amidine conjugate systems as anti-MRSA agents may include MRSA-PK as potential target. To further increase the affinity, some other H-bonding parts may be added. By doing so, another bridge with Ile361 residues on both sides can be created. Our compounds tend to violate log P limit of Lipinski, therefore some optimizations with formulation can be made. Conclusion: This study mainly includes the design, synthesis and optimization of indole-benzimidazole-amidine derivatives. Docking studies confirmed our results, since our most potent hit compound 36 created the necessary interactions between two chains of MRSA-PK. Further optimization can be considered to increase drug ability.

Metabolic Exchange with Non-Alkane-Consuming Pseudomonas stutzeri SLG510A3-8 Improves n-Alkane Biodegradation by the Alkane Degrader Dietzia sp. Strain DQ12-45-1b

ABSTRACT Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are sometimes composed of not only hydrocarbon degraders but also nonconsumers, but the synergistic mechanisms remain unknown. Here, we found that two bacterial strains isolated from Chinese oil fields, Dietzia sp. strain DQ12-45-1b and Pseudomonas stutzeri SLG510A3-8, had a synergistic effect on hexadecane (C16 compound) biodegradation, even though P. stutzeri could not utilize C16 individually. To gain a better understanding of the roles of the alkane nonconsumer P. stutzeri in the C16-degrading consortium, we reconstructed a two-species stoichiometric metabolic model, iBH1908, and integrated in silico prediction with the following in vitro validation, a comparative proteomics analysis, and extracellular metabolomic detection. Metabolic interactions between P. stutzeri and Dietzia sp. were successfully revealed to have importance in efficient C16 degradation. In the process, P. stutzeri survived on C16 metabolic intermediates from Dietzia sp., including hexadecanoate, 3-hydroxybutanoate, and α-ketoglutarate. In return, P. stutzeri reorganized its metabolic flux distribution to fed back acetate and glutamate to Dietzia sp. to enhance its C16 degradation efficiency by improving Dietzia cell accumulation and by regulating the expression of Dietzia succinate dehydrogenase. By using the synergistic microbial consortium of Dietzia sp. and P. stutzeri with the addition of the in silico-predicted key exchanged metabolites, diesel oil was effectively disposed of in 15 days with a removal fraction of 85.54% ± 6.42%, leaving small amounts of C15 to C20 isomers. Our finding provides a novel microbial assembling mode for efficient bioremediation or chemical production in the future. IMPORTANCE Many natural and synthetic microbial communities are composed of not only species whose biological properties are consistent with their corresponding communities but also ones whose chemophysical characteristics do not directly contribute to the performance of their communities. Even though the latter species are often essential to the microbial communities, their roles are unclear. Here, by investigation of an artificial two-member microbial consortium in n-alkane biodegradation, we showed that the microbial member without the n-alkane-degrading capability had a cross-feeding interaction with and metabolic regulation to the leading member for the synergistic n-alkane biodegradation. Our study improves the current understanding of microbial interactions. Because “assistant” microbes showed importance in communities in addition to the functional microbes, our findings also suggest a useful “assistant-microbe” principle in the design of microbial communities for either bioremediation or chemical production.