Gakuzo Tamura

Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable α-amylase from Aspergillus kawachii

Abstract Complementary and genomic DNAs encoding an acid-stable α-amylase (asAA) were cloned from Aspergillus kawachii IFO4308 and their nucleotide sequences were determined. The structural gene (asaA) consisted of a 1,920 bp open reading frame that encoded 640 amino acids. The gene consisted of 9 exons and 8 introns. The signal peptide was composed of 21 amino acid residues. The amino acid sequence from the N-terminus to the 479th residue shoeed 97% homology with the Aspergillus niger acid α-amylase and that from the 480th residue to the C-terminus, including the raw-starch-affinity site, i.e., the TS region, the amino acid sequence of which was rich in threonine and serine, was characteristic of the asAA. A yeast transformant carrying the cDNA produced and secreted an acid-stable α-amylase which was capable of digesting raw starch.

Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding α-glucosidase

Abstract The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding α-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at −670 nt, −596 nt and −544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; −544 to −529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; −521 to −511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase

SummaryAnalysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose. The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding β-glucuronidase (GUS), and the resultant plasmid was introduced into A. oryzae. Expression of GUS protein in the A. oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose. The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined. A comparison of the nucleotide sequence of the A. oryzae glaA promoter with those of A. oryzae amyB, encoding α-amylase, and A. niger glaA showed two regions with similar sequences. Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose.

High level expression of the synthetic human lysozyme gene in Aspergillus oryzae

SummaryAspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed of a higher level compared with the amyB gene because of its multi-copy intergration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

SummaryNorthern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding β-glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

Microbial Transformation of Sterols: Part I. Decomposition of Cholesterol by MicroorganismsPart II. Cleavage of Sterol Side Chains by Microorganisms

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Strept...

An Ascochlorin Derivative, AS-6, Reduces Insulin Resistance in the Genetically Obese Diabetic Mouse, db/db

An ascochlorin derivative, AS-6, is a new hypoglycemic agent orally active in both obese hyperinsulineroic and insulin-deficient diabetic animal models. AS-6, when given as a 0.025–0.2% admixture in the diet, dose-dependently ameliorated polydipsia, polyuria, and glycosuria in the genetically obese diabetic mouse, C57BL/KsJ db/db, while neither insulin nor tolbutamide showed any beneficial effects. The amelioration by AS-6 was associated with a marked decrease in serum glucose and triglyceride. The effects persisted at least 10 wk, accompanied by a steady decrease in drinking water consumption. The chronic treatment prevented pancreatic islet degeneration, e.g., degranulation of the β-cells, basophilic appearance of the exocrine border around the islets, and small round cell infiltration. The isolated islets from AS-6-treated mice released much more insulin in response to glucose than those from untreated controls. A significant correlation between serum immunoreactive insulin and glucose/triglyceride from both treated and untreated mice suggests that AS-6 restores sensitivity and responsiveness to insulin to the mice. In fact, the combined treatment with insulin synergistically decreased serum glucose by 50% below AS-6 treatment alone. Furthermore, the epididymal fat pad slices from AS-6-treated db/db mice increased CO2 generation and lipogenesis over the untreated controls, and the glucose metabolic rate (CO2 generation plus lipogenesis from U-[14C]-glucose) in the slices and the serum glucose level inversely correlated at r = 0.8799. These facts indicate that AS-6 reduces insulin resistance in db/db mice.

Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07– 0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the intergration position of the plasmid on the chromosome.

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.

Effect of yeast fumarase gene (FUM1) disruption on production of malic, fumaric and succinic acids in sake mash

Abstract Organic acids are essential flavor components in alcoholic beverages such as wine and sake (Japanese rice wine). The effects of fumarase deficiency on the productivity of malic, fumaric and succinic acids in Saccharomyces cerevisiae were examined using the FUM1 gene disruptant. The single nuclear gene FUM1 encoding fumarase in S. cerevisiae , DBY746, was disrupted by a site-directed one-step gene disruption technique. The fumarase activity of the isolated FUM1 disruptant was abolished. The FUM1 disruptant having a pet phenotype showed no growth on minimal medium containing glycerol as a carbon source even after prolonged incubation. The productions of organic acids by the FUM1 disruptant in YPD liquid culture containing 10% glucose and in sake mash were analyzed by HPLC. The analytical results of the FUM1 disruptant showed there were no differences in extracellular malate productivity, fumarate productivity had increased, and that succinate productivity was reduced compared with those of the control strain. In the production of sake using the FUM1 disruptant, fermentation ability as monitored by CO 2 evolution was not different from that of the control strain. These results indicate that site-directed FUM1 disruption alters organic acid production without any loss of fermentation ability in yeast.