Publications by authors named "Fumio Matsuda"

151 Publications

Comparative C-metabolic flux analysis indicates elevation of ATP regeneration, carbon dioxide, and heat production in industrial Saccharomyces cerevisiae strains.

Biotechnol J 2021 May 13:e2000438. Epub 2021 May 13.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.

Background: Various industrial Saccharomyces cerevisiae strains are used for specific processes, such as sake, wine brewing and bread making. Understanding mechanisms underlying the fermentation performance of these strains would be useful for further engineering of the S. cerevisiae metabolism. However, the relationship between the fermentation performance, intra-cellular metabolic states, and other phenotypic characteristics of industrial yeasts is still unclear. In this study, C-metabolic flux analysis of four diploid yeast strains-laboratory, sake, bread, and wine yeasts-was conducted.

Results: While the Crabtree effect was observed for all strains, the metabolic flux level of glycolysis was elevated in bread and sake yeast. Furthermore, increased flux levels of the TCA cycle were commonly observed in the three industrial strains. The specific rates of CO production, net ATP regeneration, and metabolic heat generation estimated from the metabolic flux distribution were two to three times greater than those of the laboratory strain. The elevation in metabolic heat generation was correlated with the tolerance to low-temperature stress.

Conclusion: These results indicate that the metabolic flux distribution of sake and bread yeast strains contributes to faster production of ethanol and CO . It is also suggested that the generation of metabolic heat is preferable under the actual industrial fermentation conditions.
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http://dx.doi.org/10.1002/biot.202000438DOI Listing
May 2021

Soft-sensor development for monitoring the lysine fermentation process.

J Biosci Bioeng 2021 May 3. Epub 2021 May 3.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Monitoring cell growth and target production in working fermentors is important for stabilizing high level production. In this study, we developed a novel soft sensor for estimating the concentration of a target product (lysine), substrate (sucrose), and bacterial cell in commercially working fermentors using machine learning combined with available on-line process data. The lysine concentration was accurately estimated in both linear and nonlinear models; however, the nonlinear models were also suitable for estimating the concentrations of sucrose and bacterial cells. Data enhancement by time interpolation improved the model prediction accuracy and eliminated unnecessary fluctuations. Furthermore, the soft sensor developed based on the dataset of the same process parameters in multiple fermentor tanks successfully estimated the fermentation behavior of each tank. Machine learning-based soft sensors may represent a novel monitoring system for digital transformation in the field of biotechnological processes.
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http://dx.doi.org/10.1016/j.jbiosc.2021.04.002DOI Listing
May 2021

Elevated Sporulation Efficiency in Fission Yeast Strains Isolated from .

J Fungi (Basel) 2021 Apr 29;7(5). Epub 2021 Apr 29.

Center for Biosystems Dynamics Research, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.

The fission yeast , comprising var. and var. varieties, has unique characteristics such as striking hyphal growth not seen in other species; however, information on its diversity and evolution, in particular mating and sporulation, remains limited. Here we compared the growth and mating phenotypes of 17 wild strains of , including eight var. strains newly isolated from an insect (). Unlike existing wild strains isolated from fruits/plants, the strains isolated from sporulated at high frequency even under nitrogen-abundant conditions. In addition, one of the strains from was stained by iodine vapor, although the type strain of var. is not stained. Sequence analysis further showed that the nucleotide and amino acid sequences of pheromone-related genes have diversified among the eight strains from , suggesting crossing between cells of different genetic backgrounds occurs frequently in this insect. Much of yeast ecology remains unclear, but our findings suggest that insects such as might be a good niche for mating and sporulation, and will provide a basis for the understanding of sporulation mechanisms via signal transduction, as well as the ecology and evolution of yeast.
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http://dx.doi.org/10.3390/jof7050350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146891PMC
April 2021

Treatment of RB1-intact hepatocellular carcinoma with CDK4/6 inhibitor combination therapy.

Hepatology 2021 Apr 30. Epub 2021 Apr 30.

Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, 920-1192, Japan.

Synthetic CDK4/6 inhibitors exert anti-tumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis and increased immunogenicity. These agents currently have an indication in advanced breast cancers, and are in clinical trials for many other solid tumors. Hepatocellular carcinoma (HCC) is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC, however, this is revivable as RB family members are rarely mutated or deleted in this malignancy. Loss of all Rb family members in Trp53 mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an IKKβ inhibitor Bay11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/β phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact K-Ras mutated lung and colon cancers. In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.
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http://dx.doi.org/10.1002/hep.31872DOI Listing
April 2021

Random Transfer of Genes into Reveals a Complex Background of Heat Tolerance.

J Fungi (Basel) 2021 Apr 15;7(4). Epub 2021 Apr 15.

Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.

Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, , and then introduced into . was selected because it is one of the most heat-tolerant species among yeasts. Screening of populations expressing genes at high temperatures identified 59 genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.
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http://dx.doi.org/10.3390/jof7040302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071464PMC
April 2021

Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases.

Metab Eng Commun 2021 Jun 19;12:e00169. Epub 2021 Mar 19.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. , the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.
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http://dx.doi.org/10.1016/j.mec.2021.e00169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040282PMC
June 2021

Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Metabolites 2021 Mar 26;11(4). Epub 2021 Mar 26.

Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan.

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.
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http://dx.doi.org/10.3390/metabo11040197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065654PMC
March 2021

Seed-coat protective neolignans are produced by the dirigent protein AtDP1 and the laccase AtLAC5 in Arabidopsis.

Plant Cell 2021 03;33(1):129-152

RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.
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http://dx.doi.org/10.1093/plcell/koaa014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136895PMC
March 2021

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

Commun Biol 2021 Mar 16;4(1):343. Epub 2021 Mar 16.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, Japan.

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 s). Although the growth of the parental strain almost stopped when exposed to 9000 μmol m s, 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.
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http://dx.doi.org/10.1038/s42003-021-01875-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966805PMC
March 2021

Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of sp. PCC 6803 metabolism.

Metab Eng Commun 2020 Dec 27;11:e00153. Epub 2020 Nov 27.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.

The present study attempted a computer simulation of the metabolism of a model cyanobacteria, sp. PCC 6803 (PCC 6803) to predict allosteric inhibitions that are likely to occur in photoautotrophic and mixotrophic conditions as well as in a metabolically engineered strain. PCC 6803 is a promising host for direct biochemical production from CO; however, further investigation of allosteric regulation is required for rational metabolic engineering to produce target compounds. Herein, ensemble modeling of microbial metabolism was applied to build accurate predictive models by synthesizing the results of multiple models with different parameter sets into a single score to identify plausible allosteric inhibitions. The data driven-computer simulation using metabolic flux, enzyme abundance, and metabolite concentration data successfully identified candidates for allosteric inhibition. The enzyme assay experiment using the recombinant protein confirmed isocitrate was a non-competitive inhibitor of phosphoribulokinase as a novel allosteric regulation of cyanobacteria metabolism.
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http://dx.doi.org/10.1016/j.mec.2020.e00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721636PMC
December 2020

Fake metabolomics chromatogram generation for facilitating deep learning of peak-picking neural networks.

J Biosci Bioeng 2021 Feb 10;131(2):207-212. Epub 2020 Oct 10.

Graduate School of Information Science and Technology, Osaka University, 2-1, Yamada-oka, Osaka 565-0871, Japan. Electronic address:

Finding peaks in chromatograms and determining their start and end points (peak picking) is a core task in chromatography based biotechnology. Construction of peak-picking neural networks by deep learning was, however, hampered from the preparation of exact peak-picked or "labeled" chromatograms since the exact start and end points were often unclear in overlapping peaks in real chromatograms. We present a design of a fake chromatogram generator, along with a method for deep learning of peak-picking neural networks. Fake chromatograms were generated by generation of fake peaks, random sampling of peak positions from feature distributions, and merging with real blank sample chromatograms. Information on the exact start and end points, as labeled on the fake chromatograms, were effective for training and evaluating peak-picking neural networks. The peak-picking neural networks constructed herein outperformed conventional peak-picking software and showed comparable performance with that of experienced operators for processing the widely targeted metabolome data. Results of this study indicate that generation of fake chromatograms would be crucial for developing peak-picking neural networks and a key technology for further improvement of peak picking neural networks.
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http://dx.doi.org/10.1016/j.jbiosc.2020.09.013DOI Listing
February 2021

