Metabolic Flux Analysis of the Synechocystis sp. PCC 6803 ΔnrtABCD Mutant Reveals a Mechanism for Metabolic Adaptation to Nitrogen-Limited Conditions.

Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration.

Metabolic engineering of isopropyl alcohol-producing Escherichia coli strains with 13 C-metabolic flux analysis.

Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with 13 C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. 13 C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. 13 C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO2 release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The 13 C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that 13 C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP+ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Positive effects of proline addition on the central metabolism of wild-type and lactic acid-producing Saccharomyces cerevisiae strains.

In Saccharomyces cerevisiae, proline is a stress protectant interacting with other substrate uptake systems against oxidative stress under low pH conditions. In this study, we performed metabolomics analysis to investigate the response associated with an increase in cell growth rates and maximum densities when cells were treated with proline under normal and acid stress conditions. Metabolome data show that concentrations of components of central metabolism are increased in proline-treated S. cerevisiae. No consumption of proline was observed, suggesting that proline does not act as a nutrient but regulates metabolic state and growth of cells. Treatment of lactic acid-producing yeast with proline during lactic acid bio-production improved growth rate and increased the final concentration of lactic acid.

Integrated metabolic flux and omics analysis of Synechocystis sp. PCC 6803 under mixotrophic and photoheterotrophic conditions.

Cyanobacteria have flexible metabolic capability that enables them to adapt to various environments. To investigate their underlying metabolic regulation mechanisms, we performed an integrated analysis of metabolic flux using transcriptomic and metabolomic data of a cyanobacterium Synechocystis sp. PCC 6803, under mixotrophic and photoheterotrophic conditions. The integrated analysis indicated drastic metabolic flux changes, with much smaller changes in gene expression levels and metabolite concentrations between the conditions, suggesting that the flux change was not caused mainly by the expression levels of the corresponding genes. Under photoheterotrophic conditions, created by the addition of the photosynthesis inhibitor atrazine in mixotrophic conditions, the result of metabolic flux analysis indicated the significant repression of carbon fixation and the activation of the oxidative pentose phosphate pathway (PPP). Moreover, we observed gluconeogenic activity of upstream of glycolysis, which enhanced the flux of the oxidative PPP to compensate for NADPH depletion due to the inhibition of the light reaction of photosynthesis. 'Omics' data suggested that these changes were probably caused by the repression of the gap1 gene, which functions as a control valve in the metabolic network. Since metabolic flux is the outcome of a complicated interplay of cellular components, integrating metabolic flux with other 'omics' layers can identify metabolic changes and narrow down these regulatory mechanisms more effectively.

A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

Increased 3-hydroxypropionic acid production from glycerol, by modification of central metabolism in Escherichia coli.

BACKGROUND: 3-hydroxypropionic acid (3HP) is an important chemical precursor for the production of bioplastics. Microbial production of 3HP from glycerol has previously been developed through the optimization of culture conditions and the 3HP biosynthesis pathway. In this study, a novel strategy for improving 3HP production in Escherichia coli was investigated by the modification of central metabolism based on a genome-scale metabolic model and experimental validation. RESULTS: Metabolic simulation identified the double knockout of tpiA and zwf as a candidate for improving 3HP production. A 3HP-producing strain was constructed by the expression of glycerol dehydratase and aldehyde dehydrogenase. The double knockout of tpiA and zwf increased the percentage carbon-molar yield (C-mol%) of 3HP on consumed glycerol 4.4-fold (20.1 ± 9.2 C-mol%), compared to the parental strain. Increased extracellular methylglyoxal concentrations in the ΔtpiA Δzwf strain indicated that glycerol catabolism was occurring through the methylglyoxal pathway, which converts dihydroxyacetone phosphate to pyruvate, as predicted by the metabolic model. Since the ΔtpiA Δzwf strain produced abundant 1,3-propanediol as a major byproduct (37.7 ± 13.2 C-mol%), yqhD, which encodes an enzyme involved in the production of 1,3-propanediol, was disrupted in the ΔtpiA Δzwf strain. The 3HP yield of the ΔtpiA Δzwf ΔyqhD strain (33.9 ± 1.2 C-mol%) was increased 1.7-fold further compared to the ΔtpiA Δzwf strain and by 7.4-fold compared to the parental strain. CONCLUSION: This study successfully increased 3HP production by 7.4-fold in the ΔtpiA Δzwf ΔyqhD E. coli strain by the modification of the central metabolism, based on metabolic simulation and experimental validation of engineered strains.

Correction: Anti-inflammatory effects of cold atmospheric plasma irradiation on the THP-1 human acute monocytic leukemia cell line.

[This corrects the article DOI: 10.1371/journal.pone.0292267.].

Thixotropic hydrogelators based on a cyclo(dipeptide) derivative.

Thixotropic hydrogelators have great potential in biomedical and biotechnological applications. In this study, we report new hydrogelators and their behavior during gel-sol-gel transitions. In particular, cyclo(L-O-hydroxyhexylaspartyl-L-phenylalanyl), which was synthesized with 1,6-hexanediol, formed a thermally/isothermally reversible physical gel with several solvents, including pure water, saline, alcohols, as well as 1.0 M aqueous NaCl, KCl, CaCl2, and MgCl2 solutions. TEM observations showed self-assembled fibers with diameters of 10-100 nm. FT-IR results revealed that the gels were mainly formed by hydrogen bonding and van der Waals forces; thixotropic behavior resulted from the disruption of the van der Waals forces between the alkylene chains under shearing. These results were repeatedly and reproducibly observed at room temperature, even when measurements were repeated many times.

Anti-inflammatory effects of cold atmospheric plasma irradiation on the THP-1 human acute monocytic leukemia cell line.

Cold atmospheric plasma (CAP) has been studied and clinically applied to treat chronic wounds, cancer, periodontitis, and other diseases. CAP exerts cytotoxic, bactericidal, cell-proliferative, and anti-inflammatory effects on living tissues by generating reactive species. Therefore, CAP holds promise as a treatment for diseases involving chronic inflammation and bacterial infections. However, the cellular mechanisms underlying these anti-inflammatory effects of CAP are still unclear. Thus, this study aimed to elucidate the anti-inflammatory mechanisms of CAP in vitro. The human acute monocytic leukemia cell line, THP-1, was stimulated with lipopolysaccharide and irradiated with CAP, and the cytotoxic effects of CAP were evaluated. Time-course differentiation of gene expression was analyzed, and key transcription factors were identified via transcriptome analysis. Additionally, the nuclear localization of the CAP-induced transcription factor was examined using western blotting. The results indicated that CAP showed no cytotoxic effects after less than 70 s of irradiation and significantly inhibited interleukin 6 (IL6) expression after more than 40 s of irradiation. Transcriptome analysis revealed many differentially expressed genes (DEGs) following CAP irradiation at all time points. Cluster analysis classified the DEGs into four distinct groups, each with time-dependent characteristics. Gene ontology and gene set enrichment analyses revealed CAP-induced suppression of IL6 production, other inflammatory responses, and the expression of genes related to major histocompatibility complex (MHC) class II. Transcription factor analysis suggested that nuclear factor erythroid 2-related factor 2 (NRF2), which suppresses intracellular oxidative stress, is the most activated transcription factor. Contrarily, regulatory factor X5, which regulates MHC class II expression, is the most suppressed transcription factor. Western blotting revealed the nuclear localization of NRF2 following CAP irradiation. These data suggest that CAP suppresses the inflammatory response, possibly by promoting NRF2 nuclear translocation.

Comprehensive phenotypic analysis of single-gene deletion and overexpression strains of Saccharomyces cerevisiae.

We quantified the growth behaviour of all available single-gene deletion and overexpression strains of budding yeast. Genome-wide analyses enabled the extraction of the genes and identification of the functional categories for which genetic perturbation caused the change of growth behaviour. Statistical analyses revealed defective growth for 646 deletion and 1302 overexpression strains. We classified these deleted and overexpressed genes into known functional categories, and identified several functional categories having fragility and robustness for cellular growth. We also screened the deletion and overexpression strains that exhibited a significantly higher growth rate than the strain without genetic perturbation, and found that three deletion and two overexpression strains were high-growth strains. The genes and functional categories identified in the analysis might provide useful information on designing industrially useful yeast strains.

Reconstruction and verification of a genome-scale metabolic model for Synechocystis sp. PCC6803.

In terms of generating sustainable energy resources, the prospect of producing energy and other useful materials using cyanobacteria has been attracting increasing attention since these processes require only carbon dioxide and solar energy. To establish production processes with a high productivity, in silico models to predict the metabolic activity of cyanobacteria are highly desired. In this study, we reconstructed a genome-scale metabolic model of the cyanobacterium Synechocystis sp. PCC6803, which included 465 metabolites and 493 metabolic reactions. Using this model, we performed constraint-based metabolic simulations to obtain metabolic flux profiles under various environmental conditions. We evaluated the simulated results by comparing these with experimental results from (13)C-tracer metabolic flux analyses, which were obtained under heterotrophic and mixotrophic conditions. There was a good agreement of simulation and experimental results under both conditions. Furthermore, using our model, we evaluated the production of ethanol by Synechocystis sp. PCC6803, which enabled us to estimate quantitatively how its productivity depends on the environmental conditions. The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities, and prediction of the metabolic characteristics, of Synechocystis sp. PCC6803.

Mutations in hik26 and slr1916 lead to high-light stress tolerance in Synechocystis sp. PCC6803.

