Pyruvate metabolism redirection for biological production of commodity chemicals in aerobic fungus Aspergillus oryzae.

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals.

Identification of a novel pyrithiamine resistance marker gene thiI for genome co-editing in Aspergillus oryzae.

Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism.

Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae.

Aspergillus oryzae, a filamentous fungus, can secrete large amounts of enzymes extracellularly. We constructed a genetically engineered A. oryzae that simultaneously produced cellobiohydrolase, endoglucanase, and β-glucosidase by integrating multiple copies of the genes encoding these cellulases into fungal chromosomes.

Sake lees extract improves hepatic lipid accumulation in high fat diet-fed mice.

Background: Nonalcoholic fatty liver disease (NAFLD) is increasing worldwide as one of the leading causes of chronic liver disease. Sake lees (SL) are secondary products of sake manufacturing and are considered to have beneficial effects on human health. To investigate these effects, we used high fat diet (HFD)-fed mice treated with or without the SL extract.

Enhancement of malate-production and increase in sensitivity to dimethyl succinate by mutation of the VID24 gene in Saccharomyces cerevisiae.

Malate in sake (a Japanese alcoholic beverage) is an important component for taste that is produced by yeasts during alcoholic fermentation. To date, many researchers have developed methods for breeding high-malate-producing yeasts; however, genes responsible for the high-acidity phenotype are not known. We determined the mutated gene involved in high malate production in yeast, isolated as a sensitive mutant to dimethyl succinate.

Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae.

Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast.

L-lactic acid production from starch by simultaneous saccharification and fermentation in a genetically engineered Aspergillus oryzae pure culture.

Lactic acid is a commodity chemical that can be produced biologically. Lactic acid-producing Aspergillus oryzae strains were constructed by genetic engineering. The A.

Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose.

Background: Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications.

Synthesis and functional analysis of deferriferrichrysin derivatives: application to colorimetric pH indicators.

Deferriferrichrysin belongs to the siderophore peptide family which are Fe(III)-coordinating cyclic peptides. The common structure of this family is three consecutive hydroxamate moieties, such as N(δ)-acetyl-N(δ)-hydroxy-l-ornithine (Aho). We have designed two deferriferrichrysin derivatives where three Aho residues were arranged as: cyclo(-Aho-Gly-Aho-Gly-Aho-Gly-) and cyclo(-Aho-Ser-Aho-Ser-Aho-Ser-).

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins.

Synthesis and application of an Nδ-acetyl-Nδ-hydroxyornithine analog: identification of novel metal complexes of deferriferrichrysin.

Synthesis of Fmoc-protected N(δ)-acetyl-N(δ)-(tert-butoxy)-l-ornithine has revealed it to be a metal-chelating amino-acid precursor. This protected amino acid was compatible with the preparation of ferrichrome peptides by standard Fmoc-based solid-phase peptide synthesis. Evaluation of deferriferrichrysin for metal ion chelation revealed that zirconium(IV) and titanium(IV) formed complexes with deferriferrichrysin.

Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor, SC35EK, with an N-terminal pyroglutamate capping group.

The bioorganic synthesis of an end-capped anti-HIV peptide from a recombinant protein was investigated. Cyanogen bromide-mediated cleavage of two Met-Gln sites across the target anti-HIV sequence generated an HIV-1 fusion inhibitor (SC35EK) analog bearing an N-terminal pyroglutamate (pGlu) residue and a C-terminal homoserine lactone (Hsl) residue. The end-capped peptide, pGlu-SC35EK-Hsl, had similar bioactivity and biophysical properties to the parent peptide, and an improved resistance to peptidase-mediated degradation was observed compared with the non-end-capped peptide obtained using standard recombinant technology.

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins.

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.

Reliability of rapid urinary test for antibody to Helicobacter pylori in adult patients with proteinuria.

A urinary test for detecting the anti-H. pylori antibody using immunochromatography (RAPIRAN) is considered suitable for the screening purpose. However, this may yield spurious results in the presence of proteinuria.

Wagner-Nelson method for analysing the atypical double-peaked [13CO2] excretion curve in the [13C]-octanoate gastric emptying breath test in humans.

1. In the [(13)C]-octanoic acid breath test, the time versus pulmonary [(13)CO(2)] excretion rate curve is analysed using mathematical curve-fitting techniques to calculate gastric emptying parameters. Thus, the goodness-of-fit highly influences the accuracy of the breath test.

Effect of cigarette smoking on gastric emptying of solids in Japanese smokers: a crossover study using the 13C-octanoic acid breath test.

Background: Cigarette smoking is associated with an increased risk of peptic ulcer and gastroesophageal reflux disease. Gastric emptying disorders may play a role in the development of these upper gastrointestinal diseases. Thus, studies examining a link between smoking and gastric emptying disorders have clinical relevance.

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps.

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand.