Katsunori Yoshikawa

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.

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. Copyright

Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray

To construct yeast strains showing tolerance to high salt concentration stress, we analyzed the transcriptional response to high NaCl concentration stress in the yeast Saccharomycescerevisiae using DNA microarray and compared between two yeast strains, a laboratory strain and a brewing one, which is known as a stress-tolerant strain. Gene expression dynamically changed following the addition of NaCl in both yeast strains, but the degree of change in the gene expression level in the laboratory strain was larger than that in the brewing strain. The response of gene expression to the low NaCl concentration stress was faster than that to the high NaCl concentration stress in both strains. Expressions of the genes encoding enzymes involved in carbohydrate metabolism and energy production in both strains or amino acid metabolism in the brewing strain were increased under high NaCl concentration conditions. Moreover, the genes encoding sodium ion efflux pump and copper metallothionein proteins were more highly expressed in the brewing strain than in the laboratory strain. According to the results of transcriptome analysis, candidate genes for the creation of stress-tolerant strain were selected, and the effect of overexpression of candidate genes on the tolerance to high NaCl concentration stress was evaluated. Overexpression of the GPD1 gene encoding glycerol-3-phosphate dehydrogenase, ENA1 encoding sodium ion efflux protein, and CUP1 encoding copper metallothionein conferred high salt stress tolerance to yeast cells, and our selection of candidate genes for the creation of stress-tolerant yeast strains based on the transcriptome data was validated.

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, nth1Δ, two double deletion strains, nth1Δ ath1Δ and nth1Δ nth2Δ, and the triple deletion strain nth1Δnth2Δ ath1Δ, all of which carry the nth1Δ 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. Copyright

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 13C-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.

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

Background3-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.ResultsMetabolic 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.ConclusionThis 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.

Integrated transcriptomic and metabolomic analysis of the central metabolism of Synechocystis sp. PCC 6803 under different trophic conditions

Cyanobacteria have received considerable attention as a sustainable energy resource because of their organic material production capacity using light energy and CO2 as a carbon source. Therefore, it is important to understand the cellular metabolism of cyanobacteria for metabolic engineering. In this study, to shed light on the central metabolism of cyanobacteria, we performed transcriptomic and metabolomic analyses of a glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803, which was cultured under auto- and mixotrophic conditions. Our results indicate that the oxidative pentose phosphate pathway and glycolysis are activated under mixotrophic conditions rather than autotrophic conditions. Moreover, we examined the effect of atrazine, a photosynthesis inhibitor, on the metabolism of PCC 6803 under mixotrophic conditions, which was defined as photoheterotrophic conditions, by transcriptomics and metabolomics. We demonstrated that the activity of the glycolytic pathway decreased due to the indirect effect of atrazine. In addition, the difference in transcriptional and metabolic changes between auto- and photoheterotrophic conditions could also be captured. The omics dataset reported herein provides clues for understanding the metabolism of cyanobacteria.

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.

Genome-wide expression analysis of Saccharomyces pastorianus orthologous genes using oligonucleotide microarrays.

The lager brewing yeast, Saccharomyces pastorianus, an allopolyploid species hybrid, contains 2 diverged sub-genomes; one derived from Saccharomyces cerevisiae (Sc-type) and the other from Saccharomyces bayanus (Sb-type). We analyzed the functional roles of these orthologous genes in determining the phenotypic features of S. pastorianus. We used a custom-made oligonucleotide microarray containing probes designed for both Sc-type and Sb-type ORFs for a comprehensive expression analysis of S. pastorianus in a pilot-scale fermentation. We showed a high degree of correlation between the expression levels and the expression changes for a majority of orthologous gene sets during the fermentation process. We screened the functional categories and metabolic pathways where Sc- or Sb-type genes have higher expression levels than the corresponding orthologous genes. Our data showed that, for example, pathways for sulfur metabolism, cellular import, and production of branched amino acids are dominated by Sb-type genes. This comprehensive expression analysis of orthologous genes can provide valuable insights on understanding the phenotype of S. pastorianus.

Extracting the hidden features in saline osmotic tolerance in Saccharomyces cerevisiae from DNA microarray data using the self-organizing map: biosynthesis of amino acids

During saline stress, Saccharomyces cerevisiae changes its metabolic fluxes through the direct accumulation of metabolites such as glycerol and trehalose, which in turn provide tolerance to the cell against stress. Previous research shows that the various controls at both transcriptional and translational levels decide the phenomenon of stress, but details about such transition is still not very clear. This paper attempts to extract some hidden features through the information extraction approach from DNA microarray data during transition to osmotic tolerance, which are expected to be important in directing to the tolerance stage upon encountering osmotic stress in yeast. Time course of DNA microarray data during osmotic tolerance was analyzed by computational approach ‘self-organizing map (SOM) extended with hierarchical clustering’. Since eukaryotic gene expression is governed by short regulatory sequences found upstream in promoter regions, therefore clusters containing the similar profiles obtained by SOM were further analyzed for overrepresentation of known regulatory binding sites in promoter region. It was found that apart from known and expected ‘STRE’ during osmotic stress, the ‘GCN4’ binding site is also found to be significant. Hence, it was suggested that the process of osmotic tolerance proceeds through a stage of amino acid starvation. The intracellular amino acids pool also found to be depleted during transition and restoration is faster in brewing strain than laboratory strain. Experiments involving supplementation of amino acids helps in reducing the lag time for recovery, which was found to be similar to that of brewing strain.