Assessment of Protein Content and Phosphorylation Level in sp. PCC 6803 under Various Growth Conditions Using Quantitative Phosphoproteomic Analysis.

Molecules 2020 Aug 6;25(16). Epub 2020 Aug 6.

Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.

The photosynthetic apparatus and metabolic enzymes of cyanobacteria are subject to various controls, such as transcriptional regulation and post-translational modifications, to ensure that the entire cellular system functions optimally. In particular, phosphorylation plays key roles in many cellular controls such as enzyme activity, signal transduction, and photosynthetic apparatus restructuring. Therefore, elucidating the governing functions of phosphorylation is crucial to understanding the regulatory mechanisms underlying metabolism and photosynthesis. In this study, we determined protein content and phosphorylation levels to reveal the regulation of intracellular metabolism and photosynthesis in sp. PCC 6803; for this, we obtained quantitative data of proteins and their phosphorylated forms involved in photosynthesis and metabolism under various growth conditions (photoautotrophic, mixotrophic, heterotrophic, dark, and nitrogen-deprived conditions) using targeted proteomic and phosphoproteomic analyses with nano-liquid chromatography-triple quadrupole mass spectrometry. The results indicated that in addition to the regulation of protein expression, the regulation of phosphorylation levels of cyanobacterial photosynthetic apparatus and metabolic enzymes was pivotal for adapting to changing environmental conditions. Furthermore, reduced protein levels of CpcC and altered phosphorylation levels of CpcB, ApcA, OCP, and PsbV contributed to the cellular response of the photosynthesis apparatus to nitrogen deficiency.
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http://dx.doi.org/10.3390/molecules25163582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466104PMC
August 2020

Direct and quantitative analysis of altered metabolic flux distributions and cellular ATP production pathway in fumarate hydratase-diminished cells.

Sci Rep 2020 08 3;10(1):13065. Epub 2020 Aug 3.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.

Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied C metabolic flux analysis (C-MFA) to an established cell line with diminished FH activity (FH) and parental HEK293 cells. FH cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FH cells. Consistently, FH cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FH cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.
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http://dx.doi.org/10.1038/s41598-020-70000-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400513PMC
August 2020

Data science-based modeling of the lysine fermentation process.

J Biosci Bioeng 2020 Oct 22;130(4):409-415. Epub 2020 Jul 22.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Mathematical modeling of the fermentation process is useful for understanding the influence of operating parameters on target production and control performance, depending on the situation, to stabilize the target production at a high-level. However, the previous approaches using physical modeling methods and traditional knowledge-based methods are difficult to apply on working fermentors at a commercial plant scale because they have unknown and unmeasured parameters involved in target production. This study focused on developing an ensemble learning model that can predict the amino acid fermentation process behavior based on observation values, which can be obtained from fermentation tanks and future control input. The results revealed the influence of each control input on lysine production during the culturing period. Furthermore, high-order stability, which achieved the target trajectory for lysine production, was realized using dynamic fermentation controls. Additionally, this study demonstrates that the fermentation behavior on a commercial plant scale is reproduced using the ensemble device. The ensemble learning model will provide novel control system with data-science based model of Industry 4.0 in the field of biotechnological processes.
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http://dx.doi.org/10.1016/j.jbiosc.2020.06.011DOI Listing
October 2020

Drought Stress Responses in Context-Specific Genome-Scale Metabolic Models of .

Metabolites 2020 Apr 18;10(4). Epub 2020 Apr 18.

RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan.

