Mami Okamoto

Statistical Indices for Simultaneous Large-Scale Metabolite Detections for a Single NMR Spectrum

NMR-based metabolomics has become a practical and analytical methodology for discovering novel genes, biomarkers, metabolic phenotypes, and dynamic cell behaviors in organisms. Recent developments in NMR-based metabolomics, however, have not concentrated on improvements of comprehensiveness in terms of simultaneous large-scale metabolite detections. To resolve this, we have devised and implemented a statistical index, the SpinAssign p-value, in NMR-based metabolomics for large-scale metabolite annotation and publicized this information. It enables simultaneous annotation of more than 200 candidate metabolites from the single (13)C-HSQC (heteronuclear single quantum coherence) NMR spectrum of a single sample of cell extract.

ABA Hypersensitive Germination2-1 Causes the Activation of Both Abscisic Acid and Salicylic Acid Responses in Arabidopsis

ABA and salicylic acid (SA) are believed to act antagonistically. We previously reported that an ABA-hypersensitive mutant ahg2-1, which had reduced expression of poly(A)-specific ribonuclease (PARN), exhibited pleiotropic phenotypes including unique enhanced ABA- and SA-sensitive phenotypes. In this study, we characterized the increased SA-sensitive phenotype of this mutant in detail and addressed its relationship with ABA-related and dwarf phenotypes. We found that the ahg2-1 mutant had a high endogenous SA level and an elevated resistance to bacterial pathogens. Double mutant analyses showed that Arabidopsis plants defective in the SA signaling pathway (npr1 and pad4 mutants and nahG transgenic plants) could suppress neither the ABA hypersensitivity nor the dwarf phenotypes. These results indicate that ABA-related, SA-related and dwarf phenotypes of the ahg2-1 mutant are independent of each other. To obtain more insight into the molecular basis of the effect of ahg2-1, microarray analyses were conducted not only for ahg2-1 but also for ahg2sid2 or ahg2abi1 so as to reduce the secondary effects of SA or ABA. The resulting data indicate that ahg2-1 has a unique gene expression profile, consistent with the novel phenotype of this mutant. Detailed comparison of the expression profiles of up- or down-regulated genes implied that ahg2-1 somehow affects mitochondrial function. Our data suggest that a partial loss of PARN activity affects ABA, SA and mitochondrial function independently, and that the regulation of mRNA levels is deeply implicated in diverse cellular functions.

Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium

Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR.

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45–0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = –0.95 to –0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

Toward the complete utilization of rice straw: Methane fermentation and lignin recovery by a combinational process involving mechanical milling, supporting material and nanofiltration

Rice straw was mechanically milled using a process consuming 1.9MJ/kg-biomass, and 10g/L of unmilled or milled rice straw was used as the carbon source for methane fermentation in a digester containing carbon fiber textile as the supporting material. Milling increased methane production from 226 to 419mL/L/day at an organic loading rate of 2180mg-dichromate chemical oxygen demand/L/day, corresponding to 260mLCH4/gVS. Storage of the fermentation effluent at room temperature decreased the weight of the milled rice straw residue from 3.81 to 1.00g/L. The supernatant of the effluent was subjected to nanofiltration. The black concentrates deposited on the nanofiltration membranes contained 53.0-57.9% lignin. Solution nuclear magnetic resonance showed that lignin aromatic components such as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) were retained primarily, and major lignin interunit structures such as the β-O-4-H/G unit were absent. This combinational process will aid the complete utilization of rice straw.

Changes in primary metabolism under light and dark conditions in response to overproduction of a response regulator RpaA in the unicellular cyanobacterium Synechocystis sp. PCC 6803

The study of the primary metabolism of cyanobacteria in response to light conditions is important for environmental biology because cyanobacteria are widely distributed among various ecological niches. Cyanobacteria uniquely possess circadian rhythms, with central oscillators consisting from three proteins, KaiA, KaiB, and KaiC. The two-component histidine kinase SasA/Hik8 and response regulator RpaA transduce the circadian signal from KaiABC to control gene expression. Here, we generated a strain overexpressing rpaA in a unicellular cyanobacterium Synechocystis sp. PCC 6803. The rpaA-overexpressing strain showed pleiotropic phenotypes, including slower growth, aberrant degradation of an RNA polymerase sigma factor SigE after the light-to-dark transition, and higher accumulation of sugar catabolic enzyme transcripts under dark conditions. Metabolome analysis revealed delayed glycogen degradation, decreased sugar phosphates and organic acids in the tricarboxylic acid cycle, and increased amino acids under dark conditions. The current results demonstrate that in this cyanobacterium, RpaA is a regulator of primary metabolism and involved in adaptation to changes in light conditions.

Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria

Potassium (K+) is an essential macronutrient for all living organisms including cyanobacteria. Cyanobacteria are a group of bacteria performing oxygenic photosynthesis, widely studied in basic and applied sciences. The primary metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is altered by environmental conditions, and it excretes organic acids and hydrogen under dark, anaerobic conditions. Here we demonstrated that K+ widely changes the primary carbon metabolism of this cyanobacterium. Succinate and lactate excretion from the cells incubated under dark, anaerobic conditions was enhanced in the presence of K+, while hydrogen production was repressed. The addition of K+ and the genetic manipulation of acetate kinase AckA and an RNA polymerase sigma factor SigE additively increased succinate and lactate production to 141.0 and 217.6 mg/L, which are 11 and 46 times, compared to the wild-type strain without K+, respectively. Intracellular levels of 2-oxoglutarate, succinate, fumarate, and malate increased by K+ under dark, anaerobic conditions. This study provides the evidence of the considerable effect of K+ on the biosynthesis of anionic metabolites in a unicellular cyanobacterium.

Evaluation of control mechanisms for Saccharomyces cerevisiae central metabolic reactions using metabolome data of eight single-gene deletion mutants

We performed metabolome and metabolite–metabolite correlation analyses for eight single-gene deletion mutants of Saccharomyces cerevisiae to evaluate the physiology of glucose metabolism. The irreversible enzyme reactions can become bottlenecks when intracellular metabolism is perturbed by direct interference from the central metabolic pathway by gene deletions or by a deletion of transcriptional regulator. Metabolome data reveal that transcriptional factor, gcr2, regulates the reaction that converts 3-phosphoglycerate into phosphoenolpyruvate. Metabolome data also suggest that the reaction catalyzed by pyruvate kinase makes one of the rate-limiting reactions throughout the glycolytic pathway.

Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae

To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.