Kenji Ozeki

Molecular Cloning and Characterization of a Transcriptional Activator Gene, amyR, Involved in the Amylolytic Gene Expression in Aspergillus oryzae

A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the α-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including α-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding α-glucosidase), and amyA (encoding α-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.

Translation efficiency mediated by the 5′ untranslated region greatly affects protein production in Aspergillus oryzae

We demonstrate that the 5′ untranslated region (5′UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model β-glucuronidase (GUS) expression system. Alterations in the 5′UTR resulted in an increase in GUS activity of up to eight-fold, without affecting mRNA levels. Moreover, using the most effective 5′UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.

Characterization of Aspergillus oryzae glycoside hydrolase family 43 β-xylosidase expressed in Escherichia coli

This is the first report of glycoside hydrolase family 43 beta-xylosidase from Aspergillus oryzae. To characterize this enzyme, the recombinant enzyme was expressed in Escherichia coli. Unlike known beta-xylosidases from fungal origins, the enzyme did not show substrate ambiguity and was stable at alkaline pH.

Acrylamide degradation by filamentous fungi used in food and beverage industries

Filamentous fungi (24 strains) used in food and beverage industries were investigated for acrylamide-degradation ability: Aspergillus oryzae KBN1010 showed the highest ability. Little acrylic acid was produced but no glycidamide was detected during AA degradation in roasted green tea; therefore, A. oryzae could be used for reducing the AA concentration.

Isolation of a New Lytic Enzyme for Hiochi Bacteria and Other Lactic Acid Bacteria.

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

SummaryUsing synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.

Production of Xylanase with a transformant of Aspergillus oryzae RIB40 in a Liquid-Surface Immobilization (LSI) System

Xylanase production by a XynF1 (33 kDa)-transformant of Aspergillus oryzae RIB40 was compared between submerged cultivation (SmC) and liquid-surface immobilization (LSI) systems. While the accumulation of xylanase in the SmC decreased by prolonged incubation, LSI system enabled the continuation of xylanase production to afford 4.5-fold xylanase production compared with the SmC system.

Effects of ethyl-α-d-glucoside on human dermal fibroblasts

Ethyl α-d-glucoside (α-EG) is a glycoside present in sake, Japanese rice wine. Previous studies have reported that α-EG suppresses skin roughness after ultraviolet B irradiation, transepidermal water loss, and hepatic function disorder, and has a skin moisturizing effect. In this study, 0.48 μM of α-EG was found to increase the proliferation of normal human dermal fibroblasts (NHDF) by 121.0%, and the amount of collagen I produced by NHDF increased by 159.6% at an α-EG concentration of 0.048 μM, compared to those in cells cultured without α-EG. In NHDF cultured in α-EG-supplemented medium, the expression of fibroblast growth factor I and VII mRNA increased by 148.8 and 153.1%, at an α-EG concentration of 4.8 and 0.048 μM, respectively, as measured by a quantitative reverse transcription-polymerase chain reaction. Transcript levels of type I collagen genes, COL1A1 and COL1A2, increased by 152.4 and 129.7%, respectively, and that of a type III collagen gene, COL3A1, increased by 131.8% at an α-EG concentration of 0.48 μM. These findings supported the possibility that α-EG was involved in the maintenance and improvement of skin homeostasis and moisturizing functions. When Ethyl-α-D-glucoside was added to the medium, fibroblast proliferation and Type I collagen in the medium increased, and expression levels of these genes increased.