Drought perturbs metabolism in plants and limits their growth. Because drought stress on crops affects their yields, understanding the complex adaptation mechanisms evolved by plants against drought will facilitate the development of drought-tolerant crops for agricultural use. In this study, we examined the metabolic pathways of which respond to drought stress by omics-based in silico analyses. We proposed an analysis pipeline to understand metabolism under specific conditions based on a genome-scale metabolic model (GEM). Context-specific GEMs under drought and well-watered control conditions were reconstructed using transcriptome data and examined using metabolome data. The metabolic fluxes throughout the metabolic network were estimated by flux balance analysis using the context-specific GEMs. We used in silico methods to identify an important reaction contributing to biomass production and clarified metabolic reaction responses under drought stress by comparative analysis between drought and control conditions. This proposed pipeline can be applied in other studies to understand metabolic changes under specific conditions using GEM or other available plant GEMs.
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http://dx.doi.org/10.3390/metabo10040159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241242PMC
April 2020

Effects of mutations of GID protein-coding genes on malate production and enzyme expression profiles in Saccharomyces cerevisiae.

Appl Microbiol Biotechnol 2020 Jun 4;104(11):4971-4983. Epub 2020 Apr 4.

Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan.

During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex-mediated degradation. Next, we investigated the effects of GID protein-coding genes (GID1-GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle-related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation. KEY POINTS: An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production. The protein expression profiles in the GID disruptants differed from one another.
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http://dx.doi.org/10.1007/s00253-020-10573-4DOI Listing
June 2020

Fragmentation of Dicarboxylic and Tricarboxylic Acids in the Krebs Cycle Using GC-EI-MS and GC-EI-MS/MS.

Mass Spectrom (Tokyo) 2019 30;8(1):A0073. Epub 2019 Aug 30.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, Japan.

Isotope labeling measurements using mass spectrometry can provide informative insights on the metabolic systems of various organisms. The detailed identification of carbon positions included in the fragment ions of dicarboxylic and tricarboxylic acids in central carbon metabolism is needed for precise interpretation of the metabolic states. In this study, fragment ions containing the carbon backbone cleavage of dicarboxylic and tricarboxylic in the Krebs cycle were investigated by using gas chromatography (GC)-electron ionization (EI)-MS and GC-EI-MS/MS. The positions of decarboxylation in the dicarboxylic and tricarboxylic acids were successfully identified by analyses using position-specific C-labeled standards prepared by enzymatic reactions. For example, carboxyl groups of C1 and C6 of trimethylsilyl (TMS)- and -butyldimethylsilyl (TBDMS)-derivatized malic and citric acids were primarily cleaved by EI. MS/MS analyses were also performed, and fragment ions of TBDMS-citric and α-ketoglutaric acids (αKG) with the loss of two carboxyl groups in collision-induced dissociation (CID) were observed.
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http://dx.doi.org/10.5702/massspectrometry.A0073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920549PMC
August 2019

Computational data mining method for isotopomer analysis in the quantitative assessment of metabolic reprogramming.

Sci Rep 2020 01 14;10(1):286. Epub 2020 Jan 14.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.

Measurement of metabolic flux levels using stable isotope labeling has been successfully used to investigate metabolic redirection and reprogramming in living cells or tissues. The metabolic flux ratio between two reactions can be estimated from the C-labeling patterns of a few metabolites combined with the knowledge of atom mapping in the complicated metabolic network. However, it remains unclear whether an observed change in the labeling pattern of the metabolites is sufficient evidence of a shift in flux ratio between two metabolic states. In this study, a data analysis method was developed for the quantitative assessment of metabolic reprogramming. The Metropolis-Hastings algorithm was used with an in silico metabolic model to generate a probability distribution of metabolic flux levels under a condition in which the C-labeling pattern was observed. Reanalysis of literature data demonstrated that the developed method enables analysis of metabolic redirection using whole C-labeling pattern data. Quantitative assessment by Cohen's effect size (d) enables a more detailed read-out of metabolic reprogramming information. The developed method will enable future applications of the metabolic isotopomer analysis to various targets, including cultured cells, whole tissues, and organs.
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http://dx.doi.org/10.1038/s41598-019-57146-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959353PMC
January 2020

Inter-Laboratory Comparison of Metabolite Measurements for Metabolomics Data Integration.