Increased tolerance to light stress in cyanobacteria is a desirable feature for their applications. Here, we obtained a high light tolerant (Tol) strain of Synechocystis sp. PCC6803 through an adaptive laboratory evolution, in which the cells were repeatedly sub-cultured for 52 days under high light stress conditions (7000 to 9000 µmol m-2 s-1). Although the growth of the parental strain almost stopped when exposed to 9000 µmol m-2 s-1, no growth inhibition was observed in the Tol strain. Excitation-energy flow was affected because of photosystem II damage in the parental strain under high light conditions, whereas the damage was alleviated and normal energy flow was maintained in the Tol strain. The transcriptome data indicated an increase in isiA expression in the Tol strain under high light conditions. Whole genome sequence analysis and reverse engineering revealed two mutations in hik26 and slr1916 involved in high light stress tolerance in the Tol strain.

Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae.

To examine the effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae, we constructed deletion strains of all combinations of the trehalase genes ATH1, NTH1 and NTH2 and examined their growth behaviour and intracellular trehalose accumulation under non-stress and saline-stress conditions. Saline stress was induced in yeast cells by NaCl addition at the exponential growth phase. All deletion strains showed similar specific growth rates and trehalose accumulation to their parent strain under non-stress conditions. However, under the saline stress condition, one single deletion strain, nth1Delta, two double deletion strains, nth1Delta ath1Delta and nth1Delta nth2Delta, and the triple deletion strain nth1Deltanth2Delta ath1Delta, all of which carry the nth1Delta deletion, showed increased trehalose accumulation as compared to the parent and other deletion strains. In particular, our statistical analysis revealed that the triple deletion strain showed a higher growth rate under the saline stress condition than the parent strain. Moreover, some deletion strains showed further trehalose accumulation under non-stress conditions by overexpression of the TPS1 or TPS2 genes encoding the enzymes related to trehalose biosynthesis at the mid-exponential phase. Such increased trehalose accumulation prior to NaCl addition could improve the growth of these strains under saline stress. Our results indicate that high trehalose accumulation prior to NaCl addition, rather than after NaCl addition, is necessary to achieve high growth activity under stress conditions.

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae.

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.

Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l-lactate production by Saccharomyces cerevisiae.

In order to determine whether transcriptome data obtained by DNA microarray analysis could be used to identify the genes involved in target metabolite production, we tried to identify the genes involved in L-lactate production by L-lactate-producing recombinant Saccharomyces cerevisiae strains. We obtained DNA microarray data for these strains. Plasmids carrying lactic acid bacteria, bovine, and human L-lactate dehydrogenase (LDH) genes were introduced into PDC1-disrupted S. cerevisiae strains. L-Lactate productivity of the strains harboring the human and bovine LDH genes was higher than that of the strains harboring lactic acid bacteria LDH genes. DNA microarray analysis revealed that the expression of 388 genes was significantly altered in the strains with the human and bovine LDH genes. Of these, the L-lactate productivity of human LDH-harboring deletion strains of 289 genes was compared with that of the standard and 56 randomly selected deletion strains containing the same LDH gene to validate the effectiveness of DNA microarray analysis for identifying the genes responsible for L-lactate production in the recombinant strains. Only deletion strains of the genes selected on the basis of the DNA microarray data showed significantly altered L-lactate production as compared to the standard and the randomly selected deletion strains. Our results indicated that the genes related to L-lactate production could be successfully identified by selecting the genes that exhibited significantly altered expression on DNA microarray analysis, and the effectiveness of DNA microarray analysis for identifying the genes responsible for L-lactate production was discussed.

Analysis of adaptation to high ethanol concentration in Saccharomyces cerevisiae using DNA microarray.

In industrial process, yeast cells are exposed to ethanol stress that affects the cell growth and the productivity. Thus, investigating the intracellular state of yeast cells under high ethanol concentration is important. In this study, using DNA microarray analysis, we performed comprehensive expression profiling of two strains of Saccharomyces cerevisiae, i.e., the ethanol-adapted strain that shows active growth under the ethanol stress condition and its parental strain used as the control. By comparing the expression profiles of these two strains under the ethanol stress condition, we found that the genes related to ribosomal proteins were highly up-regulated in the ethanol-adapted strain. Further, genes related to ATP synthesis in mitochondria were suggested to be important for growth under ethanol stress. We expect that the results will provide a better understanding of ethanol tolerance of yeast.

Improvement of L-lactate production by CYB2 gene disruption in a recombinant Saccharomyces cerevisiae strain under low pH condition.

Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition.

Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13C metabolic flux analysis. Cells were cultured under low (10 µmol m-2 s-1) and high light intensities (100 µmol m-2 s-1) in the presence of glucose. The flux of CO2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08-0.19 mmol gDCW-1 h-1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions.