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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. vinelandiiis 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. colishowed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandiion nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.Graphical AbstractAssembly of 17 nif-related genes into plasmids and expression in E. coli.

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.Graphical AbstractScheme of a mito-CRISPR system for generating mitochondrial DNA-deleted cells in S. cerevisiae.

ABSTRACTA co-culture platform for bioethanol production from brown macroalgae was developed, consisting of two types of engineered Saccharomyces cerevisiaestrains; alginate- and mannitol-assimilating yeast (AM1), and cellulase-displaying yeast (CDY). When the 5% (w/v) brown macroalgae Ecklonia kuromewas 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.

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 Clostridiumspecies; 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 acetobutylicumwas 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 fukuinensischitosanase-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.

ABSTRACTHypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiaecells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novoprotein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

ABSTRACTGlycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiaeto analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.

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 (IC) inhibit CPY activity. The N-terminal inhibitory reactive site of IC(the N-terminal seven amino acids of IC) 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 ICand proCPY indicated that a sequence similar to the N-terminal region of ICwas present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of IC. In the presence of peptides identical to the N-terminus of ICand 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.

Yeast cells displaying organophosphorus hydrolase (OPH), which was anchored using a lectin-like cell-wall protein (Flo1p), were used as a biocatalyst for the detection of organophosphorus compounds (OPs). The concentration of p-nitrophenol produced by the hydrolysis of the OPs paraoxon was calculated from the absorbance change at 415 nm during a 30-min reaction. The apparent Michaelis-Menten constant (K′m) for surface-displayed OPH was estimated to be approximately 50 µM, which is consistent with the value of purified OPH, and shows that cell-surface expression is a useful strategy to overcome the mass-transport resistance of substrates across the cell membrane. Notably, the long-term storage stability of the enzyme activity exceeded 40 days when cells displaying OPH were preserved at temperatures below −4°C. A fiber-optic biosensing system was also constructed using a commercial optical-fiber detection device and yeast cells with surface-displayed OPH. A linear relationship was obtained for paraoxon concentrations of up to 50 ppm (182 µM), with a detection sensitivity of 0.0043 A.U. per ppm (R2= 0.9574) and a detection limit of 5 ppm (18 µM).

The Saccharomyces cerevisiaegene GTS1is pleiotropic. GTS1induction 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 SSA1or SSA2inhibited Gts1p nuclear import. As compared to the wild type, the SSA1and SSA2double-deletion mutant showed enhancement of Gts1p-mediated heat tolerance in the stationary phase, although neither of the single deletions affected heat tolerance, irrespective of GTS1induction. 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.

In this study, a series of on-road observations were conducted to derive information necessary for safe and enjoyable driving. Four pairs of participants were chosen from among those responding to a Web survey and attending a follow-up interview. Each pair was asked to drive six routes. Three of the routes were familiar to one of the pair and new to the other, with the former serving as navigator and the latter serving as driver. For the other three routes, the roles were reversed. Three interviews were conducted, one coming after two drives in which the pair played both roles, in order to derive information considered necessary for safe and enjoyable driving by the participants who served as driver on routes unknown to them. Three kinds of information for safe and enjoyable driving were identified: 1) guidance for routing, 2) support for safe driving, and 3) provision of miscellaneous information, such as information about daily topics of interest to the driver and information about interesting things to see along the route.

Aspergillus oryzaeglucoamylases encoded by glaAand glaB, and Rhizopus oryzaeglucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiaeGRI-117-UK and laboratory yeast S. cerevisiaeMT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaAglucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzaeglucoamylase, had the highest glucoamylase activity on its cell surface.

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.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiaegene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1or ZWF1causes hypersensitivity to isobutanol but not to ethanol. By contrast, deletion of GLN3increases yeast tolerance specifically to branched-chain alcohols. Transcriptomic analyses revealed that isobutanol induces a nitrogen starvation response via GLN3and GCN4, upregulating amino acid biosynthesis and nitrogen scavenging while downregulating glycolysis, cell wall biogenesis, and membrane lipid biosynthesis. Disruption of this response by deleting GLN3is enough to enhance tolerance and boost isobutanol production 4.9-fold in engineered strains. This study illustrates how adaptive mechanisms to tolerate stress can lead to toxicity in microbial fermentations for chemical production and how genetic interventions can boost production by evading such mechanisms.

This study describes a simple and rapid analytical quantitative method for measuring 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) using liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). For a chromatographic condition, Shodex IC NI-424 column (4.6 mm i.d. x 100 mm, 5 μm) for anion analysis and an isocratic elution of 40 mM ammonium formate buffer including 0.1% formic acid (pH 3.75) at a flow rate of 0.5 mL/min was used. The column temperature was set to 40°C. In the analysis of DEH produced by exo-type alginate lyase (AlyFRB) from Falsirhodobactersp. alg1, a peak was detected with a retention time of 3.207 min. The prepared calibration curves for DEH analysis using the selected ion monitoring (SIM) mode of a mass spectrometer revealed a good linear relationship (correlation factor: 0.9998) within the test range (0.1–100 μg/mL). The limits of detection (S/N = 3) and quantification (S/N = 10) for DEH in SIM analysis were 0.008 and 0.027 μg/mL, respectively. Using the developed condition of LC/ESI/MS analysis, separation and detection of alginate unsaturated oligosaccharides were also tested. In an analysis time of about 13 min, this method was able to separate and detect an alginate unsaturated disaccharide, a trisaccharide, and a tetrasaccaride produced by poly(β-D-mannuronate) lyase, respectively. The analysis method established in this study will contribute to the quantitative and qualitative analysis of DEH, and the activity measurement of exo-type alginate lyase.