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cis-(+)-12-Oxo-phytodienoic acid (cis-OPDA) is a significant plant oxylipin, known as a biosynthetic precursor of the plant hormone jasmonoyl-l-isoleucine (JA-Ile), and a bioactive substance in plant environmental stresses. A recent study showed that a plant dioxygenase, Jasmonate Induced Dioxygenase 1 (JID1), converts cis-OPDA into an unidentified metabolite termed "modified-OPDA (mo-OPDA)" in Arabidopsis thaliana. Here, using ultra-performance liquid chromatography coupled with triple quad mass spectrometry (UPLC-MS/MS) experiment, the chemical identity of "mo-OPDA" was demonstrated and identified as a conjugate between cis-OPDA and 2-mercaptoethanol (cis-OPDA-2ME), an artifact produced by Michael addition during the JID1 digestion of cis-OPDA. However, previous reports demonstrated a decreased accumulation of cis-OPDA in the JID1-OE line, suggesting the existence of an unknown JID1-mediated mechanism regulating the level of cis-OPDA in A. thaliana., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

(3R, 7S)-jasmonoyl-L-isoleucine (JA-Ile) is a lipid-derived plant hormone that regulates plant responses, including biotic/abiotic stress adaptation. In the plant cells, JA-Ile is perceived by COI1-JAZ co-receptor by causing protein-protein interaction between COI1 and JAZ proteins to trigger gene expressions. In this study, we focused on Oryza sativa, a model monocot and an important crop, with 45 possible OsCOI-OsJAZ co-receptor pairs composed of three OsCOI homologs (OsCOI1a, OsCOI1b, and OsCOI2) and 15 OsJAZ homologs. We performed fluorescein anisotropy and pull-down assays to examine the affinity between JA-Ile and OsCOI1a/1b/2-OsJAZ1-15 co-receptor pairs. The results revealed a remarkable difference in the modes of ligand perception by OsCOI1a/1b and OsCOI2. Recently, the unique function of OsCOI2 in some of the JA-responses were revealed. Our current results will lead to the possible development of OsCOI2-selective synthetic ligand., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Ethylene (ET) and jasmonate (JA) are plant hormones that act synergistically to regulate plant development and defense against necrotrophic fungi infections, and antagonistically in response to wounds and apical hook formation. Previous studies revealed that the coordination of these responses is due to dynamic protein-protein interactions (PPI) between their master transcription factors (TFs) EIN3/EIL1 and MYC in ET and JA signaling, respectively. In addition, both TFs are activated via interactions with the same transcriptional mediator MED25, which upregulates downstream gene expression. Herein, we analyzed the PPI between EIN3/EIL1 and MED25, and as with the PPI between MYC3 and MED25, found that the short binding domain of MED25 (CMIDM) is also responsible for the interaction with EIN3/EIL1 - a finding which suggests that both TFs compete for binding with MED25. These results further inform our understanding of the coordination between the ET and JA regulatory systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. This approach is attractive for constructing a sustainable PET recycling system., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

S-Allyl-l-cysteine (SAC) has received much interest due to its beneficial effects on human health. To satisfy the increasing demand for SAC, this study aims to develop a valuable culturing method for microbial screening synthesizing SAC from readily available materials. Although tryptophan synthase is a promising enzyme for SAC synthesis, its expression in microorganisms is strictly regulated by environmental l-tryptophan. Thus, we constructed a semisynthetic medium lacking l-tryptophan using casamino acids. This medium successfully enhanced the SAC-synthesizing activity of Lactococcus lactis ssp. cremoris NBRC 100676. In addition, microorganisms with high SAC-synthesizing activity were screened by the same medium. Food-related Klebsiella pneumoniae K-15 and Pantoea agglomerans P-3 were found to have a significantly increased SAC-synthesizing activity. The SAC-producing process established in this study is shorter in duration than the conventional garlic aging method. Furthermore, this study proposes a promising alternative strategy for producing food-grade SAC by microorganisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Ammonia is critical for agricultural and chemical industries. The extracellular production of ammonia by yeast (Saccharomyces cerevisiae) using cell surface engineering can be efficient approach because yeast can avoid growth deficiencies caused by knockout of genes for ammonia assimilation. In this study, we produced ammonia outside the yeast cells by displaying an l-amino acid oxidase with a wide substrate specificity derived from Hebeloma cylindrosporum (HcLAAO) on yeast cell surfaces. The HcLAAO-displaying yeast successfully produced 12.6 m m ammonia from a mixture of 20 proteinogenic amino acids (the theoretical conversion efficiency was 63%). We also succeeded in producing ammonia from a food processing waste, soybean residues (okara) derived from tofu production. The conversion efficiency was 88.1%, a higher yield than reported in previous studies. Our study demonstrates that ammonia production outside of yeast cells is a promising strategy to utilize food processing wastes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a "mito-CRISPR system" that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Some coral-associated bacteria show protective roles for corals against pathogens. However, the distribution of coral-protecting bacteria in seawater is not well known. In addition, compared with the methods for investigating coral pathogens, few methods have been developed to detect coral-protecting bacteria. Here we prepared a simple method for detecting Ruegeria spp., some strains of which inhibit growth of the coral pathogen Vibrio coralliilyticus . We successfully obtained two Ruegeria -targeting primer sets through in silico and in vitro screening. The primer sets r38F-r30R and r445F-r446R, in addition to the newly designed universal primer set U357'F-U515'R, were evaluated in vitro using environmental DNA extracted from seawater collected in Osaka. These methods and primers should contribute to revealing the distribution of Ruegeria spp. in marine environments.

Terpene alcohol is widely used in perfumes and is known to possess antibacterial activity. Moreover, in its glycosylated form, it can be applied as a nonionic surfactant in food, and in the pharmaceutical, chemical, cosmetic, and detergent industries. Presently, chemical production of terpene glucosides is hampered by high costs and low yields. Here, we investigated the microbial glucosylation of nerol (cis-3,7-dimethylocta-2,6-dien-1-ol), a component of volatile oils, by Agrobacterium sp. M-12 isolated from soil. A microbial reaction using washed cells of Agrobacterium sp. M-12, 1 g/L of nerol, and 100 g/L of maltose under optimal conditions yielded 1.8 g/L of neryl-α-D-glucopyranoside after 72 h. The molar yield of neryl-α-D-glucopyranoside was 87.6%. Additionally, we report the successful transglucosylation of other monoterpene alcohols, such as geraniol, (-)-β-citronellol, and (-)-linalool, by Agrobacterium sp. M-12. Thus, microbial glucosylation has potential widespread applicability for efficient, low-cost production of glycosylated terpene alcohols.

A co-culture platform for bioethanol production from brown macroalgae was developed, consisting of two types of engineered Saccharomyces cerevisiae strains; alginate- and mannitol-assimilating yeast (AM1), and cellulase-displaying yeast (CDY). When the 5% (w/v) brown macroalgae Ecklonia kurome was used as the sole carbon source for this system, 2.1 g/L of ethanol was produced, along with simultaneous consumption of alginate, mannitol, and glucans.

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC-ESI-MS/MS to comprehensively identify these peptides. However, there are many parameters for LC-ESI-MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC-ESI-MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC-ESI-MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC-ESI-MS/MS systems.

In Euglena gracilis, pyruvate:NADP + oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP + oxidoreductase. In contrast, pyruvate:NADP + oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.

Jasmonates are major plant hormones involved in wounding responses. Systemic wounding responses are induced by an electrical signal derived from damaged leaves. After the signaling, jasmonic acid (JA) and jasmonoyl-l-isoleucine (JA-Ile) are translocated from wounded to undamaged leaves, but the molecular mechanism of the transport remains unclear. Here, we found that a JA-Ile transporter, GTR1, contributed to these translocations in Arabidopsis thaliana. GTR1 was expressed in and surrounding the leaf veins both of wounded and undamaged leaves. Less accumulations and translocation of JA and JA-Ile were observed in undamaged leaves of gtr1 at 30 min after wounding. Expressions of some genes related to wound responses were induced systemically in undamaged leaves of gtr1. These results suggested that GTR1 would be involved in the translocation of JA and JA-Ile in plant and may be contributed to correct positioning of JA and JA-Ile to attenuate an excessive wound response in undamaged leaves.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.

Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoyl-CoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation.

Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity.

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.

A two-dimensional high-performance liquid chromatography (2D-HPLC) system for protein separation was developed using an ion-exchange column in the first dimension and a reversed-phase monolithic column in the second dimension. The system demonstrated efficient separation of proteins in comparison with conventional systems. For proteomic analysis, proteins extracted from the cell surface of the yeast were separated by 2D-HPLC and evaluated.

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

Both the propeptide in the precursor carboxypeptidase Y (proCPY) and the mature CPY (mCPY)-specific endogenous inhibitor (I(C)) inhibit CPY activity. The N-terminal inhibitory reactive site of I(C) (the N-terminal seven amino acids of I(C)) binds to the substrate-binding site of mCPY and is essential for mCPY inhibition, but the mechanism of mCPY inhibition by the propeptide is poorly understood. In this study, sequence alignment between I(C) and proCPY indicated that a sequence similar to the N-terminal region of I(C) was present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of I(C). In the presence of peptides identical to the N-terminus of I(C) and the C-terminus of the propeptide, CPY activity was competitively inhibited. The C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.

GTS1 in yeast Saccharomyces cerevisiae is a pleiotropic gene, the expression of which affects a wide range of biological phenomena. A genome-wide transcriptional analysis identified genes upregulated in response to GTS1 induction, most of which were found to be regulated by the Tup1-Cyc8 co-repressor. Among the upregulated genes, FLO1 was indispensable for cell aggregation, one of the pleiotropic effects of GTS1. Deletion of genes encoding the chromatin remodeling complex SWI/SNF abolished FLO1 upregulation and decreased cell aggregation, although the nuclear localization of Gts1p required for cell aggregation was not affected. GTS1 induction caused chromatin remodeling at the FLO1 promoter in a SWI/SNF-dependent manner on micrococcal nuclease accessibility assay. Cyc8p was detected in Gts1p-mediated protein aggregates by agarose gel electrophoresis, indicating that Gts1p inhibited Cyc8p function. These results suggest that inhibition of the Tup1-Cyc8 complex and subsequent SWI/SNF-dependent chromatin remodeling result in Gts1p-mediated gene derepression.

The Saccharomyces cerevisiae gene GTS1 is pleiotropic. GTS1 induction produces a variety of biological phenomena represented by heat tolerance. To clarify the interaction partners of Gts1p, tandem affinity purification and immunoprecipitation were performed. Ssa1p and Ssa2p, members of the 70-kDa heat-shock protein family, were identified. Co-expression of SSA1 or SSA2 inhibited Gts1p nuclear import. As compared to the wild type, the SSA1 and SSA2 double-deletion mutant showed enhancement of Gts1p-mediated heat tolerance in the stationary phase, although neither of the single deletions affected heat tolerance, irrespective of GTS1 induction. These results indicate that the heat tolerance function of Gts1p is regulated by Ssa1p and Ssa2p. Furthermore, time-dependent production of Ssa1p and Ssa2p revealed that Gts1p controls the production of Ssa1p and Ssa2p, and that the total amounts of Ssa1p and Ssa2p are important in inhibiting the unique function of Gts1p.

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition ("click chemistry")-mediated synthesis of molecular probes. (+)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.

The retarding activity of 6-O-dodecanoyl-D-allose against rice growth was higher than that of the octanoate and the decanoate. The activities of 6-O-dodecanoyl-D-glucose, -D-mannose, and -D-galactose against rice seedlings were examined. 6-O-Dodecanoyl-D-allose exhibited the highest activity, suggesting the importance of the alpha-axial hydroxy group at C-3 of D-allose.

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser(473) residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr(308)), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5'-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.

A three-dimensional (3-D) bio-imaging technique was developed for visualizing and quantifying the 3-D distribution of yeast in frozen bread dough samples in accordance with the progress of the mixing process of the samples, applying cell-surface engineering to the surfaces of the yeast cells. The fluorescent yeast was recognized as bright spots at the wavelength of 520 nm. Frozen dough samples were sliced at intervals of 1 microm by an micro-slicer image processing system (MSIPS) equipped with a fluorescence microscope for acquiring cross-sectional images of the samples. A set of successive two-dimensional images was reconstructed to analyze the 3-D distribution of the yeast. The average shortest distance between centroids of enhanced green fluorescent protein (EGFP) yeasts was 10.7 microm at the pick-up stage, 9.7 microm at the clean-up stage, 9.0 microm at the final stage, and 10.2 microm at the over-mixing stage. The results indicated that the distribution of the yeast cells was the most uniform in the dough of white bread at the final stage, while the heterogeneous distribution at the over-mixing stage was possibly due to the destruction of the gluten network structure within the samples.

Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at -30 degrees C and sliced at 10 microm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.

A novel purification system for inactive precursor proteins without conventional time-consuming purification steps was established. We purified an inactive precursor protein, procarboxypeptidase Y (proCPY), which was displayed on the cell surface of yeast, due to difficulty of purifying it by a system in which it is produced in cells.

Albizzia saman, a leguminous plant, is known to open its leaves in the daytime and sleep at night with the leaves folded. beta-D-Glucopyranosyl 12-hydroxyjasmonate (1) was isolated as an endogenous chemical factor controlling this leafmovement. We developed a concise synthesis of optically pure (-)-1 in 9 steps from (+)-2 with a total yield of 58%. Similarly, such analogs of 1 as epi-LCF (13), enantiomer (14), and galactoside (19) were synthesized for a structure activity relationship (SAR) study. The results of this SAR study strongly suggest that the mechanism for the leaf-closing activity of 1 would be different from that of methyl jasmonate, and also suggest the involvement of a different kind of target protein which recognizes the trans-isomer of a jasmonate derivative.

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.

To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.

Carboxypeptidase Y (CPY) inhibitor (I(C)) and its homologous protein (I(C)h) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I(C) and its inhibition mode toward CPY were recently reported, but I(C)h has not been characterized. The molecular mass of I(C)h was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I(C)h is folded as a monomeric beta-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I(C)h was exclusively expressed in the early log phase, whereas I(C) was expressed in the logarithmic and stationary phase. The intracellular localization of I(C)h was different from that of I(C). These findings provide insights into the physiological functions of I(C)h.

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine alpha-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.

Chitinase B was purified from a culture medium of Ralstonia sp. A-471 by precipitation with (NH4)2SO4 and column chromatography with DEAE-Toyopearl 650 M and Sephacryl S-200. The purified enzyme was homogeneous on SDS-PAGE. The molecular weight was 45,000 by SDS-PAGE. The optimum pH was 5.0 and stable pH was from 5.0 to 10.0. In the early stage of the reaction, chitinase B produced beta-anomer of (GlcNAc)2 from the substrate (GlcNAc)6, whereas (GlcNAc)4 produced almost at equilibrium, indicating that the enzyme predominantly hydrolyzes the second glycosidic linkage from the nonreducing end of (GlcNAc)6.

Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, Km values, kcat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.

Potassium (2R,3R)-2,3,4-trihydroxy-2-methylbutanoate (1) was identified as a leaf-closing substance in the nyctinastic plant, Leucaena leucocephala. Compound 1 showed strong leaf-closing activity toward L. leucocephala and was not effective against other nyctinastic plants. The potassium ion was indispensable for the bioactivity of 1. Compound 1 gradually lost its bioactivity because of the exchange of the counter cation during isolation. A leaf-opening substance was also observed in the same plant.

In a mixture containing γ-glutamyl donor (donor) and γ-glutamyl acceptor (acceptor), the glutaminase of Pseudomonas nitroreducens IFO 12694 simultaneously catalyzed a γ-glutamyl transfer reaction and hydrolysis of the donor. The variation of the activities responding to the concentration of glutathione and glycylglycine indicated that the enzyme might be classified in a group of glutaminases that shows hydrolysis prior to transfer reaction. On the other hand, the results with glutamine and ethylamine or methylamine indicated that the enzyme was active in the transfer reaction with suppressed hydrolysis of glutamine, and suggested the possibility of using the reaction for producing γ-glutamylethylamide (theanine) or γ-glutamylmethylamide (γ-GMA). In fact, in a mixture containing high concentrations of substrates (0.7 M glutamine, 1.5 M ethylamine or methylamine) and 0.5 unit/ml glutaminase (borate buffer pH 11), 270 mM (47 g/L) theanine or 250 mM (38 g/L) γ-GMA was formed in 7 h of incubation at 30°C.

cis-p-Coumaroylagmatine (1) was isolated from Albizzia julibrissin Durazz, a nyctinastic plant, as a leaf-opening substance. The compound was quite effective for opening the plant leaves at 5×10(-6) M at night, but was not effective for other nyctinastic plants. The bioactive fraction with leaf-closing activity was also separated from the plant extract. Although the leaf-opening activity of the plant extract changed between the day and night, the content of 1 was almost constant through a 24-h day. These results suggest that the change in content of an unknown leaf-closing factor induced balance between the two leaf-movement factors through a 24-h day.

The chitinase III gene of Aeromonas sp. No. 10S-24 was expressed in Escherichia coli. Production of chitinase III by E. coli was 3-fold higher than that of chitinases in the culture supernatant of Aeromonas sp. The enzyme from E. coli was purified and characterized. The molecular weight of the chitinase III from E. coli was estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to be 55,000. This agreed with the calculated molecular weight of the mature chitinase III (54,111). However, it was different from that of the enzyme from Aeromonas sp. It showed that the chitinase III from Aeromonas sp. had an additional sugar chain, causing the higher molecular weight. Chitinase III from E. coli had almost the same enzymatic properties as chitinase III from Aeromonas sp., but the specific activity of the chitinase III from E. coli is slightly higher than that of the native chitinase III. Both enzymes hydrolyzed chitosan (80% deacetylated) well.

Six chitinases were purified from a culture supernatant of Aeromonas sp. no. 10S-24 by ammonium sulfate precipitation, DEAE-Sephadex A-50, Butyl-Toyopearl 650M, and chromatofocusing. These enzymes were most active at pH 3.5-4.5 and the optimum temperature were 50 degrees C. The molecular weights of the enzymes were 89,000 to 120,000 from SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid sequences of the enzymes were similar to that of chitinase I.

The Escherichia coli ubiA gene coding for 4-hydroxy benzoate octaprenyl transferase is thought to be a key enzyme of ubiquinone biosynthesis. Strains with ubiA disrupted were constructed by chromosomal gene replacement with the chloramphenicol resistance gene. The respiration-defective phenotype of the ubiA mutant was complemented by expression of the COQ2 gene encoding the 4-hydroxy benzoate hexaprenyl transferase of Saccharomyces cerevisiae and such strains produced ubiquinone-8. This strongly supports the idea that COQ2 catalyzes the same enzymatic reaction with UbiA and the substrate specificity of COQ2 is broad. Study of the expression of ubiA using an ubiA-lacZ fusion system showed that the ubiA expression was catabolite-repressed by glucose. This repression by glucose was obvious in the arcA mutant. ArcA is the positively acting transcriptional regulator of the oxygen regulated genes. The molecular mass of the protein product of ubiA was 32kD, found using the over-expression of the ubiA gene.

An extracellular enzyme that produces di-D-fructofuranose-2',1;2,1' dianhydride from inulin was purified from the culture broth of Arthrobacter sp. MCI2493. The molecular weight of the enzyme was 40,000 by gel filtration and SDS polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 6.0 and 50 degrees C. Using this purified enzyme, 100 g/liter inulin was converted into 60 g/liter of DFA I, nystose, and 1-F-fructofuranosyl-nystose after incubation for 30 h.

To investigate the role of signal peptides in extracellular production in Bacillus brevis, the Bacillus stearothermophilus alpha-amylase signal peptide was systematically altered and the effects were analyzed in B. brevis. Efficient extracellular production of the amylase in B. brevis was accomplished either by introducing point mutations at positions between -6 and -4 or by replacing the whole signal peptide with that of B. brevis middle wall protein.

Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.