Metabolite Changes in Indonesian Tempe Production from Raw Soybeans to Over-Fermented Tempe.

Tempe is fermented soybean from Java, Indonesia, that can serve as a functional food due to its high nutritional content and positive impact on health. Although the tempe fermentation process is known to affect its nutrient content, changes in the metabolite profile during tempe production have not been comprehensively examined. Thus, this research applied a metabolomics approach to investigate the metabolite profile in each step of tempe production, from soybean soaking to over-fermentation. Fourteen samples of raw soybeans, i.e., soaked soybeans (24 h), steamed soybeans, fungal fermented soybeans, and over-fermented soybeans (up to 72 h), were collected. Untargeted metabolomics by gas chromatography/mass spectrometry (GC-MS) was used to determine soybean transformations from various fermentation times and identify disparity-related metabolites. The results showed that soybeans samples clustered together on the basis of the different fermentation steps. The results also showed that sugar, sugar alcohol, organic acids, and amino acids, as well as fermentation time, contributed to the soybean metabolite profile transformations. During the fermentation of tempe, sugars and sugar alcohols accumulated at the beginning of the process before gradually decreasing as fermentation progressed. Specifically, at the beginning of the fermentation, gentiobiose, galactinol, and glucarate were accumulated, and several metabolites such as glutamine, 4-hydroxyphenylacetic acid, and homocysteine increased along with the progression of fermentation. In addition, notable isoflavones daidzein and genistein increased from 24 h of fermentation until 72 h. This is the first report that provides a complete description of the metabolic profile of the tempe production from soybean soaking to over-fermentation. Through this study, the dynamic changes at each step of tempe production were revealed. This information can be beneficial to the tempe industry for the improvement of product quality based on metabolite profiling.

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.

Dynamic Changes in the Bacterial Community and Metabolic Profile during Fermentation of Low-Salt Shrimp Paste (Terasi).

Low-salt shrimp paste, or terasi, is an Indonesian fermented food made from planktonic shrimp mixed with a low concentration of salt. Since high daily intake of sodium is deemed unhealthy, reduction of salt content in shrimp paste production is desired. Until now, there is no reported investigation on the bacterial population and metabolite composition of terasi during fermentation. In this study, the bacterial community of terasi was assessed using high-throughput sequencing of the 16S rRNA V3-V4 region. From this analysis, Tetragenococcus, Aloicoccus, Alkalibacillus, Atopostipes, and Alkalibacterium were found to be the dominant bacterial genus in low-salt shrimp paste. GC/MS-based metabolite profiling was also conducted to monitor the metabolite changes during shrimp paste fermentation. Results showed that acetylated amino acids increased, while glutamine levels decreased, during the fermentation of low-salt shrimp paste. At the start of shrimp paste fermentation, Tetragenococcus predominated with histamine and cadaverine accumulation. At the end of fermentation, there was an increase in 4-hydroxyphenyl acetic acid and indole-3-acetic acid levels, as well as the predominance of Atopostipes. Moreover, we found that aspartic acid increased during fermentation. Based on our findings, we recommend that fermentation of low-salt shrimp paste be done for 7 to 21 days, in order to produce shrimp paste that has high nutritional content and reduced health risk.

Metabolomics Analysis Reveals Global Metabolic Changes in the Evolved E. coli Strain with Improved Growth and 1-Butanol Production in Minimal Medium.

Production of 1-butanol from microorganisms has garnered significant interest due to its prospect as a drop-in biofuel and precursor for a variety of commercially relevant chemicals. Previously, high 1-butanol titer has been reported in Escherichia coli strain JCL166, which contains a modified clostridial 1-butanol pathway. Although conventional and metabolomics-based strain improvement strategies of E. coli strain JCL166 have been successful in improving production in rich medium, 1-butanol titer was severely limited in minimal medium. To further improve growth and consequently 1-butanol production in minimal medium, adaptive laboratory evolution (ALE) using mutD5 mutator plasmid was done on JCL166. Comparative metabolomics analysis of JCL166 and BP1 revealed global perturbations in the evolved strain BP1 compared to JCL166 (44 out of 64 metabolites), encompassing major metabolic pathways such as glycolysis, nucleotide biosynthesis, and CoA-related processes. Collectively, these metabolic changes in BP1 result in improved growth and, consequently, 1-butanol production in minimal medium. Furthermore, we found that the mutation in ihfB caused by ALE had a significant effect on the metabolome profile of the evolved strain. This study demonstrates how metabolomics was utilized for characterization of ALE-developed strains to understand the overall effect of mutations acquired through evolution.

