Multidisciplinary approach combining food metabolomics and epidemiology identifies meglutol as an important bioactive metabolite in tempe, an Indonesian fermented food.

This study introduces a multidisciplinary approach to investigate bioactive food metabolites often overlooked due to their low concentrations. We integrated an in-house food metabolite library (n = 494), a human metabolite library (n = 891) from epidemiological studies, and metabolite pharmacological databases to screen for food metabolites with potential bioactivity. We identified six potential metabolites, including meglutol (3-hydroxy-3-methylglutarate), an understudied low-density lipoprotein (LDL)-lowering compound. We further focused on meglutol as a case study to showcase the range of characterizations achievable with this approach. Green pea tempe was identified to contain the highest meglutol concentration (21.8 ± 4.6 mg/100 g). Furthermore, we identified a significant cross-sectional association between plasma meglutol (per 1-standard deviation) and lower LDL cholesterol in two Hispanic adult cohorts (n = 1,628) (ß [standard error]: -5.5 (1.6) mg/dl, P = 0.0005). These findings highlight how multidisciplinary metabolomics can serve as a systematic tool for discovering and enhancing bioactive metabolites in food, such as meglutol, with potential applications in personalized dietary approaches for disease prevention.

Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production.

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

Investigation of the characteristics of different shrimps by species and habitat using gas chromatography/mass spectrometry based metabolomics.

Approximately 6.5 million tons of shrimp are consumed annually worldwide. The price of shrimp is greatly influenced by species and habitat (e.g., farmed vs wild-caught). In recent years, false labeling has become a problem in the shrimp industry. False labeling can include species, habitat (whether farmed or wild-caught). This problem is motivated by the potential for economic benefit, and significantly reduces the consumer reliability of food. As a first step in establishing a detection method, we took a metabolomics approach to elucidate phenotypic diversity by assessing genetic differences and environmental factors. Metabolites identified by gas chromatography/mass spectrometry (GC/MS) analysis were subjected to multivariate analysis to identify metabolites that correlated with shrimp species and habitat. The characteristics based on species and habitat were observed respectively. For species, the classification approximately tended to be based on taxonomy. It suggests that species different have strong effect on metabolite profiles. In particular, the difference between Panaediae and Pandalidae was significantly observed, and some fatty acids such as palmitoleic acid and elaidic acid are abundant in Pandalidae. Among Pandalidae, Japanese tiger shrimp was characterized by metabolites related to purine metabolism. For habitat, farmed shrimp had a high amino acid content, and wild caught shrimp had a high fatty acid content. Habitat-based separation was observed in Indonesian black tiger shrimp samples, indicating that metabolites such as glycolic acid, phosphate, and pentadecanoic acid are characteristic components of natural black tiger shrimp.

Investigation of the effects of actinorhodin biosynthetic gene cluster expression and a rpoB point mutation on the metabolome of Streptomyces coelicolor M1146.

The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.

Multi-Omics Analysis of the Effect of cAMP on Actinorhodin Production in Streptomyces coelicolor.

Cyclic adenosine monophosphate (cAMP) has been known to play an important role in regulating morphological development and antibiotic production in Streptomyces coelicolor. However, the functional connection between cAMP levels and antibiotic production and the mechanism by which cAMP regulates antibiotic production remain unclear. In this study, metabolomics- and transcriptomics-based multi-omics analysis was applied to S. coelicolor strains that either produce the secondary metabolite actinorhodin (Act) or lack most secondary metabolite biosynthesis pathways including Act. Comparative multi-omics analysis of the two strains revealed that intracellular and extracellular cAMP abundance was strongly correlated with actinorhodin production. Notably, supplementation of cAMP improved cell growth and antibiotic production. Further multi-omics analysis of cAMP-supplemented S. coelicolor cultures showed an increase of guanine and the expression level of purine metabolism genes. Based on this phenomenon, supplementation with 7-methylguanine, a competitive inhibitor of reactions utilizing guanine, with or without additional cAMP supplementation, was performed. This experiment revealed that the reactions inhibited by 7-methylguanine are mediating the positive effect on growth and antibiotic production, which may occur downstream of cAMP supplementation.

Identification of Key Metabolites in Poly-γ-Glutamic Acid Production by Tuning γ-PGA Synthetase Expression.

Poly-γ-glutamic acid (γ-PGA) production is commonly achieved using glycerol, citrate, and L-glutamic acid as substrates. The constitutive expression of the γ-PGA synthetase enabled γ-PGA production with Bacillus subtilis from glucose only. The precursors for γ-PGA synthesis, D- and L-glutamate, are ubiquitous metabolites. Hence, the metabolic flux toward γ-PGA directly depends on the concentration and activity of the synthetase and thereby on its expression. To identify pathway bottlenecks and important metabolites that are highly correlated with γ-PGA production from glucose, we engineered B. subtilis strains with varying γ-PGA synthesis rates. To alter the rate of γ-PGA synthesis, the expression level was controlled by two approaches: (1) Using promoter variants from the constitutive promoter P veg and (2) Varying induction strength of the xylose inducible promoter P xyl . The variation in the metabolism caused by γ-PGA production was investigated using metabolome analysis. The xylose-induction strategy revealed that the γ-PGA production rate increased the total fluxes through metabolism indicating a driven by demand adaption of the metabolism. Metabolic bottlenecks during γ-PGA from glucose were identified by generation of a model that correlates γ-PGA production rate with intracellular metabolite levels. The generated model indicates the correlation of certain metabolites such as phosphoenolpyruvate with γ-PGA production. The identified metabolites are targets for strain improvement to achieve high level γ-PGA production from glucose.

Tailor-made poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis.

A metabolomics-based approach for the evaluation of off-tree ripening conditions and different postharvest treatments in mangosteen (Garcinia mangostana).

INTRODUCTION: Metabolomics is an important tool to support postharvest fruit development and ripening studies. Mangosteen (Garcinia mangostana L.) is a tropical fruit with high market value but has short shelf-life during postharvest handling. Several postharvest technologies have been applied to maintain mangosteen fruit quality during storage. However, there is no study to evaluate the metabolite changes that occur in different harvesting and ripening condition. Additionally, the effect of postharvest treatment using a metabolomics approach has never been studied in mangosteen. OBJECTIVES: The aims of this study were to evaluate the metabolic changes between different harvesting and ripening condition and to evaluate the effect of postharvest treatment in mangosteen. METHODS: Mangosteen ripening stage were collected with several different conditions ("natural on-tree", "random on-tree" and "off-tree"). The metabolite changes were investigated for each ripening condition. Additionally, mangosteen fruit was harvested in stage 2 and was treated with several different treatments (storage at low temperature (LT; 12.3 ± 1.4 °C) and stress inducer treatment (methyl jasmonate and salicylic acid) in comparison with control treatment (normal temperature storage) and the metabolite changes were monitored over the course of 10 days after treatment. The metabolome data obtained from gas chromatography coupled with mass spectrometry were analyzed by multivariate analysis, including hierarchical clustering analysis, principal component analysis, and partial to latent squares analysis. RESULTS: "On-tree" ripening condition showed the progression of ripening process in accordance with the accumulation of some aroma precursor metabolites in the flesh part and pectin breakdown in the peel part. Interestingly, similar trend was found in the "off-tree" ripening condition although the progression of ripening process observed through color changes occurred much faster compared to "on-tree" ripening. Additionally, low-temperature treatment is shown as the most effective treatment to prolong mangosteen shelf-life among all postharvest treatments tested in this study compared to control treatment. After postharvest treatment, a total of 71 and 65 metabolites were annotated in peel and flesh part of mangosteen, respectively. Several contributed metabolites (xylose, galactose, galacturonic acid, glucuronate, glycine, and rhamnose) were decreased after treatment in the peel part. However, low-temperature treatment did not show any significant differences compared to a room temperature treatment in the flesh part. CONCLUSIONS: Our findings clearly indicate that there is a similar trend of metabolic changes between on-tree and off-tree ripening conditions. Additionally, postharvest treatment directly or indirectly influences many metabolic processes (cell-wall degrading process, sweet-acidic taste quality) during postharvest treatment.

Metabolome analysis revealed the knockout of glyoxylate shunt as an effective strategy for improvement of 1-butanol production in transgenic Escherichia coli.

High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli.

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis.

The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D- and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h-1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.

Untargeted Metabolomics Analysis of Eggplant (Solanum melongena L.) Fruit and Its Correlation to Fruit Morphologies.

Eggplant is one of the most widely cultivated vegetables in the world and has high biodiversity in terms of fruit shape, size, and color. Therefore, fruit morphology and nutrient content become important considerations for both consumers and breeders who develop new eggplant-based products. To gain insight on the diversity of eggplant metabolites, twenty-one eggplant accessions were analyzed by untargeted metabolomics using GC-MS and LC-MS. The dataset of eggplant fruit morphologies, and metabolites specific to different eggplant fruit accessions were used for correlation analysis. Untargeted metabolomics analysis using LC-MS and GC-MS was able to detect 136 and 207 peaks, respectively. Fifty-one (51) metabolites from the LC-MS analysis and 207 metabolites from the GC-MS analysis were putatively identified, which included alkaloids, terpenes, terpenoids, fatty acids, and flavonoids. Spearman correlation analysis revealed that 14 fruit morphologies were correlated with several metabolites. This information will be very useful for the development of strategies for eggplant breeding.

Metabolic profiling of Garcinia mangostana (mangosteen) based on ripening stages.

Metabolomics is an emerging research field based on exhaustive metabolite profiling that have been proven useful to facilitate the study of postharvest fruit development and ripening. Specifically, tracking changes to the metabolome as fruit ripens should provide important clues for understanding ripening mechanisms and identify bio-markers to improve post-harvest technology of fruits. This study conducted a time-course metabolome analysis in mangosteen, an economically important tropical fruit valued for its flavor. Mangosteen is a climacteric fruit that requires an important plant hormone ethylene to regulate ripening processes and rate. We first categorized mangosteen samples in different ripening stages based on color changes, an established indicator of ripening. Using gas chromatography/mass spectrometry, small hydrophilic metabolites were profiled from non-ripened to fully ripened (ripening stages 0-6). These metabolites were then correlated with color changes to verify their involvement mangosteen ripening. Our results suggest that the increase of 2-aminoisobutyric acid, psicose, and several amino acids (phenylalanine, valine, isoleucine, serine, and tyrosine) showed a correlation with the progression of mangosteen ripening. This is the first report of the application of non-targeted metabolomics in mangosteen.

Orthogonal partial least squares/projections to latent structures regression-based metabolomics approach for identification of gene targets for improvement of 1-butanol production in Escherichia coli.

Metabolomics is the comprehensive analysis of metabolites in biological systems that uses multivariate analyses such as principal component analysis (PCA) or partial least squares/projections to latent structures regression (PLSR) to understand the metabolome state and extract important information from biological systems. In this study, orthogonal PLSR (OPLSR) model-based metabolomics approach was applied to 1-butanol producing Escherichia coli to facilitate in strain improvement strategies. Here, metabolite data obtained by liquid chromatography/mass spectrometry (LC/MS) was used to construct an OPLSR model to correlate metabolite changes with 1-butanol production and rationally identify gene targets for strain improvement. Using this approach, acetyl-CoA was determined as the rate-limiting step of the pathway while free CoA was found to be insufficient for 1-butanol production. By resolving the problems addressed by the OPLSR model, higher 1-butanol productivity was achieved. In this study, the usefulness of OPLSR-based metabolomics approach for understanding the whole metabolome state and determining the most relevant metabolites was demonstrated. Moreover, it was able to provide valuable insights for selection of rational gene targets for strain improvement.

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli.

High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.

Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production.

Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using 13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.

Metabolic distance estimation based on principle component analysis of metabolic turnover.

Visualization of metabolic dynamism is important for various types of metabolic studies including studies on optimization of bio-production processes and studies of metabolism-related diseases. Many methodologies have been developed for metabolic studies. Among these, metabolic turnover analysis (MTA) is often used to analyze metabolic dynamics. MTA involves observation of changes in the isotopomer ratio of metabolites over time following introduction of isotope-labeled substrates. MTA has several advantages compared with (13)C-metabolic flux analysis, including the diversity of applicable samples, the variety of isotope tracers, and the wide range of target pathways. However, MTA produces highly complex data from which mining useful information becomes difficult. For easy understanding of MTA data, a new approach was developed using principal component analysis (PCA). The resulting PCA score plot visualizes the metabolic distance, which is defined as distance between metabolites on the real metabolic map. And the score plot gives us some hints of interesting metabolism for further study. We used this method to analyze the central metabolism of Saccharomyces cerevisiae under moderated aerobic conditions, and time course data for 77 isotopomers of 14 metabolites were obtained. The PCA score plot for this dataset represented a metabolic map and indicated interesting phenomena such as activity of fumarate reductase under aerated condition. These findings show the importance of a multivariate analysis to MTA. In addition, because the approach is not biased, this method has potential application for analysis of less-studied pathways and organisms.

Current metabolomics: technological advances.

Metabolomics, the global quantitative assessment of metabolites in a biological system, has played a pivotal role in various fields of science in the post-genomic era. Metabolites are the result of the interaction of the system's genome with its environment and are not merely the end product of gene expression, but also form part of the regulatory system in an integrated manner. Therefore, metabolomics is often considered a powerful tool to provide an instantaneous snapshot of the physiology of a cell. The power of metabolomics lies on the acquisition of analytical data in which metabolites in a cellular system are quantified, and the extraction of the most meaningful elements of the data by using various data analysis tool. In this review, we discuss the latest development of analytical techniques and data analyses methods in metabolomics study.

Current metabolomics: practical applications.

The field of metabolomics continues to grow rapidly over the last decade and has been proven to be a powerful technology in predicting and explaining complex phenotypes in diverse biological systems. Metabolomics complements other omics, such as transcriptomics and proteomics and since it is a 'downstream' result of gene expression, changes in the metabolome is considered to best reflect the activities of the cell at a functional level. Thus far, metabolomics might be the sole technology capable of detecting complex, biologically essential changes. As one of the omics technology, metabolomics has exciting applications in varied fields, including medical science, synthetic biology, medicine, and predictive modeling of plant, animal and microbial systems. In addition, integrated applications with genomics, transcriptomics, and proteomics provide greater understanding of global system biology. In this review, we discuss recent applications of metabolomics in microbiology, plant, animal, food, and medical science.

Farinomalein, a maleimide-bearing compound from the entomopathogenic fungus Paecilomyces farinosus.

A new maleimide-bearing compound, farinomalein (1), was isolated from the entomopathogenic fungus Paecilomyces farinosus HF599. The structure was determined on the basis of spectroscopic analyses and chemical conversion. Compound 1 showed potent activity (5 mug/disk) against the plant pathogenic Phytophthora sojae P6497.