Metabolites 2019 Oct 31;9(11). Epub 2019 Oct 31.

Department of Lipidomics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Background: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory.

Methods: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory.

Results: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference.

Conclusion: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization.
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http://dx.doi.org/10.3390/metabo9110257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918145PMC
October 2019

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae.

Microb Cell Fact 2019 Oct 15;18(1):177. Epub 2019 Oct 15.

Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Background: Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae.

Results: The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production.

Conclusion: These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.
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http://dx.doi.org/10.1186/s12934-019-1226-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794801PMC
October 2019

Comparison of metabolic profiles of yeasts based on the difference of the Crabtree positive and negative.

J Biosci Bioeng 2020 Jan 16;129(1):52-58. Epub 2019 Sep 16.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

The Crabtree effect involves energy management in which yeasts utilize glycolysis as the terminal electron acceptor instead of oxygen, despite the presence of sufficient dissolved oxygen, when oxygen concentrations exceed a certain limit. The Crabtree effect is detrimental to bakery yeast production, because it results in lower cellular glucose yields. Batch culture of Saccharomyces cerevisiae, a Crabtree positive yeast, decreased the cell yield of glucose and produced large amounts of ethanol despite a high specific glucose consumption rate compared to Candida utilis, a Crabtree negative yeast. This study investigated the effect of these characteristics on metabolite levels. We performed metabolome analysis of both yeasts during each growth phase of batch culture using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Principle component analysis of metabolome data indicated that the Crabtree effect affected metabolites related to NADH synthesis in central metabolism. The amount of these metabolites in S. cerevisiae was lower than that in C. utilis. However, to maintain the specific glucose consumption rate at high levels, yeasts must avoid depletion of NAD, which is essential for glucose utilization. Our results indicated that NADH was oxidized by converting acetaldehyde to ethanol in S. cerevisiae, which is in accordance with previous reports. Therefore, the specific NADH production rates of S. cerevisiae and C. utilis did not show a difference. This study suggested that NAD/NADH ratio is disrupted by the Crabtree effect, which in turn influenced central metabolism and that S. cerevisiae maintained the NAD/NADH ratio by producing ethanol.
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http://dx.doi.org/10.1016/j.jbiosc.2019.07.007DOI Listing
January 2020

Survival of membrane-damaged Escherichia coli in a cytosol-mimicking solution.

J Biosci Bioeng 2019 Nov 7;128(5):558-563. Epub 2019 Jun 7.

Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Frontier Bioscience, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Arts and Science, Komaba Institute for Science, Universal Biology Institute, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. Electronic address:

Selective permeability of cell membrane is critically important for cell survival. The damage caused to cell membrane by pore-forming antimicrobial peptides may result in the loss of selective permeability and leakage of intracellular molecules, eventually leading to cell death. Here, we examined whether the membrane-damaged Escherichia coli cells survive in a cytosol-mimicking solution (CMS), which compensates for the lethal leakage of intracellular molecules. We prepared a CMS comprising 34 low molecular weight compounds from the cytosol and found that the cells were able to grow in CMS even in the presence of a pore-forming peptide, melittin. We confirmed that the melittin-treated cells lost selective membrane permeability by staining with membrane-impermeable dyes, propidium iodide and SYTOX green. Some stained cells maintained the colony formation ability in CMS. These results provide an evidence that E. coli cells can at least partially survive in the CMS even after the temporary impairment of membrane selective permeability. This study demonstrates a technique that allows temporal loss of the selective permeability of the cell membrane while maintaining the viability of cells that may be useful for the introduction of membrane-impermeable molecules into E. coli cells.
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http://dx.doi.org/10.1016/j.jbiosc.2019.05.005DOI Listing
November 2019

A Fusion Method to Develop an Expanded Artificial Genomic RNA Replicable by Qβ Replicase.