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.

Metabolic repair through emergence of new pathways in Escherichia coli.

Escherichia coli can derive all essential metabolites and cofactors through a highly evolved metabolic system. Damage of pathways may affect cell growth and physiology, but the strategies by which damaged metabolic pathways can be circumvented remain intriguing. Here, we use a ΔpanD (encoding for aspartate 1-decarboxylase) strain of E. coli that is unable to produce the ß-alanine required for CoA biosynthesis to demonstrate that metabolic systems can overcome pathway damage by extensively rerouting metabolic pathways and modifying existing enzymes for unnatural functions. Using directed cell evolution, rewiring and repurposing of uracil metabolism allowed formation of an alternative ß-alanine biosynthetic pathway. After this pathway was deleted, a second was evolved that used a gain-of-function mutation on ornithine decarboxylase (SpeC) to alter reaction and substrate specificity toward an oxidative decarboxylation-deamination reaction. After deletion of both pathways, yet another independent pathway emerged using polyamine biosynthesis, demonstrating the vast capacity of metabolic repair.

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.

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.

Suppression mechanism of the calcium sensitivity in Saccharomyces cerevisiae ptp2Δmsg5Δ double disruptant involves a novel HOG-independent function of Ssk2, transcription factor Msn2 and the protein kinase A component Bcy1.

In Saccharomyces cerevisiae, disruption of both protein phosphatase genes, PTP2 and MSG5, causes calcium sensitivity while additional disruption of protein kinase genes BCK1, MKK1, SLT2, MCK1, YAK1 and SSK2 confers calcium tolerance. Although the roles of BCK1, MKK1 and SLT2 have been characterized recently, the mechanism of suppression of the calcium sensitivity by SSK2 disruption is poorly understood. In this study, genetic analysis revealed a novel, high osmolarity glycerol (HOG)-independent suppressor function of Ssk2 in relation to the Ptp2 and Msg5-mediated calcium signaling. Through microarray analysis, we identified 19 genes with distinct pattern of expression that is likely involved in the calcium sensitive phenotype of the ptp2Δmsg5Δ double disruptant. Furthermore, we found msn2Δ and bcy1Δ as suppressors of the calcium sensitive phenotype. Our results suggest the interrelationship of a HOG-independent function of Ssk2, transcription factor Msn2, protein kinase A-related protein Bcy1 and 19 rise and fall genes as responsible for the suppression mechanism of the ptp2Δmsg5Δ double disruptant by ssk2Δ disruption.

Functionally redundant protein phosphatase genes PTP2 and MSG5 co-regulate the calcium signaling pathway in Saccharomyces cerevisiae upon exposure to high extracellular calcium concentration.

Reversible phosphorylation is one of the key post-translational modifications for the regulation of many essential cellular processes. We have previously reported that the disruption of two protein phosphatase (PPase) genes, PTP2 and MSG5, causes calcium sensitivity indicating that functional redundancy exists between the two PPases in response to high extracellular calcium. In this paper, we found that the inactivation of calcineurin by the disruption of the calcineurin regulatory subunit, CNB1 or treatment with a calcineurin inhibitor, FK506, can suppress the calcium-sensitive phenotype of the ptp2Δmsg5Δ double disruptant. In the wake of a calcium-induced, calcineurin-driven signaling pathway activation, the calcium sensitivity of the ptp2Δmsg5Δ double disruptant can be suppressed by regulating the SLT2 pathway through the disruption of the major kinases in the SLT2 signal cascade that include BCK1, MKK1 and SLT2. Also, we show that PTP2 and MSG5 are key regulatory PPases that prevent over-activation of the calcium-induced signaling cascade under the parallel control of the SLT2 and calcineurin pathways.

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Deltaptp2 Deltamsg5 protein phosphatase double disruptant.

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Cas). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1-S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Deltabck1, Deltamkk1, Deltaslt2/Deltampk1, Deltamck1, Deltassk2 and Deltayak1) suppressed the Cas-phenotype of the Deltaptp2 Deltamsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Deltassk2 and Deltayak1 but not Deltabck1, Deltamkk1, Deltaslt2 and Deltamck1 was able to suppress the delayed G1-S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1-S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Deltaptp2 Deltamsg5 double disruptant.