Chembiochem 2019 09 5;20(18):2331-2335. Epub 2019 Aug 5.

Department of Life Science, Graduate School of Arts and Science, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan.

RNA-based genomes are used to synthesize artificial cells that harbor genome replication systems. Previously, continuous replication of an artificial genomic RNA that encoded an RNA replicase was demonstrated. The next important challenge is to expand such genomes by increasing the number of encoded genes. However, technical difficulties are encountered during such expansions because the introduction of new genes disrupts the secondary structure of RNA and makes RNA nonreplicable through replicase. Herein, a fusion method that enables the construction of a longer RNA from two replicable RNAs, while retaining replication capability, is proposed. Two replicable RNAs that encode different genes at various positions are fused, and a new parameter, the unreplicable index, which negatively correlates with the replication ability of the fused RNAs better than that of the previous parameter, is determined. The unreplicable index represents the expected value of the number of G or C nucleotides that are unpaired in both the template and complementary strands. It is also observed that some of the constructed fused RNAs replicate efficiently by using the internally translated replicase. The method proposed herein could contribute to the development of an expanded RNA genome that can be used for the purpose of artificial cell synthesis.
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http://dx.doi.org/10.1002/cbic.201900120DOI Listing
September 2019

Time-resolved analysis of short term metabolic adaptation at dark transition in Synechocystis sp. PCC 6803.

J Biosci Bioeng 2019 Oct 10;128(4):424-428. Epub 2019 Apr 10.

Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

In photosynthetic organisms, such as cyanobacteria, ATP and NADPH are generated through the light reaction, and then are used for CO fixation in the dark reaction. As light intensity always fluctuates under natural conditions, balancing the cofactor regeneration and consumption is essential to maintain active CO fixation as well as for metabolic engineering of strains that produce biochemicals. In this study, a time-resolved metabolome analysis of Synechocystis sp. PCC 6803 (PCC6803) was conducted to investigate a metabolic adaptation at 0-15 min after a sudden shift from light to dark conditions. Rapid accumulation of sedoheptulose 7-phosphate, ribulose 5-phosphate, xylulose 5-phosphate, and 6-phosphogluconate suggested that the central metabolism of PCC6803 was regulated by inactivation of phosphoribulokinase and activation of glucose-6-phosphate dehydrogenase (G6PDH) probably via the redox regulation. The culture and metabolic profile of the Δzwf strain lacking G6PDH showed that the role of G6PDH in regeneration of NADPH could be complemented by the activation of isocitrate dehydrogenase in the TCA cycle, indicating the importance of the rapid regulation of NADPH regeneration after the shift to dark conditions. The mechanism underlying metabolic regulation is also useful for metabolic engineering of PCC6803, as the Δzwf strain produced higher amount of organic acids than wild type.
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http://dx.doi.org/10.1016/j.jbiosc.2019.03.016DOI Listing
October 2019

Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli.

Biotechnol Lett 2019 Jul 5;41(6-7):743-751. Epub 2019 Apr 5.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Objectives: Fine-tuning of enzyme expression at low levels is an important challenge for metabolic engineers. Here, theophylline-inducible riboswitch for translational regulation was evaluated. The background expression, translation rate, and time delay for its induction was reported.

Results: To evaluate the effect of the amount of mRNA on its translation rate, transcription of the riboswitch RNA with red fluorescent protein (RFP) was controlled by the lac system with addition of isopropyl β-D-1-thiogalactopyranoside in Escherichia coli. Regardless of the amount of riboswitch mRNA, the translation of RFP was completely suppressed without theophylline during both growth and stationary phases. Furthermore, a strong positive correlation between theophylline concentration (0 to 1 mM) and specific RFP production rate was observed. The specific RFP production rate with the riboswitch was approximately 2.3% of that without the riboswitch. Furthermore, 60 min of time delay for RFP expression was observed after adding theophylline during the stationary phase.

Conclusion: Theophylline-inducible riboswitch precisely controls protein translation at low expression levels with significantly low background expression. It can emerge as a powerful tool for fine tuning of enzyme expression.
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http://dx.doi.org/10.1007/s10529-019-02672-8DOI Listing
July 2019

Transomics data-driven, ensemble kinetic modeling for system-level understanding and engineering of the cyanobacteria central metabolism.

Metab Eng 2019 03 8;52:273-283. Epub 2019 Jan 8.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

In silico kinetic modeling is an essential tool for rationally designing metabolically engineered organisms based on a system-level understanding of their regulatory mechanisms. However, an estimation of enzyme parameters has been a bottleneck in the computer simulation of metabolic dynamics. In this study, the ensemble-modeling approach was integrated with the transomics data to construct kinetic models. Kinetic metabolic models of a photosynthetic bacterium, Synechocystis sp. PCC 6803, were constructed to identify engineering targets for improving ethanol production based on an understanding of metabolic regulatory systems. A kinetic model ensemble was constructed by randomly sampling parameters, and the best 100 models were selected by comparing predicted metabolic state with a measured dataset, including metabolic flux, metabolite concentrations, and protein abundance data. Metabolic control analysis using the model ensemble revealed that a large pool size of 3-phosphoglycerate could be a metabolic buffer responsible for the stability of the Calvin-Benson cycle, and also identified that phosphoglycerate kinase (PGK) is a promising engineering target to improve a pyruvate supply such as for ethanol production. Overexpression of PGK in the metabolically engineered PCC 6803 strain showed that the specific ethanol production rate and ethanol titers at 48 h were 1.23- and 1.37-fold greater than that of the control strain. PGK is useful for future metabolic engineering since pyruvate is a common precursor for the biosynthesis of various chemicals.
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http://dx.doi.org/10.1016/j.ymben.2019.01.004DOI Listing
March 2019

Magnesium starvation improves production of malonyl-CoA-derived metabolites in Escherichia coli.

Metab Eng 2019 03 6;52:215-223. Epub 2018 Dec 6.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Starvation of essential nutrients, such as nitrogen, sulfur, magnesium, and phosphorus, leads cells into stationary phase and potentially enhances target metabolite production because cells do not consume carbon for the biomass synthesis. The overall metabolic behavior changes depend on the type of nutrient starvation in Escherichia coli. In the present study, we determined the optimum nutrient starvation type for producing malonyl-CoA-derived metabolites such as 3-hydroxypropionic acid (3HP) and naringenin in E. coli. For 3HP production, high production titer (2.3 or 2.0 mM) and high specific production rate (0.14 or 0.28 mmol gCDW h) was observed under sulfur or magnesium starvation, whereas almost no 3HP production was detected under nitrogen or phosphorus starvation. Metabolic profiling analysis revealed that the intracellular malonyl-CoA concentration was significantly increased under the 3HP producing conditions. This accumulation should contribute to the 3HP production because malonyl-CoA is a precursor of 3HP. Strong positive correlation (r = 0.95) between intracellular concentrations of ATP and malonyl-CoA indicates that the ATP level is important for malonyl-CoA synthesis due to the ATP requirement by acetyl-CoA carboxylase. For naringenin production, magnesium starvation led to the highest production titer (144 ± 15 μM) and specific productivity (127 ± 21 μmol gCDW). These results demonstrated that magnesium starvation is a useful approach to improve the metabolic state of strains engineered for the production of malonyl-CoA derivatives.
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http://dx.doi.org/10.1016/j.ymben.2018.12.002DOI Listing
March 2019

Editorial overview: Recent progress in analytical technologies for design-build-test-learn cycle in biotechnology.

Curr Opin Biotechnol 2018 12 2;54:145-147. Epub 2018 Oct 2.

Analytical Biotechnology Laboratory, Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.copbio.2018.09.009DOI Listing
December 2018

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

J Biosci Bioeng 2019 Mar 28;127(3):394-402. Epub 2018 Sep 28.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.
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http://dx.doi.org/10.1016/j.jbiosc.2018.09.001DOI Listing
March 2019